Comparative analysis of thermophilic and mesophilic proteins using Protein Energy Networks

Background: Thermophilic proteins sustain themselves and function at higher temperatures. Despite their structural and functional similarities with their mesophilic homologues, they show enhanced stability. Various comparative studies at genomic, protein sequence and structure levels, and experimental works highlight the different factors and dominant interacting forces contributing to this increased stability. Methods: In this comparative structure based study, we have used interaction energies between amino acids, to generate structure networks called as Protein Energy Networks (PENs). These PENs are used to compute network, sub-graph, and node specific parameters. These parameters are then compared between the thermophile-mesophile homologues. Results: The results show an increased number of clusters and low energy cliques in thermophiles as the main contributing factors for their enhanced stability. Further more, we see an increase in the number of hubs in thermophiles. We also observe no community of electrostatic cliques forming in PENs. Conclusion: In this study we were able to take an energy based network approach, to identify the factors responsible for enhanced stability of thermophiles, by comparative analysis. We were able to point out that the sub-graph parameters are the prominent contributing factors. The thermophiles have a better-packed hydrophobic core. We have also discussed how thermophiles, although increasing stability through higher connectivity retains conformational flexibility, from a cliques and communities perspective.

Blood proteins of the silkworm Bombyx mori L

The qualitative and quantitative aspects of the proteins of the silkworm blood were studied by the technique of agarophoresis. The blood of larvae at the final stage revealed the presence of six different protein zones. Considerable differences in the patterns were observed at different stages of growth. There was an increase in the total nitrogen of the blood up to the 5th instar and then came a sudden decrease in the one-day old pupae. Nitrogen concentration was at its highest in egg 1 stage and the electrophoretic pattern closely corresponded to the final larval pattern. Results indicate to the involvement of silk glands in the synthesis and breakdown of a protein designated as protein 5.

Sterol-binding proteins in late endosomes : Regulation of endosome motility and lipid metabolism

Despite its bad reputation in the mass media, cholesterol is an indispensable constituent of cellular membranes and vertebrate life. It is, however, also potentially lethal as it may accumulate in the arterial intima causing atherosclerosis or elsewhere in the body due to inherited conditions. Studying cholesterol in cells, and research on how the cell biology of cholesterol affects on system level is essential for a better understanding of the disease states associated with cholesterol and for the development of new therapies for these conditions. On its way to the cell, exogenous cholesterol traverses through endosomes, transport vesicles involved in internalizing material to cells, and needs to be transported out of this compartment. This endosomal pool of cholesterol is important for understanding both the common disorders of metabolism and the more rare hereditary disorders of cholesterol metabolism. The study of cholesterol in cells has been hampered by the lack of bright fluorescent sterol analogs that would resemble cholesterol enough to be used in cellular studies. In the first study of my thesis, we present a new sterol analog, Boron-Dipyrromethene (BODIPY)-cholesterol for visualizing sterols in living cells and organism. This fluorescent cholesterol derivative is shown to behave similarly to cholesterol both by atomic scale computer simulations and biochemical experiments. We characterize its localization inside different types of living cells and show that it can be used to study sterol trafficking in living organisms. Two sterol binding proteins associated with the endosomal membrane; the Niemann-Pick type C disease protein 1 (NPC1) and the Oxysterol Binding Protein Related Protein 1 (ORP1) are the subjects of the rest of this study. Sensing cholesterol on endosomes, transporting lipids away from this compartment and the effects these lipids play on cellular metabolism are considered. In the second study we characterize how the NPC1 protein affects lipid metabolism. We show that this cholesterol binding protein affects synthesis of triglycerides and that genetic polymorphisms or a genetic defect in the NPC1 gene affect triglyceride on the whole body level. These effects take place via regulation of carbon fluxes to different lipid classes in cells. In the third part we characterize the effects of another endosomal sterol binding protein, ORP1L on the function and motility of endosomes. Specifically we elucidate how a mutation in the ability of ORP1L to bind sterols affects its behavior in cells, and how a change in ORP1L levels in cells affects the localization, degradative capacity and motility of endosomes. In addition we show that ORP1L manipulations affect cholesterol balance also in macrophages, a cell type important for the development of atherosclerosis.

Proteins in Wastewater and Wastewater Sludges

Although there is some information on the total amounts of proteins in wastewater and sludges1"3 and on the amino acids in them,4-11 especially in activated sludge,12 there is almost no evidence on the nature of the proteins in these materials. A knowledge of the nature of proteins in wastewater, sludges, and similar substances would be useful not only for determining the pollutional effects on the environment and the changes in the protein structures during decomposition or treatment, but also for determining the possible usage of the resulting materials in agriculture,13 includ ing animal nutrition.

Mycobacterium tuberculosis UvrD1 and UvrA Proteins Suppress DNA Strand Exchange Promoted by Cognate and Noncognate RecA Proteins

DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coil RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

Differential synthesis of proteins by T4 phage and its head protein amber mutant

There was no difference in the incorporation of S-35 label into proteins of T4 and amber B17 phage grown on Escherichia coli B. The head protein peak was absent in the polyacrylamide gel electrophoretic profile of the S-35 labeled proteins of amber B17 grown on non-permissive host, E.coli B. However, an increase of 15–70% in the synthesis of other phage proteins of amber B17 over that of T4 phage was observed. The lysozyme activity increased by two fold in amber B17 in comparison with that of T4 phage grown on E.coli B. These results imply that in the absence of head protein synthesis by amber mutant there was an increase in the synthesis of other phage proteins.

Export of proteins across membranes: The helix reversion hypothesis

A model is presented which explains the biological role of the leader peptide in protein export. Along the lines of this model, the conformational changes of a protein with environment serves as a general mechanism for translocation. The leader peptide in the cytoplasm takes a hairpin like conformation which reverts to an extended helix upon integration into the membrane. The essential features of this model are in accord with recent results of protein export.

Characterization of viral proteins ofOryctes baculovirus and comparison between two geographical isolates

BacilliformOryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infectedOryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reportedOryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins stained positive for glycosylation. Comparison between the viral proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

Mycobacterium tuberculosis UvrD1 and UvrA Proteins Suppress DNA Strand Exchange Promoted by Cognate and Noncognate RecA Proteins

DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coil RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine, Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments, The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in Delta C-p and positive values of both Delta Delta H degrees and aas of folding. Enthalpic effects were dominant, and the sign of Delta Delta S is the opposite of that expected from the hydrophobic effect. Values of Delta Delta G degrees and Delta Delta H degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.

The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins

Formylation of the initiator tRNA is essential for normal growth of Escherichia coil, The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon, However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation, In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient, The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms.