Becoming Vegan: The Complete Guide

1 Month Vegan Challenge

This is simply a vegan-challenge program where individuals will go vegan for a month by the end of which there are noticeable changes in the body and health of the individuals partaking in the program. Some of these changes include increased energy, loss of weight, freshness, a lighter and improved skin. According to the creator of this program, there are many good reasons why you should partake in this program which lasts a full month including gain a boosted health status, expanding your choices of foods, and the general environmental impact. On another side of this challenge is the emotional aspect which the author explains that you gain willpower and personal development when you start to eat food from the less cruel method of preparation and develop compassion for the animals. There is no information given for the author of this product even on the product's website, but from all from well-known celebrities like Zac Efron, Alicia Silverstone and Liam Hemsworth, the author is a vegan and program works and it inspires individuals to see the advantages of the vegan lifestyle. Read more here...

1 Month Vegan Challenge Summary

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Detecting the Proteins in Question

How well an immunoprecipitation will work depends on a variety of factors, the most important being the affinity of the antibody to the antigenic site on the protein. Antibody affinity can vary over a wide range, but for an immunoprecip-tiation to work efficiently the affinity of the antibody to the antigen should be at least 107 mol-1 to 109 mol-1 (1). The simplest way to improve the detection of an antibody-antigen complex is to increase the concentration of the antibody and the antigen. This will only be effective under conditions in which the antibody is not saturating, which must be empirically determined by doing a titration of the amount of antibody for a given amount of antigen in a given reaction volume. When the quantity of antibody is limited, it is easiest to reduce the reaction volume. When an epitope-tag is used, it is also possible to improve detection by inserting multiple copies of the tag onto the protein to allow for multivalent binding by the antibody. To determine...

Main Types Of Stress Protein

Since the functions of stress proteins are only now becoming defined, a variety of names exist in the literature. A convenient terminology is based on their migration during electrophoresis on sodium dodecyl sulphate polyacrylamide gels, and on this basis, four main groups can be recognized namely hsp 90, hsp 70, hsp 60, and the small hsps (or hsps 15-30). These groups all contain conserved protein families, and the numbers represent rounded-off values for the apparent relative molecular masses of their subunits for example, the hsp 70 group contains highly related proteins of subunit size 66-78 kDa. The small hsps are especially prominent in plants which contain four multigene families (24). The fifth group is used to encompass a mixture of less well-studied stress proteins that are unrelated in sequence to either one another or to the other groups. The hsp 60 proteins are often called the chaperonins (cpns), since they were the first group identified as functioning as molecular...

Immunoprecipitation of Proteins

Antibody specific to the protein of interest. Polyclonal antisera, ascites fluid, and culture supernatant of a hybridoma that secretes a MAb can all be used for immunoprecipitation. Use approx 1 ig of antibody per im-munoprecipitation. Increase this amount several-fold for antibodies with low affinity to antigen. This has to be determined empirically. Antibody suppliers can be located in the BiosupplyNet Source Book (www.biosup-plynet.com) published yearly in collaboration with Cold Spring Harbor 2. Protein A Sepharose and or Protein G Sepharose. Recipe can be scaled up or down. Hydrate 1.5 g of protein A or G Sepharose beads in 30 mL of 50 mM Tris-HCl, pH 7.5, for 1-2 h on ice. Pellet beads by gravity or very gentle cen-trifugation (1 min at 1000 g) and then wash four times with immunoprecipitation buffer that lacks (the expensive) protease inhibitor mix and contains 1 mM sodium azide. Resuspend the beads in 15 mL of this same buffer to yield a final slurry concentration of approx...

Other Types of Stress Protein

This group is a ragbag of unrelated stress proteins, and only two known to act as molecular chaperones in polypeptide folding (DnaJ and GrpE) are considered here. For information about others such as hsp 75, hsp 104, hsp 110, Ion, lysU, rpoD, Clp, and ubiquitin, reference 21 is a good starting point. The genes encoding the DnaJ and GrpE proteins were discovered, together with that for the DnaK protein, because mutations in any of them block replication of phage lambda DNA in E. coli (52). Subsequent in vitro studies showed that these three unrelated proteins act together in the presence of ATP in dissociating DnaB helicase from a tight inactive complex with other proteins the released active helicase then initiates the unwinding of the DNA double helix at the origin of replication (78). This ability of these three proteins to disassemble protein complexes is aptly described by the term molecular crowbar (13). A family of proteins sharing some similarity to the 70 aminoterminal...

The Unique Role of Heat Shock Proteins in Infections

Parasitic microorganisms have played a tremendous role in the evolution of humans (1). Parasites are microbes which live at the expense of their mammalian hosts. Although some of these parasites interact with their hosts loosely, others achieve intimate contact, thus causing stable infection. As a direct or indirect consequence, the host is harmed and clinical disease evolves. Whether infection progresses in a stable or an abortive form and, as a corollary, whether disease develops or not is markedly influenced by the host immune system. This immune system has the capacity to identify small molecular entities which are characteristic of foreign invaders. Thus, the immune system is first able to distinguish its own molecular entities (termed self) from those of foreign intruders (termed nonself ), and second, it is able to differentiate specifically among the enormous diversity of microbial pathogens. The whole molecule to which the immune system responds is an antigen, and the small...

The atomistic description of globular proteins the tertiary structure

During the research that eventually led A. Fleming to the discovery of the antibiotic penicillin, he found (in 1922) that a substance in his own nasal mucus (he was having a cold at the time ) also killed bacteria. This substance is the protein lysozyme. It catalyses the cleavage of a particular bond in the cell wall of 'gram-negative' bacteria (a large class that includes E. coli), leading to the swelling and bursting ('lysis', hence lysis enzyme lysozyme) of cells. Hen egg white lysozyme has the distinction of being the first enzyme Proteins are polypeptides, or random polymers made up of amino acid monomers (or, in biological parlance, 'residues') see Figure 1. Different amino acids are distinguished by their 'side chains' (R, R', etc., in Figure 1) 20 occur naturally. Hen egg white lysozyme has 129 residues and a molecular weight of Mw 14400. The sequence of residues in lysozyme (the protein's 'primary structure') and its three-dimensional structure obtained by Phillips and his...

Coarsegraining level 3 Proteins as colloids

Globular proteins in their native state are rather compact objects their interiors approach close packing densities. This compactness is a reflection of the high degree of internal ordering (into secondary structural motifs). The radius of gyration of a typical globular protein is considerably smaller than that of a synthetic polymer of comparable molecular weight but lacking internal structure. Thus, it is possible to think of a globular protein as a hard colloidal particle.8 (Nowadays, one may be tempted to call proteins 'nanocolloids' ) 5.1 Dilute protein solutions Colloid scientists today have an impressive array of experimental tools for characterising particles. Even given the atomic details that x-ray crystallography (and nuclear magnetic resonance spectroscopy) can reveal, the tools of colloids science can still be used profitably to study dilute protein solutions. For instance, a careful combination of data from dynamic light scattering (which measures the protein's...

Pct And Protein Stability

The behavior of biosystems under high hydrostatic pressure is governed by Le Chatelier's principle, which predicts that pressure favors processes accompanied by negative volume changes and that, conversely, pressure inhibits processes accompanied by positive volume changes. As with general chemical reactions, a pressure-mediated reaction is governed by the Gibbs free energy change (AG), which is a function of pressure and temperature, for example, d(AG) (AV)dp - (AS)dT. When temperature is constant, volume changes determine the signs of free energy change or equilibrium of reactions under pressure. In cases of proteins and other biopolymers, because the packing of the atoms is not always perfect, Kauzmann proposed that the partial molar volume (V.) of a protein in solution is composed of three components atoms, cavities, and hydration (Equation 1.1).8 Pressure and temperature give rise to changes in cavities and hydration, but not to volume change of atoms. Thus, at constant...

Myelin basic proteins

There are at least 6 isoforms of MBP, whose relative susceptibilities to proteolysis have not been explored, although this could represent another level of control in the developing CNS. The P1 basic protein of PNS myelin is identical to the 18Kd MBP from CNS myelin while the tryptic peptide map of the 14Kd P2 protein is unique18. The P2 protein does however have similarities to the CNS MBP, in that it is highly basic, easily extracted with acid and digestible by the same proteolytic enzymes.

Argonaute Proteins Structure and Function

The Ago gene family is a conserved class of highly basic proteins found in all organisms with a functional RNA silencing machinery. Besides playing a role in RNA silencing they perform other vital biological functions such as germline stem cell development, nuclear division, and centromeric heterochromatin formation during early embryogenesis in Drosophila (Deshpande et al. 2005). Ago2-null mice are embryonic lethal, highlighting their biological role in development. Structural studies of Ago proteins, especially Ago2, have contributed to the unraveling of mechanistic steps involved in RNA silencing and a greater understanding of its biological role (Parker et al. 2005). The Ago proteins have a characteristic PAZ domain of about 130 amino acids and a C-terminal PIWI domain of about 300 amino acids. The PAZ domain is a RNA binding motif and plays a role in binding the 2-nt overhangs at the 3' end of the single-stranded siRNA, while the C-terminal PIWI domain is involved in the...

Drosophila AGO Proteins Unique Features and Crucial Role in RNAi

A series of biochemical experiments performed with Drosophila embryo lysates has generated a wealth of information about the RNAi mechanism in Drosophila and the crucial role played by DmAGO proteins (Matranga et al. 2005 Miyoshi et al. 2005). Peptide and nucleotide sequence analysis revealed that DmAgol closely resembles hAgo2. This comparison was further validated by biochemical experiments showing that DmAgol has the slicer activity by virtue of its association with the guide strand siRNA, thereby mediating cleavage of the target mRNA. In vitro studies using recombinant full-length DmAgol and its various truncated forms showed that the PAZ domain is dispensable for target cleavage, while the PIWI domain and the amino acids in the adjacent domain are the chief determinants of the slicer activity. It is entirely possible that, in vivo, the PAZ domain might be enhancing the RNAi mechanism by virtue of different protein-protein interactions. Thus, in Drosophila both AGO1 and AGO2 have...

Proteinase Inhibitors Of Inflammatory Demyelination

From the evidence that increased neutral and acid proteolytic activities are associated with demyelinating lesions in the CNS and PNS, probably originating from the macrophages in the cellular infiltrate, proteinase inhibitors have been tested both in the experimental model, EAE, and in MS. Proteolytic enzymes may be necessary at several steps of the sensitization process in the development of EAE including the activation of the immunologic process, the invasion of cells into the brain parenchyma, as well as the final dissolution of myelin. The first reports in the literature indicating some degree of success with proteolytic inhibitors in suppressing EAE showed that pepstatin, an inhibitor of acid proteinase will suppress the clinical signs as well as the incidence of lesions of EAE in the Lewis rats 8 0. In another report amino caproic acid, an inhibitor of plasminogen activator, given in large amounts suppressed both the paralysis and lesions of EAE and in Lewis rats81 . importance...

Targeting PCatenin Protein

APC tumor-suppressor function is lost in the majority of colorectal cancers and up to 50 of colorectal cancers with wild-type APC harbor activating P-catenin mutations. In consequence, regulation of P-catenin degradation is severely impaired and P-catenin-mediated signaling is activated. Consistent with the critical function of APC in colorectal carcinogenesis, reexpression of APC in the APC-deficient colorectal cancer cell line HT29 led as a proof of principle to the reduction of P-catenin level, growth arrest, and induction of apoptosis (168). Later studies demonstrated that the central third of the APC protein, which contains the P-catenin binding sites, is sufficient to downregulate P-catenin and to suppress P-catenin-mediated transcription (169). Infection of colorectal cancer cells with an adenovirus expressing this mini-APC fragment resulted in growth inhibition and activation of apoptosis. Another approach to downregulate P-catenin in cell lines focuses on the ubiquitination...

Proteins And Proteolytic Enzymes Of Myelin

Since this chapter deals essentially with the role of proteolytic enzymes in myelin breakdown, it is important to briefly review the structural proteins of CNS and PNS myelin which together with lipids maintain the integrity of the myelin sheath. Several important CNS proteinases partially responsible for destabilizing the myelin sheath in diseases will also be reviewed. Myelin is largely composed of lipids (70 ) and proteins (30 ). The two major proteins of myelin are myelin basic protein (MBP) and proteolipid protein (PLP) which constitute 30 and 50 of all myelin proteins, respectively. PLP (24kD molecular weight) is tightly bound to lipids while MBP (18kD molecular weight) is also complexed with lipids, but with less affinity than PLP16. Minor myelin proteins with important roles include MAG (myelin-associated glycoprotein) MOG (myelin oligodendrocyte-specific glycoprotein), MOBP (myelin oligodendrocyte-specific basic protein), and DM-20 of the PLP family of proteins. Proteinases,...

Six Protein Core Complex Mediates Telomere End Protection

This telomeric t-loop is bound and stabilized by a number of telomere-specific binding proteins that form a six-protein complex, termed telosome (Liu et al. 2004) or shelterin (de Lange 2005). It is postulated that this highly regulated telomere structure serves three essential functions (1) protecting natural chromosomal DNA ends from being inappropriately recognized as double-stranded breaks (DSBs) and therefore initiating an inappropriate DNA damage response (DDR), (2) protecting chromosomal ends from inappropriate enzymatic degradation, and (3) preventing chromosomal end-to-end fusion. Three sequence-specific DNA binding proteins are recruited to chromosomal ends the duplex telomere-binding proteins TRF1 and TRF2 and the single-stranded TTAGGG repeat binding protein POT1 (Protection Of Telomeres 1). These proteins are interconnected by three additional proteins TIN2, TPP1, and RAP1. TPP1 heterodimerizes with POT1 to augment its association with telomeres (Wang et al. 2007, Xin et...

Measuring Half Lives of Proteins in Mammalian Cells by Pulse Chase Analysis

This section describes a standard procedure for measuring half-lives of proteins in adherent tissue culture cells. The same principle applies to nonadherent cells, although the initial metabolic labeling procedure will be slightly different (see Note 1). 1. For measuring the half-lives of proteins exogenously expressed via transient transfection, plate 2 X 106 HeLa cells on one to two 10 cm dishes, and incubate in CO2 incubator overnight. For measuring half-lives of endogenous proteins, plate equal number of cells in five 100 mm dishes and culture for 1 d. Enough cells should be plated on each dish so they reach 60-80 confluency on the second day, which is optimal for pulse labeling (see Note 2). 3. Add 100 Ci of Express-35S protein-labeling mix to each 100-mm plate (see Note 3). Incubate the cells for 30 min at 37 C with shaking every 5-10 min. 6. For all other time points, add 3 mL chase medium containing prewarmed DMEM, 10 FCS, 3 mM L-methionine, and 1 mM L-cysteine to each plate....

Measuring the Half Life of Yeast Proteins

This section describes two commonly used methods to determine the half-lives of proteins in yeast pulse-chase analysis and promoter turn-off. Pulse-chase analysis is generally used for determining the turnover rate of endogenous proteins or proteins expressed from constitutive promoters on plasmids. Promoter turnoff is a simple procedure used to measure the stability of proteins exogenously expressed from inducible promoters on plasmids. 1. Grow yeast cells overnight to stationary phase in either YPD for endogenous proteins or synthetic complete medium lacking the amino acid corresponding to the selectable marker on the transformed plasmid. 4. Add 100-500 iCi of Express-35S protein labeling mix per mL of cell culture and shake at 30 C for 10 min. See Note 10 for measuring half-life in temperature sensitive mutant yeast cells. 5. Determine the protein concentrations by the Bradford assay or the DC protein assay (Bio-Rad) (see step 11 of Subheading 3.1.2.). 7. Perform...

Purification of Expressed vSrc Protein

This section gives detailed protocols for preparing yeast cell extracts and recovering the induced v-Src protein by immunoprecipitation. 5. Add 20 iL 2X SDS-PAGE sample buffer to one tube, mix with the bead by vortexing, heat for 5 min at 100 C. Then separate the sample by SDS-PAGE and analyze by Western blot to ascertain that the Src protein was properly expressed and affinity-purified. The anti-v-Src 327 MAb can also be used for the Western analysis.

Soluble TCR and MHC Proteins

In this study, we have used the 2B4 TCR, which recognizes peptide residues 81-104 from pigeon cytochrome c complexed to the murine MHC class II, I-Ek (26-28). Soluble TCR and MHC proteins can be prepared using various expression systems. Several of these expression systems, which include bacterial, yeast (Pichia pastoris), and Drosophila, have been found to yield appreciable quantities of soluble TCR proteins in our hands (19,29,30), although no technique has worked well for all TCRs. The 2B4 TCR described here was expressed using Pichia pastoris expression vectors pPIC9 and pPIC9K (Invitrogen, San Diego, CA) (19). The methods for the production of active MHC class II proteins in soluble forms are highly reproducible. Currently, the most commonly used methods include insect cell expression of truncated proteins (31,32) and Escherichia coli expression and refolding from inclusion bodies (33). In the study described here, soluble Class II I-Ek proteins were purified from Triton X-100...

Assaying Src Protein Tyrosine Kinase Activity

The first protocol makes use of a small synthetic peptide substrate. Such pep-tides can be designed as optimized substrates for any protein kinase of interest, thus enabling precise determination of kinetic parameters (17). The oligopep-tide includes basic residues allowing its separation from nucleotides and free phosphate present in the kinase reaction mixture through its ability to bind tightly to phosphocellulose paper (18). 1. For each reaction, prepare 10 iL of 3X concentrated protein kinase assay buffer (3XPKB) 50 mM HEPES, pH 7.4, 24 mM MgCl2, 6 mM MnCl2, 300 iM Na3VO4, 60 iM ATP, 15 ig mL leupeptin, 15 ig mL aprotinin, and 5 iCi y-32P ATP (see Notes 1 and 2). Use the kinase assay buffer stock solutions and mix thoroughly by pipeting after adding each compound. Be sure to account for radioactive decay when adding the y-32P ATP. The y-32P ATP should be added last from this step you must exercise radioactive safety precautions. 3.3.2. Protein-Tyrosine Kinase Assay Using...

Proteins in Disease

The heterotrimeric G proteins are involved in various diseases through mechanisms involving modifications of Ga-subunits, genetic changes and conceivably by acting as transducers of signals implicated in pathogenetic processes, which, in some cases, may involve quantitative changes in certain subgroups of G proteins. However, it is difficult to define how tight the observed alterations in G protein levels are connected to the disease mechanisms i.e., whether they only reflect secondary changes or if they are integrated in significant patho-genetic steps. The disease groups covered here, that could have been even more extended, represent main clinical areas wherere there is evidence, although to a variable extent, for an involvement of G proteins.

Dicer Proteins in Different Organisms

Dicers are large multidomain proteins found in most eukaryotes (e.g., animals, plants, and Schizosaccharomyces pombe, but not in Saccharomyces cerevisiae). Metazoan and plant Dicer proteins generally contain ATPase helicase, DUF283, PAZ, two RNase III, and a dsRBD, but Dicers of lower eukaryotes frequently have a less complex domain organization (Fig. 1). The PAZ, dsRBD, and RNase III domains are involved in dsRNA binding and cleavage. The PAZ domain is also found in PPD (PAZ and Piwi domain) or Argonaute proteins that are also involved in RNAi and miRNA pathways. The presence of the helicase ATPase domain could explain the observation that the generation of siRNAs by the C. elegans Dicer and one of the two Drosophila Dicers is stimulated by addition of ATP (Bernstein et al. 2001 Ketting et al. 2001 Liu et al. 2003 Nykanen et al. 2001). However, ATP has no significant effect on the activity of the mammalian enzyme even though it contains the ATPase helicase domain (Zhang et al. 2002)....

FPLC Purification of Proteins

It is essential that highly purified proteins be used for BIAcore binding kinetics determination. All purified proteins to be used in BIAcore binding assays are first subjected to size-exclusion chro-matography. This step is important in removing protein aggregates that might be present and is performed as close to the BIAcore binding assays as possible, preferably the same day or the night before. Although larger aggregates tend not to bind, dimers or trimers can cause potential complexity in the nature of binding. A representative example of separation of aggregates from active MHC proteins over a Superdex HR200 column is shown in Fig.1.

Immobilization of Proteins

In all BIAcore measurements of biomolecular interaction, a protein is selected for coupling to the sensor chip ('the ligand'), while the binding partner ('the analyte') is pumped over the ligand surface. The first step in SPR measurements of protein-protein binding interaction studies is, therefore, the selection of immobilization chemistry. For most protein ligands, the most commonly used coupling chemistry is amine coupling. However, it is advisable to use other, more favorable chemistry, which may allow a site-directed approach to surface immobilization (see Note 1). Some of these coupling chemistries (e.g, thiol, aldehyde, biotin-streptavidin) are useful in preparing a more homogenous ligand surface and often help to avoid some of the pitfalls of amine coupling. Other experimental design and analysis considerations, such as mass transport, rebinding, and so on, are highlighted in a recent issue of this series (35). For studying the trimolecular interactions between TCR, MHC, and...

ArgonautesPPD Proteins

Another group of well-characterized Dicer partners is represented by PPD or Argonaute proteins. Members of the PPD protein family contain two signature domains a PAZ domain in the center and a PIWI domain at the carboxyl terminus (Carmell et al. 2002 Tolia and Joshua-Tor 2007). Genetic and biochemical studies have indicated that PPD proteins are involved in control of stem cell differentiation, tissue development (Carmell et al. 2002), and chromatin modification (Verdel et al. 2004 Irvine et al. 2006). PPD proteins can be divided into two subgroups those that are homologous to the Arabidopsis Argonaute-1 and are ubiquitously expressed, and those that are most similar to the Drosophila Piwi, expressed in germline stem cells (Carmell et al. 2002 Tolia and Joshua-Tor 2007). These subgroups are referred to as Argonaute (Ago) and Piwi proteins. Different Ago proteins have been identified as components of the RISC in different organisms (Tabara et al. 1999 Hammond et al. 2001 Caudy and...

Protein Identification In Seldi Array Research

Regardless of study design, once a protein of interest has been detected, protein characterization efforts often ensue. Proteins are characterized by identifying post-translational modifications, providing primary sequence information, and, ultimately, by elucidating protein identity. As a typical starting point, proteins are chemically reduced and then exposed to endoprotease digestion. The resultant peptides may also be treated with dephosphorylating, deglycosylating, or exoproteolytic enzymes. At each step along the way, MS detection is performed to monitor changes in peptide

PGlycoprotein MDR1 PGP

Fig. 10.2 Mechanisms of penetration through the blood-brain barrier. 1 transcellular lipophilic pathway 2 paracel-lular aqueous pathway 3 transport proteins 4 specific receptor-mediated endocytosis 5 efflux pumps Fig. 10.2 Mechanisms of penetration through the blood-brain barrier. 1 transcellular lipophilic pathway 2 paracel-lular aqueous pathway 3 transport proteins 4 specific receptor-mediated endocytosis 5 efflux pumps

Fusion Proteins in the Expression Vectors

In many cases, different fusion proteins lead to substantially different expression levels and solubility of the target proteins. Thioredoxin, protein B G1 domain, and maltose-binding protein (MBP) are known to promote high expression as well as high solubility of the target proteins. Protein B G1 domain is small ( 56 residues) and does not cause severe complication of spectral analysis and, hence, can be used as a sample solubility enhancer of the target proteins or protein complexes. However, caution must be paid since fusion proteins may prevent folding or induce misfolding of the target proteins, particularly when the N-terminal regions of the proteins are important interior components of protein structures. In these cases, proteins are usually found in insoluble inclusion bodies or look unfolded as judged from the NMR spectra. To avoid this problem, vectors without fusions such as pET3a (Table 3.1) can be exploited.

Optimization of Protein Expression

When proteins or domains have low expression level and low solubility, a number of expression vectors need to be explored for systematic and sometimes time-consuming optimization. The first thing to do to increase protein expression is to choose appropriate cell lines. The most common cell lines (strains) for bacterial protein expression are BL21(DE3), BL21(DE3) pLYS, HMS, etc., which are commercially available. The cDNA containing the target protein is usually transformed into the above strains on Day 1. The next day, one colony is picked from each transformation and grown in a 5 ml culture for each strain. Cell density (ODgoonm) is checked after a few hours and 1 mM IPTG (isopropyl-1-thio- -D-galactoside) is added (typical for the first time but can be varied, see below) when the OD is approximately 0.6 to induce protein expression (1 ml of culture is sampled before addition of IPTG). The cells are harvested after 3-4 hr and spun down at 10,000 g. The pellets are lysed by sonication...

Overexpression Of Isotopelabeled Proteins

After optimization of protein expression, the next step is to overexpress in large scale isotope-labeled proteins for NMR studies. Heteronuclear multidimensional NMR experiments for structure determination of medium-sized protein requires that the target proteins or domains be uniformly 15N and or 13C-labeled. This is done by growing cell cultures in minimal media in which 15NH4Cl and or 13C glucose are the only sources for nitrogen and carbon atoms. The standard recipe for the minimal media is shown in Table 3.2 and the recipe for Trace Element Solution in Table 3.3. The most common cell lines for protein expression in minimal media are BL21(DE3), BL21(DE3) pLYS, HMS, etc., which are commercially available. Note that protein expression levels are usually lower in minimal media as compared to rich LB media. Hence, it is recommended that different cell lines be used to explore optimum expression. Due to the expensive cost of 13C glucose, it is recommended to optimize glucose usage at...

Iistress Proteins In The Normal

Most cells within the normal CNS express constitutive forms of stress proteins. This was demonstrated in our laboratory using a panel of 20 monoclonal antibodies with specificities either for the 65-kDa stress protein of mycobacteria, the human 70 72-kDa stress protein, or the human 60-kDa stress protein. A rabbit polyvalent antibody to the Escherichia coli stress proteins was also used. These antibodies were used to immunocytochemically stain normal CNS (1). Patterns of staining varied with the particular antibody. Most monoclonals to the 65-kDa mycobacterial stress protein stained neuronal, glial, and microglial cell bodies. Similar staining patterns were seen with the polyvalent rabbit antiserum. Little, if any, staining was noted of oligodendrocytes with any of the monoclonals tested. Three monoclonals bound to normal central and peripheral nervous system myelin. One such monoclonal was IH9, specific for an epitope of the Mycobacterium leprae 65-kDa stress protein. Another...

Purification Of Isotopelabeled Proteins

Purification of isotope-labeled proteins is a key step and probably the most time-consuming step for NMR sample preparation. The procedures and tips for purifying the isotope-labeled proteins are the same as for non-labeled proteins described in many textbooks and the literature. If the labeled proteins contain fusions, fusion-targeted affinity columns will be the first step for purification followed by protease cleavage and gel filtration. This procedure typically works if the protein behaves during the process however, protease cleavage sometimes can be a tricky process. Excess amounts of protease or over-digestion by protease can lead to non-specific cleavage and hence optimization is usually required. When proteins are not fused, the chemical structure and physical properties of the proteins are the two key parameters used to develop the most efficient purification protocols. Isoelectric point ( pI), pH stability, and charge density are important properties to be exploited during...

Biomarker Amplification Via Carrier Protein Sequestration Underpinnings Of The Mass Spectral Information

Based on the need for identification, our own laboratory research efforts have centered on the identification and sequencing of the underlying discriminatory information that exists in the mass range profiled by direct MS profiling work. Some have argued that only high-abundance molecules are represented in the mass spectral read-out, and that, as such, these molecules can be only nonspecific epiphenomena.33 Relevant to this concern, we are beginning to understand some of the mechanisms by which low-abundance biomarkers can be amplified biologically to detectable concentrations. As we sought to understand the source and identity of the molecules, we realized, and experimentally demonstrated, that a vast majority of the LMW biomarkers under study were actually complexed with high-abundance circulating carrier proteins.8'30'3435 Accumulation of LMW biomarkers in association with circulating carrier proteins greatly amplifies the total serum plasma concentration of the measurable...

Ivstress Proteins As Markers Of Nervous System Injury

Stress protein expression has been used as a marker to define the extent of injury caused by particular experimental or natural disease processes. For example, it was used to define regions of ischemia (3,6-8), hyperthermia (8-12), and trauma (8,13) in several experimental systems. It was also used to identify injured cells in patients with Alzheimer's disease (10,14-16) and other neurodegenerative diseases (10,14,16). Expression of stress proteins in affected cells frequently occurred in the absence of other anatomical changes, suggesting that stress protein expression may be a sensitive marker of cell injury. The functions of stress proteins in injured nervous system are not clear, but they may play a protective role. For example, Lowenstein and coworkers (17) noted that induction of hsp 72 in cultured rat cerebellar granular cells protected them from the damaging effects of the excitotoxin glutamate.

Preparation of Protein Peptide Complexes

The contact surface contributing to the interactions of high affinity and specificity often involves 30 or less amino acid residues from each protein of the complex (de Vos et al, 1992 Song and Ni, 1998). Often this contact surface is located in a single continuous fragment of one of the proteins, which can be identified by mutation and deletion experiments. Therefore, fragments can be chemically synthesized in large amounts and studied by two-dimensional1H NMR experiments due to their small molecular size (Wuthrich, 1986). Samples for protein-peptide complexes are commonly prepared from isotopically labeled protein and unlabeled peptide according to the following procedure, since the availability of labeled peptide is often prohibited by the expense of chemical synthesis from labeled amino acids and the difficulty of biosynthesis due to peptide instability during its expression and purification (Huth et al., 1997 Newlon et al, 1997). Preparation of the complexes is done by titrating...

Preparation of Protein Protein Complexes

Protein-protein interactions play an essential role at various levels in information flow associated with various biological processes, such as gene transcription and translation, cell growth and differentiation, neurotransmission, and immune response. The interactions frequently lead to changes in the shape or dynamics as well as the chemical or physical properties of proteins involved. Solution NMR spectroscopy provides a powerful tool to characterize these interactions at the atomic level and at near physiological conditions. With the use of isotopic labeling, the structures of many protein complexes in the 40 kDa total molecular mass regime have to be determined (Clore and Gronenborn, 1998). The development of novel NMR techniques and sample preparation has been further increasing the mass size available for the structural determination of protein complexes. Furthermore, NMR has been utilized to quickly identify the binding sites of the complexes based on the results of chemical...

Domain Structure and Function of RNase III Proteins

Table 1 Three-dimensional structures of RNase III proteins Table 1 Three-dimensional structures of RNase III proteins Protein Protein Data Bank (Berman et al. 2000) cThe 5' terminal RNA hairpin of Snr47 precursor dCoordinates are not available ePrimary reference is not available Fig. 2 A-C Schematic view showing the crystal structures of A Gi-Dicer Mn2+ (PDB entry 2FFL, Table 1), B Aa-RNase III dsRNA Mg2+ (2EZ6, Table 1), and C Hs-Agol-PAZ dsRNA (1SI3). Proteins are illustrated as surface representations and ribbon diagrams (helices as spirals, P-strands as arrows. and loops as pipes), RNA as rod (backbone) and sticks (bases), and metal ions as spheres. The proteins are color-coded on the basis of their domain structures (Fig. 1A) platform in black, PAZ in green, connector in red, endoND in cyan or yellow, spacer in pink, and dsRBD in gray. The Mn2+ ions are gray and Mg2+ are black. The dsRNA strands are in red and blue, and the stem-loop in gray. The orientations of the three...

PH Bile Salts and Proteins for Biorelevance pH Adjustment

Proteins or Micellar Additives for the Creation of Sink Conditions In order to preserve sink conditions in the static in vitro models, it has been proposed to include additives in the receiver compartment that are able to decrease the free drug concentration (Figure 5). Among all the proposed alternatives, the ideal option should be suitable for high-throughput screening and not increase the workload associated with the Caco-2 transport assay. The inclusion of serum albumin (used as such or as present in culture medium) in the receiver compartment has been demonstrated to modify the transport properties of drugs (Mathieu et al., 1999 Walgren and Walle, 1999 Aungst et al., 2000 Yamashita et al., 2000 Krishna et al., 2001 Deferme et al., 2002 Saha and Kou 2002 Demirbas and Stavchansky, 2003 Neuhoff, 2005). The presence of albumin in the basolateral compartment can promote drug partitioning from the cell monolayer into the basolateral compartment in addition, it can also prevent Figure...

Chemical Nature Of Protein Streaking

These smears and streaks are proteins cross-linked through the formation of intramolecular and intermolecular disulfide bridges (R S-S-R when R1 R2 represents intramolecular disulfides) following the oxidation of the cysteinyl thiol groups (-SH).5,6 Therefore utilizing a reductant in sample solubilization buffer, such as the most commonly used dithiothreitol (DTT) or its isomer dithioerythritol (DTE), is a common practice to reduce disulfide bond and to help unfolding and disaggregating the protein prior to the first dimension IEF. However DTT and DTE are weak acids that can migrate toward the anode during IEF, leading to the loss of the reducing agent from the basic portion of the IPG strip.7,8 In an environment lacking a reducing agent, the highly reactive cysteinyl thiol groups tend to cross-link again, leading to the spontaneous restoration of disulfide bridges and therefore the presence of a highly streaky basic end (pH 7). The electrochemical oxidation at cathode may also...

Stress Protein Gene Transfer Into Tumor Cells

Work over the past decade has demonstrated the importance of heat shock proteins in the immune response to many infectious agents. Heat shock proteins have also been implicated in a variety of autoimmune conditions and situations where inappropriate immune responses are occurring, such as the immuno-pathologically mediated nerve damage seen in leprosy (13) and in thryoid follicles of patients with Graves' disease (14). Among the explanations for such findings was the possibility that high levels of heat shock proteins in a particular cell or tissue could be increasing or altering processing and or presentation of self-molecules such that they were much more likely to be recognized by the immune system. This hypothesis was tested by transfecting the macrophage cell line J774 with the mycobacterial hsp-65 gene J774 cells are weakly immunogenic tumor cells of BALB c origin and are also derived from antigen-present-ing cells, and hence they provide an ideal model for testing the...

Protein Immobilization On Solid Supports

The fabrication of protein and antibody arrays is routinely based on the manual or robotic printing of samples onto a solid phase. The physicochemical parameters of the surface are important to ensure a high sensitivity in binding assays. Choice of surface is determined by the need for a low background, reproducibility of detected fluorescent signals, binding capacity, and protein structure preservation. A wide variety of materials have been developed and tested to immobilize proteins, and a comprehensive list of supports and chemical approaches to functionalize various surfaces is described elsewhere 7 . A simple way to anchor protein molecules is by physical adsorption by noncovalent hydrogen bonding or electrostatic, hydrophobic, and van der Waals interactions with flat or porous supports. The traditional glass slide coated with polylysine, widely used for the generation of DNA arrays, has been found to be suitable for protein and antibody arrays 8 . Noncovalent but high-affinity...

Washing Protein ASepharose Immune Complexes

Pellet the Protein A-sepharose-bound immune complexes by centrifuging at 4000 g for 2 min at 4 C. 2. Wash the Protein A-sepharose-bound immune complexes with 500 pL of each solution twice with ice-cold IP wash buffer 1, once with ice-cold IP wash buffer 2, and twice with ice-cold 1X TE buffer. 3. Transfer the Protein A-sepharose immune complex pellets to new 1.5-mL microfuge tubes with first wash of IP wash buffer 1. This step is critical for reducing background. 5. Pellet the Protein A-sepharose at 4000 g for 2 min at 4 C, and discard the supernatant (see Note 22).

Protein Profiling With Total Protein Arrays

Total protein arrays, also referred to as reverse phase protein arrays, consist of immobilized extracts of cells, tissues, or sera that are probed using a given antibody, followed by detection with a labeled secondary antibody (fig. 10.2C). This format allows numerous protein samples to be analyzed simultaneously with one or two antibodies under the same experimental conditions 24,27,53 . Such protein arrays were used to study cancer progression from normal prostate epithelium to intra-epithelial neoplasia and to invasive prostate cancer 24 . A remarkable reduction was observed in the ratio of phosphorylated ERK to total ERK during the longitudinal progression of the cancer. In a similar way, protein microarrays were employed to FIGURE 10.3 (See color insert.) Two-color protein profiling in cell lysates from breast tumor cell lines MDA MB-231 and SKBR3. Analytes were labeled with IRDye 800CW (red) and Alexa Fluor 680 (green). Circled numbers refer to position in the array of...

Expression Of Host Stress Proteins

Mammalian cells normally express immunogenic hsp peptides which are recognized in MHC-restricted manner by autoreactive CD4 T cells under conditions of syngeneic mixed lymphocyte culture (83). The expression of mammalian hsp molecules in infected macrophages is of keen interest in the light of the report that enhanced hsp 60 expression closely correlated with the host protective immunity following immunization with Toxoplasma gondii (84). In the case of mycobacterial infection, it was suggested that macrophages producing reactive oxygen metabolites may protect themselves from damage by enhanced synthesis of stress proteins (85). However, hsp 70 synthesis in human B cells was found to be stimulated following heat shock but not by peroxide treatment (86) moreover, heat stress failed to protect the cells from the adverse effects of peroxide treatment on antigen processing. So far, only one study has reported an increased production of the heat-inducible, but not of the constitutive, form...

Autoassembling Protein Microarrays

Snyder and coworkers used conventional gene cloning and recombinant protein expression with affinity purification methods to prepare the first whole-proteome array, composed of 5,800 of 6,200 yeast open reading frames 12 . Its functional exploitation allowed new calmodulin targets and phospholipid-binding proteins to be identified. However, there appears to be an alternative to the costly and time-consuming strategy of gene cloning in cells that might be widely used for other proteomes. Prokaryotic and eukaryotic cell-free systems for protein synthesis were developed a long time ago 86-88 . The best systems, with improved biosynthetic and energetic parameters, provide quite a high yield of many protein families and the production of toxic and more soluble proteins. Tagged proteins produced in cell-free systems and purified by affinity were used to prepare arrays and to study protein-protein and protein-DNA interactions, thereby significantly shortening an array-based binding protocol...

Mutations in Mitochondrial Proteins Indirectly Affecting OXPHOS

Showing us that we should broaden our perspective of what defects can cause mitochondrial energy failure. Several different diseases could be put in this group, of which Freidreich's ataxia, (caused by a mutation in fratax-in, a protein involved in iron homeostasis) and autosomal dominant optic atrophy, (caused by mutations in OPA1, dynamin-related guanosine triphosphatases) are probably the most common ones 43 .

Enmr Of Proteins In Biological Buffer Solutions

In the earlier days of ENMR, when large glass tubes were used as sample chambers, the study of proteins in biological buffer solutions of high salt concentration was impossible due to the heat-induced convection. For such protein solutions, the electric conductivity (a) is high and the electric field (EdC IJaA) is too low to drive the electrophoretic motion of proteins in the large U-tube glass chambers of fixed cross-sectional area (A). The high electric field required for this purpose would be accompanied by a relatively large electric current (Ie) and, subsequently, by large heat-induced convection that would prevent accurate ENMR measurements of protein migration. To control sample temperature, heat is removed in ENMR experiments by cooling air or liquid outside the sample tube 11,23,34 . Since heat is more effectively removed at the edge of the tube rather than the tube center, a temperature gradient is established, which in turn produces a density gradient to cause bulk...

Proteins and Higher Order Complexes

RdRPs have been shown to interact with a number of proteins produced by either the virus or the host, particularly during the initiation of RNA replication. In the Picornaviridae, a 22-amino-acid virally encoded initiator protein called VPg (virion protein genome linked) is uridylylated by the RdRP as an initial step in replication (Lee et al. 1977 Nomoto et al. 1977 Paul et al. 1998). The structure of the FMDV RdRP-VPg complex reveals interactions between VPg and the RdRP active-site cleft that position the side-chain hydroxyl group of Tyr 3 in VPg near the a-phosphate moiety of the uridine triphosphate (UTP) cosubstrate (Ferrer-Orta et al. 2006). In combination with mutational studies (Boerner et al. 2005 Lyle et al. 2002 Pathak et al. 2002), this structure reveals a number of residues in the active site cleft involved with the binding of VPg and with the uridylylation reaction involved with the initiation of RNA synthesis. Higher-order complexes involving proteins and RNA...

Analysing Protein Domains

Analysis of proteins by experts enables classification to be driven by expert knowledge, which draws on the collective knowledge in the community. Experts can interpret the information from the biological literature and apply it to the observed results. This is, however, a time-consuming process and many academic institutions cannot support large teams of bioinformaticians required for such activities. The alternative choice is automated classification. This tends to be quicker, but the level of detail is often reduced, which means proteins are often only classified into broad functional classes. For example, taking the top BLAST hit as a basis for classification of an unknown protein can infer relationships between the unknown protein and previously characterized proteins, allowing the new sequences to be annotated as 'similar to' a characterized protein. This has value, but it also has intrinsic problems. One of the largest problems is that the databases of characterized sequences...

Classifying Proteins into Families

Many proteins are assemblies of sequence motifs and domains. Each domain or motif might have a separate function within the protein, such as catalysis or regulation, but it is the overall composition that gives each protein its specific function. Recognition of domain and motif composition is a powerful bioinformatics technique which can be employed in the classification of proteins. There are many tools dedicated to discovering protein features and functional domains and motifs (hereafter referred to as p-domains). Examples include PROSITE 9 and Pfam 10 . These tools each employ different methods of analysis to detect sequence features and p-domains, for example, PROSITE uses simple pattern-matching to single motifs, whereas Pfam uses hidden markov models (HMMs). Researchers routinely use many different p-domain detection tools together to build up a consensus of results. To facilitate this process, InterPro encapsulates many of these tools, and allows scientists to perform analyses...

The Protein Phosphatase Family

The enzymes primarily involved in catalyzing phosphorylation events can be divided into two families, protein kinases and protein phosphatases. Kinases are involved in the phosphorylation of the amino acids serine, threonine and tyrosine 16 and phosphatases are involved in the removal of phosphates from these residues. It is the careful balance between these two opposing reactions that controls the phosphorylation state of a multitude of biological molecules and ultimately controls almost all biological processes Protein phosphatases all perform the same chemical reaction in the cell, the removal of a phosphate group, but the phosphatases are diverse in biological function and catalytic activity. They can be broadly divided into two subfamilies, the serine threonine phosphatases and the tyrosine phosphatases. Recent reviews on the protein phosphatase family ( 18 , 19 and 20 ) focus on either one or the other. There have been extensive studies into the characterisation of each in the...

Activation of the protein kinase C pathway

Agonists, such as bradykinin, bind to G protein-coupled receptors that consequently lead to the release of IP3 (producing an elevation in intracellular Ca2+) and dia-cylglycerol. Release of Ca2+ and liberation of diacylglycerol results in the activation of conventional subtypes of protein kinase C (PKC). Early studies using phorbol esters to directly activate PKC showed that this pathway was involved in the stimulation of primary afferents 29, 30 as well as isolated sensory neurons 31 . Later work by Schepelmann et al. 32 demonstrated that phorbol esters could directly excite primary afferents innervating the knee joint, but also, phorbol esters lead to a sensitization of the response to passive movement of the joint. Consistent with these observations, Barber and Vasko 33 found that low concentrations of the phorbol ester, PDBu, enhanced the release of neuropeptides from isolated sensory neurons. Taken together, these observations suggested that activation of PKC played an important...

Bone morphogenetic proteins

Zhang and Bradley21 demonstrated the central role of bone morphogenetic protein-2 (BMP2) in mammalian cardiogenesis. In a murine embryonic model the generation of a homozygous null mutation for BMP2 led to abnormalities in cardiac development. Full cardiac differentiation of non-cardiogenic tissue by administration of soluble BMP2 or BMP4 to explant cultures was shown by Schultheiss et al.22 Data has been produced for the discreet roles of BMP2 and BMP4 in the developing mouse heart. It appears that the formation of the atrioventricular junction and valves is linked to BMP2, with BMP4 involved in the development of the outflow tract myocardium and of the endocardial cushion.23 Furthermore the exposure of precardiac mesoderm to Noggin, a known BMP antagonist, resulted in inhibition of cardiac differentiation.22 Even though BMPs play a crucial role in both processes of mesodermal induction24 and cardiomyocyte differentiation,21 there seems to be a transient but essential inhibitory...

Proteinprotein interaction databases

To date, over 50,000 protein-protein interactions have been reported in the literature and catalogued into various databases 27 . Among these databases are the Database of Interacting Proteins (DIP) 28 , the Biomolec-ular Interaction Network Database (BIND) 29 , and the Molecular Interactions Database (MINT) 30 . Additionally, a number of web servers have arisen to catalog both known and putative protein pathways. These servers include the Kyoto Encyclopedia of Genes and Genomes (KEGG) 31 , the Encyclopedia of E. coli Genes and Metabolism (EcoCyc) 32 , and the Munich Information Center for Protein Sequences (MIPS) 33 . Together these databases and web servers provide a useful source for investigating proteinprotein interactions in organisms ranging from E. coli to human.

Computational methods to identify protein linkages

In addition to biochemical methods to identify linked proteins, a number of computational methods have been developed to identify functionally linked proteins, including the Rosetta Stone 8 , Phylogenetic Profile 11 , conserved Gene Neighbor 14, 15 , and Operon Gene Cluster 13, 34 methods. Each of these methods utilizes genomic sequence information garnered from genome sequencing efforts. Currently there are over 300 completed genomes available 35, 36 , and over 1,000 ongoing genome sequencing efforts 35 . Together these efforts provide us with a tremendous amount of information regarding not only the genetic blueprint of hundreds of organisms, but also facilitate the computational inference of protein linkages and protein networks.

Stress Proteins in Inflammatory Liver Disease

Only very few studies have thus far dealt with the expression of heat shock proteins (hsps) in normal and inflamed liver, and little is known to date on specific disease associations and immune responses to hsps in liver diseases. These studies have almost exclusively concentrated on the 60 kDa heat shock protein, hsp 60. The acute phase response in systemic inflammatory conditions is centered on the liver, where protein synthesis is rapidly adjusted to the need of the endangered organism. It is likely that this massive response is associated with a marked increase in the expression of stress proteins, but data on this are not yet available. Using freshly isolated and cultured guinea pig hepatocytes, Kupffer cells, and sinusoidal endothelial cells, we were able to attain more information on the distribution of hsp 60 in the normal liver. Western blot analysis of solubilized proteins from these cells showed marked expression in normal hepatocytes, significant expression in Kupffer...

Using Global Methylation Inhibitors as a Tool to Study Protein Methylation

Proteins in wild-type (+ +) and Prmt4 mutant ( ) MEF cell lines were labeled in vivo with L- methy -3H methionine. Immuno-precipitations (IP) were performed with aPABPl (a PRMT4 substrate) and aSam68 (a PRMT1 substrate) antibodies. The 3H-labeled proteins were visualized by fluorography. The molecular mass markers are shown on the left in kDa. Fig. 3. In vivo methylation assay. Proteins in wild-type (+ +) and Prmt4 mutant ( ) MEF cell lines were labeled in vivo with L- methy -3H methionine. Immuno-precipitations (IP) were performed with aPABPl (a PRMT4 substrate) and aSam68 (a PRMT1 substrate) antibodies. The 3H-labeled proteins were visualized by fluorography. The molecular mass markers are shown on the left in kDa. Sinefungin is a commonly used AdoMet analogue and adenosine dialdehyde (AdOx) is often used as an AdoHcy hydrolase inhibitor. Because demethylase activity is very low or absent in cells (see Note 5), endogenous protein substrates are...

The evolutionary capacity of proteins

One of the remarkable properties of proteins is the redundancy of sequence space with respect to structure space. There are numerous sequences that fold into the same shape. An obvious question is how large 'numerous' is, and in this section we attempt to address this problem (Meyerguz et al. 2004). More concretely, we compute the entity we name 'structure capacity' the number of sequences that a particular protein can accommodate up to an energy E. We consider protein sequences that improve on the stability of the native structure, i.e., sequences that are more stable than the native sequence of a particular (experimentally determined) protein structure. We find an exponentially large number of 'better' (more stable) sequences. The observation that one may improve stability (in a considerable way) compared to the natural sequence is perhaps not that surprising, since protein sequences are not optimised for structural stability only. True biological sequences are subject to...

Computing temperatures for all protein folds

We have computed the number of sequences for all relevant free-energy differences N (AF). This function has a strong (exponential) dependence on the protein length, which is easy to rationalise. The total number of possible sequences is exponential in length (20L). The actual number of accepted sequences is expected to grow as ML (M 20) (still grows exponentially with the length). Every length extension of the Figure 7. Computing sequence capacity for five proteins of the same length (150 amino acids), from the set of 3660 proteins that we analyse are shown in detail. The proteins are (from left to right) 1f3g, 1nul, lash, 1br1, 1bbr. Figure 7. Computing sequence capacity for five proteins of the same length (150 amino acids), from the set of 3660 proteins that we analyse are shown in detail. The proteins are (from left to right) 1f3g, 1nul, lash, 1br1, 1bbr. protein molecule and the addition of a new structural site will allow a few more amino acids (per site) to be accommodated,...

Retinoid Processing Proteins in the Apical RPE

Hypothesis for a retinoid-processing complex in apical RPE. Left A model representation of apical RPE process filled with actin fibers cross-linked to each other and to the plasma membrane. Right Enlarged view of the model in the left with identified protein components labeled. Figure 66.3. Hypothesis for a retinoid-processing complex in apical RPE. Left A model representation of apical RPE process filled with actin fibers cross-linked to each other and to the plasma membrane. Right Enlarged view of the model in the left with identified protein components labeled. logical analyses of the bead-bound RPE apical membranes support a highly purified preparation. Proteins were recovered from the beads, separated by SDS-PAGE, gel bands excised, digested in situ with trypsin and proteins identified by LC MS MS. CRALBP, EBP50, RDH5 and ezrin were among the proteins bound to the lectin beads.23 These findings further support the proposed retinoid-processing complex in the RPE. Many...

Definition of Protein Solubility

Protein solubility is a thermodynamic characteristic of the protein solvent system defined as the concentration of soluble protein in equilibrium with the solid phase at a given pH, temperature, and solvent composition (Flynn, 1984 Arakawa and Timasheff, 1985 Middaugh and Volkin, 1992). For practical purposes, solubility of proteins can be defined as the maximum amount of protein that remains in a visibly clear solution (i.e. does not show protein precipitates, crystals, gels, or hazy soluble aggregates), or does not sediment at 30,000 g cen-trifugation for 30 min (Schein, 1990 Ducruix and Reis-Kautt, 1990).

Hydration effects and the dynamical transition 21 The dynamical transition in proteins

Various experimental techniques such as neutron scattering, Mossbauer spectroscopy and x-ray scattering have shown the presence of a temperature-dependent transition in protein protein and solvent at same temperature protein held at 180K water held at 180K protein held at 80K water held at 80K protein and solvent at same temperature protein held at 180K water held at 180K protein held at 80K water held at 80K of the protein non-hydrogen atoms for differ- dynamics at around 180-220 K (Doster et al. 1989, Dunn et al. 2000, Parak et al. 1981, Tilton et al. 1992). In this temperature range the dynamics of proteins, as represented by the mean-square displacement, (u2), of the protein atoms, change from harmonic behaviour below the transition temperature to anharmonic behaviour above. This dynamical transition has also been seen using molecular dynamics (MD) simulation techniques (Smith et al. 1990, Hayward and Smith 2002, Bizzarri et al. 2000). Figure 1 shows such a simulation, in which a...

Ligandprotein Complexes

As NMR spectroscopy has been widely used to determine the structures and dynamics of molecules ranging from synthetic compounds to macro biomolecules, it has become a powerful approach for studying the interactions between proteins (and or nucleic acids) and ligands. The interactions can be studied by observing a change in NMR phenomena (signal) that is induced by the binding. For this purpose, a variety of pulse sequences has been implemented to observe changes in chemical shifts, mobility, relaxation properties, and NOEs, etc. Some of the methods make full use of the difference in mass between protein and ligands, such as methods measuring the diffusion and relaxation of ligands, whereas others observe binding-induced changes such as chemical shifts, NOE, and 1H exchange rate.

Solvent caging of protein dynamics

Figure 1 also presents the protein fluctuations calculated from the dual heatbath simulations, performed fixing the temperature of one component below the dynamical transition while varying the temperature of the other component. Fixing the solvent temperature at 80 K or 180 K suppresses the dynamical transition, the protein (u2) increasing linearly with temperature up to 300 K. Therefore, low temperature solvent cages the protein dynamics. Figure 1 also shows that holding the protein temperature constant at 80 K or 180 K and varying the solvent temperature also abolishes the dynamical transition behaviour in the protein. In summary then, holding either component at a low temperature suppresses the protein dynamical transition. Cold (80 K and 180 K) solvent is seen to effectively cage protein dynamics over the whole range of protein temperatures examined (from 80 K up to 180 K). This indicates the important role of solvent in influencing protein dynamics.

Pdes Interacts With Isoprenylated Protein

Summary of PDES interacting proteins. Interacting Proteins A group of small GTPases in the Ras superfamily were shown to interact specifically with PDES. Most of these GTPases have a common feature, that is, a CAAX box motif in their C-terminal amino acid sequence which signals posttranslational modification resulting in cleavage of -AAX polypeptides followed by carboxymethylation and prenylation (prenyl-thioether formation) of the cysteine residue. The last residue of the CAAX box specifies whether the added prenyl chains are either farnesyl (Ci5 moieties) or geranylgeranyl (C20 moieties). Prenylation is a step necessary for the targeting of proteins to membrane, and interaction of PDES with prenylated proteins relies on the prenyl lipid chain. The binding of PDES to PDE in vitro is mediated by its prenylated C-terminal (Cook et al., 2000). The y2h screening in our lab identified rhodopsin kinase (GRK1) as another interacting member. Mammalian GRK1s are farnesylated...

Dynamical transition and protein function

Protein function is dependent on protein flexibility. As during the dynamical transition there is a significant increase in flexibility, a loss of function might be expected below the dynamical transition temperature. A number of studies have indeed shown that at least some proteins cease to function below dynamical transition (Daniel et al. 2003). However, the temperature dependence of motions in a cryosolution of the enzyme glutamate dehydrogenase when examined and compared with enzyme activity (Daniel et al. 1999) showed that the enzyme activity remains below the measured picosecond-timescale dynamical transition at 220 K with no significant deviation of activity from Arrhenius behaviour down to 190 K. These results suggest that there exists a range of temperatures (190-220 K) at which the enzyme rate limiting step does not require, and is not affected by the anharmonic motions taking place on the picosecond timescale. Another important aspect of enzyme activity is the hydration...

Ubiquitinlike proteins

Two types of ubiquitin-like (Ubl) proteins have been identified type 1 and type-2 Ubls28. Type 1 Ubls, such as SUMO1 (small Ub-related modifier) andNEDD8 (neural precursor cell-expressed developmentally down-regulated gene), are small and are covalently attached to proteins in a manner similar to ubiquitination, although they require their own enzymatic components29. Some SUMO1-modified proteins seem to assist nuclear translocation of other proteins28. NEDD8-protein interaction is important in cell cycle regulation28. Type-2 Ubls, such as RAD23, Parkin and ElonginB, are not ligated to other proteins. Instead, they occur as fusion proteins with a ubiquitin-like domain located at their N-terminus, in the central portion, or at the C-terminus. The physiological significance of these fusion proteins remains uncertain, although they may function in DNA repair (RAD23) or as ubiquitin ligases (ElonginB).28

Ontology Initialization for Proteins

We now need ontology support for analysing protein information (Req. 2), just as for organisms. iName and orlglrt of the protein Protein name 5.3.1 The Swiss-Prot Protein Database The UniProt Knowledge Base 3 is a set of two protein databases, Swiss-Prot10 and TrEMBL. Both hold entries about proteins appearing in published works, including information about protein functions, their domain structure, associated organisms, post-translational modifications, variants, among others. Swiss-Prot, which consisted of 228,670 entries as of 2006-07-02, contains manually-annotated records wich information extracted from literature and curator-evaluated computational analysis, 11 while TrEMBL is populated by automatic analysis tools. In the Mutation Miner system, we use the manually curated Swiss-Prot database to gain reliable grounding (see Section 4.2) of proteins found in biological documents (Req. 4). Figure 13-6 shows the Swiss-Prot entry for a variant of the xyianase 2 protein. The entries...

Highsensitivity Esims Analysis Of Protein Tryptic Digests Using Ultra Low Id Polystyrenedivinylbenzene Monolithic

The high mass sensitivity identification of large numbers of peptides from protein digests is one of the major goals of proteomics. A main characteristic of nanocolumns is high mass sensitivity, as a result of the decreased dilution of the chromatographic band.45,48 Nanoflow LC, using commercially available 75 and 100 m i.d. reversed-phase columns, offers the advantages of high resolution, high mass sensitivity, and low sample and mobile phase consumption. However, analysis of a limited amount of sample (e.g., immunoprecipitated protein complexes, laser capture microdissected cells, 2D gel spots) can still be challenging with the above columns. For a fixed limited amount of sample injected, columns with smaller inner diameter can decrease chromatographic band dilution45,48 and thus increase the signal for concentration-sensitive ESI-MS.49 Theoretically, downscaling from conventional nano-LC columns of 75 to 20 m i.d. should result in a gain in sensitivity of (d1 d2)2 14 (for the same...

Protonation reactions in proteins

Electrostatic interactions are important for understanding biochemical systems. Acid-base reactions create or destruct unit charges in biomolecules and can thus be fundamental for their function. Together with association reactions and chemical modifications such as phosphorylations, acid-base reactions are the main cause of changes in protein properties. Protonation or deprotonation of titratable groups can cause changes in binding affinities, enzymatic activities, and structural properties. Moreover, very often protonations or deprotonations are the key events in enzymatic reactions. The reduction or oxidation of redox-active groups has a similar importance. In particular, the reduction of disulfide bonds can cause unfolding or functionally important conformational transitions. Consequently, the function of most proteins depends crucially on the pH and on the redox potential of the solution. For example, acidic denaturation of proteins in the stomach is a prerequisite for protein...

Coupling between conformational and protonation state changes in membrane proteins

Many membrane proteins transport electrons and protons across a membrane (Ullmann 2001). Protonatable groups play a prominent role in these reactions, because they can either function as proton acceptors or donors in proton transfer reactions or they can influence the redox potential of adjacent redox-active groups. The titration behaviour of protonatable groups in proteins can often considerably deviate from the behaviour of isolated compounds in aqueous solution. This deviation is caused by interactions of the protonatable group with other charges in the protein and also by changes in the dielectric environment of the titratable group when the group is transferred from aqueous solution into the protein (Ullmann and Knapp 1999, Beroza and Case 1998, Briggs and Antosiewicz 1999, Sham et al. 1997). The situation can be even more complicated because owing to the fact that the charge of protonable residues depends on pH, their interaction is pH-dependent. This can lead to titration...

Analysis of conformational changes in proteins

Proteins often have multiple stable macrostates. The conformations associated with one macrostate correspond to a certain biological function. Understanding the transition between these macrostates is important to comprehend the interplay between the protein in question and its environment and can even help to understand malfunctions that lead to diseases like cancer. While these conformational transitions are usually too fast to be measured experimentally, they also occur too rarely to observe them by running standard molecular dynamics simulations. They thus pose a difficult challenge to theoretical molecular biophysicists. In this final section, we will briefly summarise computational methods which have been proposed to analyse conformational changes in macro-molecules and identify possible reaction pathways. In particular we will be interested in the analysis of complex transitions in proteins such as the conformational switch in Ras p21 (Figure 6). Computationally, the problem of...

Stability of Peptide and Protein Pharmaceuticals

Chapters 2, 3, and 4 concerned the stability of pharmaceuticals containing pharmacologically active ingredients of relatively low molecular weight. This chapter addresses the stability of peptide and protein drugs. Peptides and proteins can undergo some of the same degradation processes seen in small molecules. However, the stability of protein and peptide pharmaceuticals can be affected by additional reactions that alter their tertiary or higher structures. Like drugs of low molecular weight, peptides and proteins undergo chemical degradation pathways such as hydrolysis and racemization. Depending on their molecular weight, they are also susceptible to physical degradation by denaturation, aggregation, and precipitation. Because of the complicated degradation mechanisms, it is generally more difficult to predict the stability of peptide and protein pharmaceuticals. Chemical and physical properties of peptides and proteins have been studied extensively. The thermodynamics of protein...

Degradation of Peptide and Protein Pharmaceuticals

Degradation observed with peptide protein pharmaceuticals is classified into chemical and physical mechanisms. The former involve changes in covalent bonds, and the latter involve changes in noncovalent interactions such as hydrophobic bonding associations. For a specific peptide protein, degradation usually includes both chemical and physical pathways as well as interactive pathways that might result when a molecule undergoes intermolecular disulfide exchange accompanied by precipitation. This section outlines each of the major chemical and physical degradation pathways.

Telomere Binding Proteins and Disease

Abstract An increasingly growing number of human diseases including cancer and age-related diseases have been associated to telomeric dysfunction. The progressive telemore loss that occurs with aging in humans is proposed to contribute to the patho-biology of age-related diseases. Evidence for this comes from the study of mouse models that reproduce the corresponding full-blown human pathology only when in the setting of critically short telemores. Moreover, telomerase itself, as well as several telomere-binding proteins, has been found altered in human pathologies. Here, we will review recent findings suggesting that the length and function of telomeres are a biological determinant in the development of certain human diseases associated with aging, particularly from the perspective of mouse models for telomere dysfunction.

Proteinenergy Malnutrition

In acute starvation, the nervous system sustains itself first on glucose derived from alanine, then on ketone bodies from the breakdown of fats. This process continues until fat is depleted, and then catabolism returns to visceral proteins. Death results from cardiac muscle resorption and eventual cardiac failure. In chronic PEM, the nervous system adapts poorly, and a retarded rate of brain growth, hypomyelination, and slowed conduction velocities of peripheral nerves results. Traditionally, PEM has been viewed as either a primary protein deficiency (kwashiorkor), or an energy deficiency (marasmus). Kwashiorkor occurs in children older than 18 months and is often associated with diarrheal illnesses or other infectious diseases. Owing to the severe protein deficiency, circulating serum proteins are inadequate to maintain normal oncotic pressure, leading to marked peripheral edema. Marasmus occurs in children younger than 1 year of age and is not accompanied by edema. PEM may cause...

Biogenesis Of Ubiquitin Protein Inclusions Aggresomes

The hallmark of many neurodegenerative diseases is the presence of intraneuronal inclusions consisting of ubiquitin protein conjugates. The mechanisms leading to formation of such abnormal aggregates remain unclear and their role in the progression of the disease has yet to be elucidated123. It is possible that inclusions arise from a cellular attempt to compartmentalize accumulated proteins, and prevent their interference with normal cell function. Their presence may also confer cytotoxic effects that can contribute to cellular damage associated with neurodegeneration. Aggregate size may be a pivotal determinant in their toxicity92. As the ubiquitin protein aggregates expand they may confer fatal effects by chokin the cell as the cytosolic or nuclear space is ultimately filled by the abnormal aggregates 124. The fact that many components of the ubiquitin proteasome pathway, such ubiquitin C-terminal hydrolases and 26S proteasome subunits, are found together with ubiquitin conjugates...

Degradation in Peptide and Protein Formulations

Degradation of peptides and proteins in formulations is complex because various factors may be involved in the degradation. Therefore, it is usually not easy to elucidate degradation mechanisms. Analytical methods such as electrophoresis and gel permeation chromatogra-phy are useful in assessing the stability of peptide and protein formulations and in studying degradation mechanisms.828 829 Peptide mapping procedures were used to elucidate the mechanism of degradation of monoclonal antibody formulations containing polysorbate 80 they indicated that the major routes of degradation were deamidation, oxidation, and formation of cross-linkages.830 Temperature-gradient gel electrophoresis is useful for investigating whether protein denaturation is reversible or irreversible.831 Quasi-elastic light scattering is useful for determining changes in size distribution upon peptide or protein aggregation.832 Adsorption of peptides and proteins onto the walls of containers has been reported for...

Detection of Phosphoproteins

When considering the protein load for 2D gels, it is advisable to start with a load appropriate for a Colloidal Coomassie stain, which is similar to the sensitivity of Pro-Q Diamond phosphoprotein gel stain for singly phosphorylated proteins. We routinely load 150 to 250 g of protein of a cell lysate for large format 2D gel (4-7 or 3-10 IPG strips). After running the 2D polyacrylamide gels, it is important to fix the gels in 50 methanol, 10 acetic acid overnight. All steps are conducted with gentle agitation (50 rpm). Complete removal of SDS ensures specific staining especially if a lower grade SDS is used. In some cases a second fix for 1 hour might be needed. The next day the fixative is washed out with three 15-minute washes in dH20. The washes can be extended for up to 30 minutes each without negative influence on the final results. The gels are then stained for 1.5 to 2 hours in Pro-Q Diamond dye solution followed by three 30-minute destainings. Before imaging, it is important to...

Factors Affecting the Degradation of Peptide and Protein Drugs

Degradation of peptide and protein drugs is affected by various factors. Chemical degradation of peptide and protein drugs is often dependent on pH, buffer components and concentration, and the presence of excipients.839-845 As with small-molecule drugs, stabilization by incorporation into microspheres has been observed with peptide and protein drugs.846 This section describes mainly the factors affecting physical degradation such as denaturation and aggregation, which are specific to peptide and protein drugs. Although physical degradation may occur during formulation processing, including steps such as stirring, filtration, dilution, pressure loading, freezing, and drying, this section focuses on degradation during storage of peptide and protein pharmaceuticals.

Detection of Glycoproteins

For gel imaging a UV light source is used for excitation in combination with a 520 nm band pass filter or a Wratten 9 longpass filter. Even though the stain binds covalently to carbohydrates, proteins can still be identified by mass spectrometry, especially, if the carbohydrate moiety is cleaved of before trypsin digestion using PNGaseF, for example, for N-linked carbohydrates.

Protein Protein Complexes

Protein-protein interactions play an essential role at various levels of the information flow associated with various biological processes such as gene transcription and translation, cell growth and differentiation, neurotransmission, and immune responses. The interactions frequently lead to changes in shape and dynamics as well as in the chemical or physical properties of the proteins involved. Solution NMR spectroscopy provides a powerful tool to characterize these interactions at the atomic level and at near-physiological conditions. With the use of isotopic labeling, structures of many protein complexes in the 40 kDa total molecular mass regime have been determined (Clore and Gronenborn, 1998). The development of novel NMR techniques and sample preparation has been increasing the mass size further for the structural determination of protein complexes. Furthermore, NMR has been utilized to quickly identify the binding sites of the complexes based on the results of chemical shift...

Protein Peptide Complexes

The contact surface contributing to the interactions of high affinity and specificity generally involves 30 or less amino acid residues from each protein of the complex (de Vos et al., 1992 Song and Ni, 1998). Frequently, this contact surface is located in a single continuous fragment of one of the proteins, which can be identified by mutation and deletion experiments. Therefore, fragments can be chemically synthesized in large amount and studied by 1H NMR experiments owing to their small molecular size (Wuthrich, 1986). In the study of protein-peptide complexes, samples prepared according to the procedure discussed in Chapter 3 for isotopic-labeled protein and unlabeled peptide are most commonly used since the availability of labeled peptide is prohibited by the expense of chemical synthesis from labeled amino acids and the difficulty of biosynthesis due to peptide stability problems during expression and purification. Data collection and resonance assignment for the complex can be...

Quantitative Description of Peptide and Protein Degradation

Chemical degradation of peptide and protein pharmaceuticals can also be analyzed kinetically in the same manner as for small-molecule drugs. Specifically, chemical degradation of small peptides in aqueous solutions generally conforms to simple first-order kinetics. For example, first-order kinetics have been reported for the hydrolysis in aqueous solution of secretin, which has 27 amino acid residues (Fig. 205).795 Deamidation, hydrolysis, and epimerization of an LH-RH antagonist having 10 amino acid residues (Fig. 206),802 deamidation of calcitonin, having 32 amino acid residues (Fig. 207),876 and degradation of gonadorelin877 and growth hormone-releasing hexapeptide878 also follow first-order kinetics. Kinetic analysis has even been attempted for the degradation of peptide and protein pharmaceuticals for which the mechanism and pathways are unknown. Apparent inactivation of a-chymotrypsin and bromelain in aqueous solutions was described by monoexponential The kinetics of solid-state...

Rpe Microvilli Proteins And Function

Recently, we have improved a method to isolate RPE apical microvilli. The procedure relies on the binding of N-acetylglucosamine and sialic acid-containing glycoconjugates present in abundance on the RPE apical surface16 to the WGA lectin conjugated to agarose beads. Mass interactions of the surface glycoconjugates with the immobilized lectin on the bead allow for the detachment of the RPE microvilli upon physical removal of the WGA beads. The RPE isolated microvilli are resolved by SDS-PAGE, in gel digested with trypsin, and peptides extracted and analyzed by mass spectrometry.317 This procedure was done in mice eyecups with the RPE exposed and it has resulted in the identification of over 283 proteins, distributed over functional categories such as retinoid-metabolizing, cytoskeletal, enzymes, extracellular matrix components, membrane proteins and transporters, among others. A summary of selected proteins identified by this method is presented in Table 72.1 and has been recently...

Temperature Dependence of the Degradation Rate of Peptide and Protein Drugs

Stability prediction for peptide and protein drugs under accelerated testing conditions is possible if the temperature dependence of the degradation rate is determined and found to be well behaved. The temperature dependence can often be represented by the Arrhenius equation, as was seen with small-molecule drugs. Linear Arrhenius plots and the values of apparent activation energy calculated from the slopes have been reported for chemical degradation of various peptides in aqueous solutions. Values of approximately 20 kcal mol

Theory Of Spin Relaxation In Proteins

The relaxation rates of proteins are affected primarily by dipolar interactions and chemical shift anisotropy (CSA). For 2H labeled proteins, the 2H quadrupolar interaction also contributes to the relaxation rates. The overall relaxation rates are the linear combination of all rates of the interactions. The relaxation rates can be expressed in terms of the combination of spectral density functions (Abragam, 1961 Kay etal., 1989). For an isolated XH spin system, the relaxation rate constants of the X spin (15N or 13C) caused by the dipolar interaction of the X spin with the 1H spin and by the magnetic shielding arising from the CSA interaction of the X spin

Protein Dynamics From Snapshots to Movies

Although NMR is playing a vital role in structural proteomics efforts, its main contribution may be in a related effort. In the actual proteome as it exists in the cell, proteins are not static structures but rather are constantly in motion and NMR is able to characterize protein motions on various timescales. Proteins are constantly in motion, with entire domains and regions of secondary structure and even sidechains moving, to accommodate binding events as well as changes in structure needed for the protein to accomplish its function as a receptor, enzyme, ion channel, molecular motor, and so forth. Dynamic motion is vital to protein function, which depends on alterations in 3D structure in response to specific molecular interactions with other proteins or with small molecule ligands the focus of chemical proteomic studies. Multidimensional NMR methods, combined with isotope labeling, can provide access to dynamic information for virtually every atom in a protein.22 Typical...

CoTransformation and CoExpression of Fusion Proteins

Both green and unpigmented plant tissues are suitable for BRET experiments. However, it is critical that the pigmentation level is the same between tissue co-expressing the RLUC-and YFP-tagged proteins and tissue expressing the RLUC-only control. It is important to include the RLUC-tagged protein alone as a negative control in every experiment.

Heat Shock Proteins As Carrier Molecules For Conjugated Bcell Epitopes Avoid The Need For Adjuvants

PPD is composed of a plethora of mycobacterial epitopes, and repeated analysis of the preparation have failed to characterise it chemically. (10). In the definition of which mycobacterial antigen(s) present in the PPD were mediating the carrier effect observed, three observations were made. The PPD portion of the conjugate could not be replaced by T-cell epitopes without homologies with known mycobacterial antigens. Second, only mice previously primed with live BCG, but not those that had received heat-killed or sonicated BCG, produced anti-(NANP) antibodies after immunization with the PPD-peptide conjugate in the absence of adjuvants. Finally, antipeptide IgG antibodies could also be induced following immunization with the PPD-peptide conjugate without adjuvants in mice sensitized with other live intracellular parasites of the macrophage, such as Salmonella typhimurium and Leishmania tropica (18). Taken together, these data suggested that the priming was being mediated by...

Moduration of Neurite Outgrowth by a Recombinant TGN Protein and RNAi in Retinal Explant Cultures

Addition of the recombinant TGN protein induced a large number of explants with long and thick neurites after 2 days (Fig. 73.2b,d), as compared with the control culture (Fig. 73.2a,d). The neurite outgrowth of the culture containing recombinant TGN protein was evoked in 50 of the explants during 2 days of culture whereas neurite outgrowth of the Figure 73.2. Explant culture of adult goldfish retinas treated with recombinant TGN protein and TGN-specific RNAi. (a) No addition. (b) Recombinant TGN protein (4 lg ml). (c) TGN-specific siRNA (100 pmol ml). (d) Graphical representation of the neurite outgrowth for 2 days of culture. Note a suppression of neurite outgrowth by TGN-specific siRNA (**p 0.01) and an enhancement of neurite outgrowth by recombinant TGN (*p 0.01) compared with control cultures. The values represent the mean SD in five independent experiments. Scale bar 200 m. Figure 73.2. Explant culture of adult goldfish retinas treated with recombinant TGN protein and...

Analysis of proteinRNA associations in rRNA processing

Since all the experimental evidence available indicates that metazoan rRNA processing events are not autocatalytic, there is considerable interest in learning about the cellular components that are responsible for carrying out the processing reactions. A number of methods are available for exploring the association of proteins with pre-rRNA substrates. These are described in the following sections as applied to the 5' ETS processing in mouse. 3. Dilute the stock 20mg ml heparin solution to 0.35mg ml with H20. Gently add 2 pi of 0.35 mg ml heparin to the incubation mixture (step 2) to remove loosely associated proteins. Do not mix the contents of the microcentrifuge tube after the heparin addition. Figure 5 illustrates the use of Northern blot analysis following the gel-shift assay. Here the presence of small nuclear RNAs was shown using an unlabelled rRNA substrate and a radiolabeled hybridization probe for the snRNA. One example is the U3 snRNP which is present in complexes formed on...

Cell Surface Proteins

Bacterial cell-surface proteins play a role in host-parasite interactions and adaptive response to host defense systems. Cell-surface proteins also represent candidates for vaccine development and serve as useful markers for discrimination of closely related species or strains. As previously described, the Wolbachia surface protein gene exhibits a high level of sequence diversity between species, and has been used as a standard gene for strain identification. Genome sequencing of Wolbachia wMel identified two additional paralogues (named wspB and wspC) in the genome (12). Wu et al. showed that wspB evolves faster than wsp, and proposed that wspB may be an additional marker for discriminating between closely related Wolbachia strains. The A. marginale genome contains two large superfamilies containing immunodominant proteins (56-member msp2 superfamily and 9-member msp1 superfamily). As previously described, recombination between functional genes and pseudogenes contributes to the...

Separating Protein Signals Without Physical Separation

Multidimensional electrophoretic NMR (nD-ENMR) was initially designed to study 3D structures of coexisting proteins and protein conformations in solution without physical separation of the protein components. By applying a DC electric pulse, the NMR signals of the proteins can be resolved in a new dimension of electrophoretic flow at different resonant frequencies proportional to their electrophoretic migration rates (Figure 27.2).21 In a 3D-ENMR experiment, for example, the overlapping two-dimensional (2D) NMR spectra of different molecules can be resolved into different subspectral planes as if they were independently obtained from pure protein solutions. Each subspectrum of the component protein gives the NMR parameters of chemical shifts, spin J-coupling constants, or dipolar coupling parameters for sequential and stereospecific structural assignments. The feasibility of separating protein signals in the electrophoretic flow dimension by ENMR was demonstrated using a protein...

Modulation of Intracellular Proteins

Heat shock protein 90 (Hsp90) is a molecular chaperone induced under stress conditions. It supports the proper folding, stability, and function of target proteins involved in cell growth and survival, i.e., protein kinases and hormone receptors (Neckers 2002). Hsp90 inhibitors cause the inactivation, destabilization, and eventual degradation of Hsp90 target proteins (Neckers 2002) and have thus reached attention as potential antitumoral drugs. In Flt3-ITD-positive AML, Flt3-ITD is a direct target of Hsp90 (Minami et al. 2002). Flt3-ITD transformed 32D cells, but not parenteral 32D cells, exhibited marked growth inhibition and apoptosis after treatment with Hsp90 inhibitors (Minami et al. 2002). Co-treatment of the Hsp90 inhibitor (17AAG) with either the FLT-3 kinase inhibitor PKC412 (George et al. 2004) or the histone deacetylase inhibitor LBH589 (George et al. 2005) had synergistic cytotoxic effects in human Flt-ITD-positive leu-kemic cells. In addition, 17AAG demonstrated...

Determination Of Protein Active Pockets

Since NMR is a major tool for high-resolution structural determination of proteins, ENMR has an unique advantage for proteomic studies not only can a group of proteins be identified as disease markers (as in SELDI-TOF-MS and 2D electrophoresis methods), but also the identified proteins can be characterized for their chemical identification with detailed structural information. In addition, ENMR experiments can be designed to visualize protein conformational changes during protein interactions in the presence of the other proteomic molecules. By applying a DC electric field, multicomponent protein interactions can be studied using established intermolecular NOE-based screening methods for reactive residues in polypeptide chains, or other structural mapping methods based on altered chemical shifts, dynamic parameters, and residual dipole coupling patterns.17 For example, ENMR can detect drug-binding affinities to different proteins in the same solution by comparing the interacting...

Matrixrepresented protein networks genome maps

An alternative approach to represent genome-wide protein networks is shown in Figure 4. In this approach, each linked pair of proteins is indicated as a single point on a two dimensional matrix, corresponding to the position of the genes on the chromosome 50 . Each axis of the graph represents a monotonically ordered list of genes, starting at the origin of replication and proceeding along the chromosome. The M. tuberculosis genome has approximately 4,000 genes, as indicated on the x and y axis of the matrix in Figure 4c. Each point on this graph indicates a computationally inferred protein linkage between two proteins 50 . Figure 4a depicts a zoomed in region of the map, representing only the first 50 genes. The point at coordinate x 1, y 5 represents a linkage between the 1st gene on the M. tuberculosis chromosome (Rv0001, dnaA) and the 5th gene on The representation of protein networks as two dimensional genome maps reveals certain characteristics that are not observable using...

Other Proteins Interacting with Dicer

Several other proteins have been found to interact with Dicer. In C. elegans, the RNA-helicase-related protein DRH-1, which is required for RNAi, was found to interact with Rde-4 and Dicer (Tabara et al. 2002). FMRP, an mRNA-binding protein involved in the pathogenesis of fragile X syndrome, has been shown to interact with Dicer and Ago-1 in mammalian cells (Jin et al. 2004), and Drosophila dFXR, a fly ortholog of FMRP, interacts with Dicer-1 and Ago-2 (Caudy et al. 2002 Ishizuka et al. 2002). In mammalian male germ cells, Dicer was shown to interact with mouse vasa homolog (MVH), with both proteins localizing to the P body-related structure known as a chromatoid body (Kotaja et al. 2006). A major proteomic effort was undertaken to characterize proteins interacting with Dicer in C. elegans (Duchaine et al. 2006). A total of 108 candidate proteins were identified. The authors focused on the top 20 proteins most reproducibly co-purifying with Dicer. They were divided into five groups...

Proteins Important Intracellular Buffers

Proteins are among the most plentiful buffers in the body because of their high concentrations, especially within the cells. Approximately 60 to 70 per cent of the total chemical buffering of the body fluids is inside the cells, and most of this results from the intracellular proteins. However, except for the red blood cells, the slowness with which H+ and HCO3- move through the cell membranes often delays for several hours the maximum ability of the intracellular proteins to buffer extracellular acid-base abnormalities. In addition to the high concentration of proteins in the cells, another factor that contributes to their buffering power is the fact that the pKs of many of these protein systems are fairly close to 7.4.

Case Study Proteinbrowser

Biological databases are growing rapidly. Currently there is much effort spent on annotating these databases with terms from controlled, hierarchical vocabularies such as the Gene Ontology. It is often useful to be able to retrieve all entries from a database, which are annotated with a given term from the ontology. The ProteinBrowser use-case shows how one typically needs to join data from different sources. The starting point is the Gene Ontology (GO), from which a hierarchy of terms is obtained. Using the Gene Ontology Annotation (GOA) database, the user can link GO terms to the UniProt identifiers of proteins that have been annotated with biological processes, molecular functions, and cellular components. After choosing a specific protein, the user can, remotely, query additional information from the UniProt database, for example the sequence of the protein. In turn, the PDB database can be remotely queried for still additional information. Finally, using the PubMed identifier of...