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.

Drosophila indirect flight muscle specific Act88F actin mutants as a model system for studying congenital myopathies of the human ACTA1 skeletal muscle actin gene

Most human ACTA1 skeletal actin gene mutations cause dominant, congenital myopathies often with severely reduced muscle function and neonatal mortality. High sequence conservation of actin means many mutated ACTA1 residues are identical to those in the Drosophila Act88F, an indirect flight muscle specific sarcomeric actin. Four known Act88F mutations occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for similar muscle phenotypes. Mutant homozygotes show phenotypes ranging from a lack of myofibrils to almost normal sarcomeres at eclosion. Aberrant Z-disc-like structures and serial Z-disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all four Act88F mutants. These electron-dense structures show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. We conclude that the Drosophila indirect flight muscles provide a good model system for studying ACTA1 mutations.

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.

Vitamin A and thyroxine carrier proteins in chicken plasma Steady-state control of the plasma level of free retinol-binding protein and free thyroxine

1. 1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occuring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. 6. The thyroxine-binding proteins were found to be two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz.,prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

Prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoEM structures

Background: Dengue virus along with the other members of the flaviviridae family has reemerged as deadly human pathogens. Understanding the mechanistic details of these infections can be highly rewarding in developing effective antivirals. During maturation of the virus inside the host cell, the coat proteins E and M undergo conformational changes, altering the morphology of the viral coat. However, due to low resolution nature of the available 3-D structures of viral assemblies, the atomic details of these changes are still elusive. Results: In the present analysis, starting from C alpha positions of low resolution cryo electron microscopic structures the residue level details of protein-protein interaction interfaces of dengue virus coat proteins have been predicted. By comparing the preexisting structures of virus in different phases of life cycle, the changes taking place in these predicted protein-protein interaction interfaces were followed as a function of maturation process of the virus. Besides changing the current notion about the presence of only homodimers in the mature viral coat, the present analysis indicated presence of a proline-rich motif at the protein-protein interaction interface of the coat protein. Investigating the conservation status of these seemingly functionally crucial residues across other members of flaviviridae family enabled dissecting common mechanisms used for infections by these viruses. Conclusions: Thus, using computational approach the present analysis has provided better insights into the preexisting low resolution structures of virus assemblies, the findings of which can be made use of in designing effective antivirals against these deadly human pathogens.