Protein Molecular Weight Markers Research Articles

Effect of Milk Sources on Whey Protein and Fractions of Casein

Aim: Milk from Cow, Buffalo and goat was used for the study of isolation and fractionation of casein and whey proteins. Buffalo and goat milk were subjected to physicochemical attribute studies and a detailed protein profile namely total protein, casein fractions α-, β- and κ casein and whey protein were separated by urea fractionation method. The aim of this study was to compare various casein fractions isolated from cows, Buffalo and Goat milk. The method of separating casein into its fractions is based on the solubility of the individual components in urea the solution. The separation of casein fractions was carried out in dissolving molar concentration of urea and the yield of fractions and total protein percentages were calculated. Buffalo and goat milk have comparatively higher total protein and whey protein content as compared to cow milk. Maximum whey proteins found in goat milk (20.58%) whereas cow milk had the lowest whey protein (19.29%). Among casein fractions, α fraction (16.64%) was found maximum in Buffalo milk followed by cow (14.92%) and goat milk (5.42%). The goat milk showed the highest level of β casein (17.81%) whereas, the lowest concentration of β casein was found in cow milk (9.38%). Study Design: A significant contribution to the total milk production of India comes from buffalo milk and goat milk. Despite that, Buffalo milk and goat milk are not being utilized for many products because of difference in composition of different species milk and their inherent problems associated in the production of products. Place and Duration of Study: Karnataka Veterinary Animal and Fisheries Sciences University (KVAFSU), Dairy Science College, Hebbal, Bangalore Karnataka, India Materials: The Goat milk samples were collected from Sinchana Goat and Sheep farm, Marenahalli village (Bengaluru Rural Dist) and Buffalo milk was obtained from Country Delight Pvt. Ltd., J. P. Nagar, Bengaluru, Karnataka. Cow milk used in this investigation was collected from SEDP, Dairy Science College, Hebbal, Bengalore.. All the glassware used was soaked in chromic acid solution, repeatedly washed with water, rinsed with distilled water and dried before use. For microbiological analysis dried test tubes, conical flasks, pipettes were cotton plugged and sterilized in a hot air oven. The chemicals and reagents used in this study were mainly of analytical grade procured from Prince Laboratory Company Pvt. Limited, Bangalore. The protein molecular weight markers used for the electrophoretic study were procured from Bangalore Genei Pvt. Ltd. Methodology: Standard urea fractionation method adopted by Hipp et al was used for isolation and fractionation of caseins. Skim milk was prepared by subjecting whole milk for centrifugation, the skim milk obtained was acidified using 1 % hydrochloric acid at 200 C to obtain casein precipitate. Obtained whole casein was dissolved in 3.3 M urea at pH 7.5 and adjusted to pH 4.6 which precipitates the bulk of α casein and leaving k-caseins into soluble filtrates. Again the supernatant was adjusted to pH 4.9 diluted to 1.0 M urea and warmed to 300 C precipitating the β-caeisn. Further, addition of ammonium sulphate was used to isolate k-casein from the precipitate. Results: The sources of protein had a significant influence on the total casein, casein fractions and whey protein content of different species. The highest quantity of total caseins (34.30g/l) and whey protein (8.87 g/l) were observed in buffalo milk followed by cow (28.52 g/l) and lowest was observed in goat milk (28.45 g/l). Total protein, casein and whey protein contents were greatly affected by the source of milk. Conclusion: The significant effect of source of milk on total protein, casein, its fractions and their yield was observed. highest yield of casein (54.31%) was observed in cow milk than buffalo milk (48.95 %) and lower yield was found in goat milk. Amongst the three species, highest per cent of β casein was noted in goat milk (54.05 %) followed by buffalo (36.03%) and cow milk (34.14%). Recommendation: Rasagulla will be consumed universally by all age groups globally. Thus, the consumption of Rasagulla helps in providing overall nutritional requirements. However, the study has been conducted on a pilot scale. Moreover, commercial production may necessitate huge cost on enzymatic modification of proteins and separation peptides for value addition.

Enzymatic Modification of Cow, Buffalo and Goat Milk Proteins and their Effect on Anti-Oxidant Activity and Digestibility

Aim: Buffalo milk and goat milk were enzymatically hydrolyzed to have 5, 10 and 15 per cent DH to improve the techno-functional properties of milk proteins in order to utilize in production of channa based dairy products. Study Design: A significant contribution to total milk production of India comes from buffalo milk and followed by cow and goat milk. Inspite of that, Buffalo milk and goat milk are not being utilized for many products in view of their inherent problems associated in production of Chhana based dairy products. Buffalo milk and goat milk were enzymatically hydrolyzed to have 5, 10 and 15 per cent DH to improve the techno-functional properties of milk proteins in order to utilize in production of products. Place and Duration of Study: Karnataka Veterinary Animal and Fisheries Sciences University (KVAFSU), Dairy Science College, Hebbal, Bangalore Karnataka, India Methodology: The Goat milk samples were collected from Sinchana Goat and sheep farm, Marenahalli village (Bengaluru Rural Dist) and Buffalo milk was obtained from country Delight Pvt. Ltd., J. P. Nagar, Bengaluru, Karnataka. Cow milk used in this investigation was collected from SEDP, Dairy Science College, Hebbal, Bangalore. Commercially available pure Neutrase enzyme was purchased from DSM Nutritional Products India Pvt. Ltd, Bangalore. All the glassware used were soaked in chromic acid solution, repeatedly washed with water, rinsed with distilled water and dried before use. For microbiological analysis dried test tubes, conical flask, pipettes were cotton plugged and sterilized in hot air oven. The chemicals and reagents used in this study were mainly of analytical grade procured from Prince Laboratory Company Pvt limited, Bangalore. The protein molecular weight markers were used for the electrophoretic study were procured from Bangalore Genei Pvt Ltd. Results: Highest protein content was noted in buffalo casein hydrolysate (83.0 %) than cow (81.0 %) and goat (81.16 %) and lowest nitrogen and ash content was observed in goat casein hydrolysate (13.65 %) and (15.20 %) respectively. (Akshya pl include few lines of enzymatic modification on anti-oxidant property and digestibility copy from discussions) Conclusion: Though buffalo milk contributes nearly half of the total milk production of India and goat milk carries both nutritional and therapeutic benefits, the potential utilization of both these milks are very much limited due to the inherent problems associated with them. This investigation has shown that by enzymatically hydrolyzing buffalo milk and goat milk to 5 per cent DH results significant improvement in physico-chemical and functional attributes Rasagulla which is on par with cow’s milk protein hydrolysates. Recommendation: Scavenging activity or anti-oxidant activity and Digestibility of hydrolysates has been studied as influenced by the extent of enzymatic modification in this study.

The Penn State Protein Ladder system for inexpensive protein molecular weight markers

We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10–50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. We have also constructed plasmids to express 150 and 250 kD proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification.

Critical Aspects to Produce Low-Cost Protein Molecular Weight Marker: A Review

Proteomic studies usually begin with characterizing protein profile, like the molecular weight of protein, which can be done by the SDS-PAGE technique followed by Western Blot. These methods need a standard protein called molecular weight marker (MWM). In this review, the important, basic aspects of either recombinant or native MWM production was discussed, including the type and effect of dyes can be used for preparing a prestained MWM. Moreover, buffer and polyols used in the formulation can also affect the quality and stability. Also, another adjuvant may be needed to increase the robustness of MWM to lower the risk of protease contamination that can breakdown the protein inside the MWM during storage. Understanding those critical aspects will help to produce/formulate a good quality of MWM.

Characterization of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough

Sulfate‐reducing bacteria play a key role in the global carbon and sulfur cycles; a role realized through complex competitive or syntrophic metabolic relationships. These relationships are established in response to sulfate levels as well as concentrations of other nutrients such as short chain fatty acids (SCFAs). For some prokaryotes, SCFA production or consumption requires an ATP‐dependent reaction catalyzed by a SCFA kinase. In this study, we have determined the biochemical properties of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough heterologously expressed in E. coli. Our experiments characterize optimal conditions for activity as well as kinetic parameters for six SCFA substrates and the nucleotide cofactor ATP. These findings have implications on structure and function relationships as well as the potential physiological role of this enzyme.Support or Funding InformationThe authors would like to thank the Scott and Alice Thomson Fellowship Program for financial support of MB. Further, the authors thank the UW‐Parkside Committee on Research and Creative Activity and the College of Natural and Health Sciences for financial support of this project.Enzymatic Characterization of DvBuk.(A) DvBuk purity assessment was visualized by resolving 10 μg of purified DvBuk on 10% (w/v) polyacrylamide gel and stained with Coomassie Blue. Lane 1: Protein molecular weight markers, Lane 2: recombinant His‐tagged DvBuk. Enzyme activity (%) at various (B) pH and (C) temperature values. (D) Saturation kinetics of DvBuk with butyrate as substrate. (E) Relative activities of DvBuk in the presence of various SCFA substrates.Figure 1Substrate Preference of DvBuk.(A) Relative activities of DvBuk in the presence of various SCFA substrates (relative activities normalized to observed specific activity in the presence of 200 mM valerate = 9.7 U mg−1). (B) Kinetic constants for DvBuk using various SCFA substrates.Figure 2

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

Biochemical Characterization of Bread Wheat (<i>Triticum aestivum</i> L.) Genotypes Based on SDS-PAGE

Genetic diversity of twelve Indian wheat genotypes was assessed on the basis of seed storage protein profiling on 10 and 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Out of twelve genotypes, six genotypes are resistant to salinity, whereas remaining are susceptible. Genetic diversity was calculated using Shannon index, Nei’s index and unweighted pair group method with arithmetic mean (UPGMA) cluster analysis by constructing an electrogram of fragments of proteins, which were used to calculate Jaccard’s similarity coefficients between these varieties. Seed storage protein analyzed by 15 subunits ranging from 14.3-97.4 KDa protein molecular weight marker (PMW-M). The similarity coefficient ranged from 29% to 100%. The detected variation could be used to provide useful information on individual enzymes or transporters, activity as well as modification of structural protein, protein-protein interactions and stress dependent protein movement and can be used to classify adapted cultivars to improve the efficiency of wheat breeding programmes.

Blotting from PhastGel to Membranes by Ultrasound.

Ultrasound based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and 14C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

Vibrio cholerae O395 Outer Membrane Vesicles Modulate Intestinal Epithelial Cells in a NOD1 Protein-dependent Manner and Induce Dendritic Cell-mediated Th2/Th17 Cell Responses

Like other Gram-negative pathogens, Vibrio cholerae, the causative agent of the diarrheal disease cholera, secretes outer membrane vesicles (OMVs). OMVs are complex entities composed of a subset of envelope lipid and protein components and play a role in the delivery of effector molecules to host cells. We previously showed that V. cholerae O395 cells secrete OMVs that are internalized by host cells, but their role in pathogenesis has not been well elucidated. In the present study, we evaluated the interaction of OMVs with intestinal epithelial cells. These vesicles induced expression of proinflammatory cytokines such as IL-8 and GM-CSF and chemokines such as CCL2, CCL20, and thymic stromal lymphopoietin in epithelial cells through activation of MAPK and NF-κB pathways in NOD1-dependent manner. Epithelial cells stimulated with OMVs activated dendritic cells (DCs) in a direct co-culture system. Activated DCs expressed high levels of co-stimulatory molecules; released inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-23 and chemokines CCL22 and CCL17; and subsequently primed CD4(+) T cells leading to IL-4, IL-13, and IL-17 expression. These results suggest that V. cholerae O395 OMVs modulate the epithelial proinflammatory response and activate DCs, which promote T cell polarization toward an inflammatory Th2/Th17 response.

A Study of Fibrin Zymography Method for the Assay of Plasminogen Activators

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most important and widely used technology which is mainly used to analyze the protein molecular weight. Fibrin zymography based on the SDS-PAGE is the best method for qualitative analysis of unknown plasminogen activators (PAs), especially for the analysis of molecular weight. In electrophoresis technique, molecular weight marker is the most important factor. However, it is difficult to detect protein molecular weight markers in fibrin zymography. In this study, some important factors, such as concentrations of fibrinogen and plasminogen, are discussed. Our results provide an efficient and convenient method which can clearly exhibit the dark blue bands of protein molecular weight (MW) markers and the transparent bands of PAs against the light blue background on one gel at the same time, and show high sensitivity.

Study of Heat Shock Protein: In Housefly.

Insects and other animals, including humans, produce heat-shock proteins in response to extremely high temperatures. Hsp is vital in withstanding stress conditions which insects normally thrive, the aim of our work was to study Hsps and find out the molecular weight of Hsps in Musca domestica. Houseflies were collected. The samples prepared were loaded on SDS gel along with protein molecular weight marker and study of Hsps was done by using silver staining technique. The test samples which was exposed to heat shock at 42oC & 45oC were compared with control which was prepared by growing larvae at optimum temperature i.e. 26°C-28°C. It was found that survival rate was not decrease along with increase in temperature. Thus, Hsp’s play very important role when Hsp’s expressed through stress conditions. Hsps was increased when cells were exposed to elevated temperature. A band of approximately 70 kDa was seen to be expressed in all the Hsps as against control sample.

A docile method of isolation and purification of riboflavin binding protein (Rfbp) from peahen (Pavo cristatus) and domestic fowl (Gallus gallus)

Riboflavin binding protein (Rfbp) was isolated from domestic fowl (Gallus gallus) and peahen (Pavo cristatus) egg-white and egg-yolk. The protein was purified in two steps, DEAE-Sephadex A-50 ion exchange chromatography and eluted with phosphate buffer pH 7.3 containing 0.5 M sodium chloride. The final purification of protein was achieved on Sephadex G-100. The purity of the protein was judged on cylindrical and slab gel electrophoresis, SDS-PAGE technique. Sephadex G-100 fraction Rfbp moved as a single band both on the Slab and Cylindrical gels. Comparison of the mobility of Rfbp with that of the standard molecular weight marker proteins revealed with that the Rfbp had a molecular weight close to 29,000 kd. Interestingly, hen egg-white Rfbp and peahen egg- white, yolk Rfbp had the same molecular weight as revealed by the SDS-PAGE. This is a novel approach for the study of riboflavin binding protein purified from different avian eggs in two steps and studied electrophoretic characterization with standard molecular weight marker.

A new method for preparation of an etched porous joint for capillary electrophoresis and its pore‐size evaluation

In this work, an improved approach was developed for the construction of a hydrofluoric acid etched capillary porous joint, and the pore size of the porous joint was investigated. Several etching factors such as reagents concentration and etching temperature were optimized. Results showed that moderate temperature with low hydrofluoric acid concentration was helpful for proportional etching. Low-range protein molecular weight marker labeled with acridinium ester was used as pore-size reference for pore-size evaluation of the joint. A homemade CE with chemiluminescence detection system was used for the analysis of the acridinium ester labeled protein molecular weight marker, and the results showed that only the small molecular peptides can pass through the etched porous joint and the big size proteins cannot. The penetration of protein whose molecular mass was larger than 6500 Da was a signal of over-etching.

Three Consecutive Arginines Are Important for the Mycobacterial Peptide Deformylase Enzyme Activity

Genes encoding the peptide deformylase enzyme (def) are present in all eubacteria and are involved in the deformylation of the N-formyl group of newly synthesized polypeptides during protein synthesis. We compared the amino acid sequences of this enzyme in different mycobacterial species and found that they are highly conserved (76% homology with 62% identity); however, when this comparison was extended to other eubacterial homologs, it emerged that the mycobacterial proteins have an insertion region containing three consecutive arginine residues (residues 77-79 in Mycobacterium tuberculosis peptide deformylase (mPDF)). Here, we demonstrate that these three arginines are important for the activity of mPDF. Circular dichroism studies of wild-type mPDF and of mPDF containing individual conservative substitutions (R77K, R78K, or R79K) or combined substitutions incorporated into a triple mutant (R77K/R78K/R79K) indicate that such mutations cause mPDF to undergo structural alterations. Molecular modeling of mPDF suggests that the three arginines are distal to the active site. Molecular dynamics simulations of wild-type and mutant mPDF structures indicate that the arginines may be involved in the stabilization of substrate binding pocket residues for their proper interaction with peptide(s). Treatment with 5'-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against different regions of def from M. tuberculosis inhibits growth of Mycobacterium smegmatis in culture. Taken together, these results hold out the possibility of future design of novel mycobacteria-specific PDF inhibitors.

Profiling the Enzymatic Properties and Inhibition of Human Complement Factor B

Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4- to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.

Solution structure of FK506 binding domain (FKBD) of Plasmodium falciparum FK506 binding protein 35 (PfFKBP35)

Plasmodium falciparum is the most lethal pathogen among four species of Plasmodium that infect human beings.1 The immunosuppressive drug FK506 shows an antimalarial effect,2 suggesting that the parasite may contain a potential FK506 binding protein (FKBP) as the molecular target of the drug. Database analysis revealed the presence of a putative peptidylprolyl cis-trans isomerase (PPIase) (Swiss-Prot/TremBL ID Q8I4V_PLAF7) in Plasmodium falciparum. Recent studies have named it Plasmodium falciparum FKBP35 (PfFKBP35; GeneBank accession no. AAN36539) as a member of the FKBP family in the parasite.3,4 PfFKBP35 shows a high similarity to FKBP12 in the catalytic core domain [40% sequence identity, Fig. 1(A)], whereas the overall structural architecture resembles the multiple tetratricopeptide repeat-containing FKBP family including FKBP38, FKBP51, and FKBP52.7 FKBP family, which is a part of a larger superfamily of immunophilins exerting roles as chaperones and possessing PPIase, plays important roles in several cellular processes including protein folding and suppressing T-cell activation upon binding the immunosuppressive ligands.8 Currently, little is know about how the growth of the parasite is inhibited by FK506. To better understand the biological function of PfFKBP35 and also provide a basis of the molecular interaction between PfFKBP35 and the ligand, in this study, we have determined the solution structure of the core isomerase domain PfFKBP35 (referred hereafter as FKBD). The structure shows the similar folds as seen in the other FKBP family proteins. Differences were seen in the regions of b3/4 and b5/6 loops, which are important for ligand-binding and PPIase activities. MATERIALS AND METHODS

Biochemical Characterization of VlmL, a Seryl-tRNA Synthetase Encoded by the Valanimycin Biosynthetic Gene Cluster

Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed.

Modulation of the Viral ATPase Activity by the Portal Protein Correlates with DNA Packaging Efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple.