3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%–19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.     

Sequence-specific NMR assignments of the trp repressor from Escherichia coli using three-dimensional 15N/1H heteronuclear techniques.

Sequence-specific 15N and 1H assignments for the trp holorepressor from Escherichia coli are reported. The trp repressor consists of two identical 107-residue subunits which are highly helical in the crystal state [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L. & Sigler, P. B. (1985) Nature 317, 782-786]. The high helical content and the relatively large size of the protein (Mr = 25,000) make it difficult to assign even the main-chain resonances by conventional homonuclear two-dimensional NMR methods. However, we have now assigned the main-chain resonances of 94% of the residues by using three-dimensional 15N/1H heteronuclear experiments on a sample of protein uniformly labelled with 15N. The additional resolution obtained by spreading out the signals into three dimensions proved indispensable in making these assignments. In particular, we have been able to resolve signals from residues in the N-terminal region of the A helix for the first time in solution. The observed NOE results confirm that the repressor is highly helical in solution, and contains no extended chain conformations.     

Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins.

The Rab escort protein (REP) is an essential component of the heterotrimeric enzyme Rab geranylgeranyl transferase that modifies the carboxy-terminal cysteines of the Ras-like small G proteins belonging to the Rab/Ypt family. Deletions in the human CHM locus, encoding one of the two REPs known in humans, result in a retinal degenerative syndrome called choroideremia. The only known yeast homologue of the choroideremia gene product is encoded by an essential gene called MRS6. Besides three structurally conserved regions (SCRs) previously detected in the amino-terminal half of REPs and RabGDIs, three other regions in the carboxy-terminal domain (RCR 1-3) are here identified as being characteristic of REPs alone. We have performed the first mutational analysis of a REP protein to experimentally define the regions functionally important for Rab/Ypt protein binding, making use of the genetic system of the yeast Saccharomyces cerevisiae. This analysis has shown that the SCRs are necessary but not sufficient for Ypt1p binding by the yeast REP, the carboxy-terminal region also being required.     

Genetic and structural analysis of a virulence determinant in polyomavirus VP1.

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.     

Cloning and characterization of senC, a gene involved in both aerobic respiration and photosynthesis gene expression in Rhodobacter capsulatus.

The purple nonsulfur photosynthetic eubacterium Rhodobacter capsulatus is a versatile organism that can obtain cellular energy by several means, including the capture of light energy for photosynthesis as well as the use of light-independent respiration, in which molecular oxygen serves as a terminal electron acceptor. In this study, we have identified and characterized a novel gene, senC, mutations in which affect respiration as well as the induction of photosynthesis gene expression. The protein coded by senC exhibits 33% sequence identity to the yeast nucleus-encoded protein SCO1, which is thought to be a mitochondrion-associated cytochrome c oxidase assembly factor. Like yeast SCO1, SenC is required for optimal cytochrome c oxidase activity in aerobically grown R. capsulatus cells. We further show that senC is required for maximal induction from the puf and puh operons, which encode the structural polypeptides of the light-harvesting and reaction center complexes.     

Impact of moisture on thermally induced denaturation and decomposition of lyophilized bovine somatotropin

The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via differential scanning calorimetry were evaluated with respect to moisture content. The transition peak temperature of rbSt decreased with increasing moisture from 161°C in the dry state to a plateau of 65°C at 28% moisture, which is similar to that of rbSt in solution. Using high performance liquid chromatography, this irreversible endothermic transition consisted primarily of unfolding, hydrophobic aggregation, and some covalent modifications. In the dry state, covalent modifications, including polymerization into compounds of higher molecular weight, were more prominent, while in the presence of moisture, hydrophobic aggregation was most prominent. The irreversibility and scan rate dependence of the endothermic phenomena supports the kinetic nature of the transition rather than a simple equilibrium between globular and unfolded states. The apparent activation energy, for the net transition (i.e., unfolding, hydrophobic aggregation, and covalent modifications) was 57 kcal/mol for rbSt at 9.9% moisture. The observed enthalpy of the transition increased, decreased, then approximately leveled off as a function of increasing moisture content. This can be explained by the increasingly significant contribution of the exothermic aggregation at higher moisture contents. © 1995 John Wiley & Sons, Inc.     

Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components.

The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria. To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects. Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype. Each of these mutants was defective in chemotactic response. Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response. In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface. When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity. Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant. These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade.