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Biomedical subjects

V Ramakrishnan

Publications and source records attributed to V Ramakrishnan.

At least 91 records · Page 5Linked to original sources
PubMed1999

Crystal structure of the conserved subdomain of human protein SRP54M at 2.1 A resolution: evidence for the mechanism of signal peptide binding.

Protein SRP54 is an integral part of the mammalian signal recognition particle (SRP), a cytosolic ribonucleoprotein complex which associates with ribosomes and serves to recognize, bind, and transport proteins destined for the membrane or secretion. The methionine-rich M-domain of protein SRP54 (SRP54M) binds the SRP RNA and the signal peptide as the nascent protein emerges from the ribosome. A focal point of this critical cellular function is the detailed understanding of how different hydrophobic signal peptides are recognized efficiently and transported specifically, despite considerable variation in sequence. We have solved the crystal structure of a conserved functional subdomain of the human SRP54 protein (hSRP54m) at 2.1 A resolution showing a predominantly alpha helical protein with a large fraction of the structure available for binding. RNA binding is predicted to occur in the vicinity of helices 4 to 6. The N-terminal helix extends significantly from the core of the structure into a large but constricted hydrophobic groove of an adjacent molecule, thus revealing molecular details of possible interactions between alpha helical signal peptides and human SRP54.

Amino Acid Sequence↗
PubMed1999

Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution.

The 30S ribosomal subunit binds messenger RNA and the anticodon stem-loop of transfer RNA during protein synthesis. A crystallographic analysis of the structure of the subunit from the bacterium Thermus thermophilus is presented. At a resolution of 5.5 A, the phosphate backbone of the ribosomal RNA is visible, as are the alpha-helices of the ribosomal proteins, enabling double-helical regions of RNA to be identified throughout the subunit, all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map, and the fold of the entire central domain of the small-subunit ribosomal RNA to be determined.

Bacterial Proteins↗
PubMed1999

A detailed view of a ribosomal active site: the structure of the L11-RNA complex.

We report the crystal structure of a 58 nucleotide fragment of 23S ribosomal RNA bound to ribosomal protein L11. This highly conserved ribonucleoprotein domain is the target for the thiostrepton family of antibiotics that disrupt elongation factor function. The highly compact RNA has both familiar and novel structural motifs. While the C-terminal domain of L11 binds RNA tightly, the N-terminal domain makes only limited contacts with RNA and is proposed to function as a switch that reversibly associates with an adjacent region of RNA. The sites of mutations conferring resistance to thiostrepton and micrococcin line a narrow cleft between the RNA and the N-terminal domain. These antibiotics are proposed to bind in this cleft, locking the putative switch and interfering with the function of elongation factors.

Amino Acid Sequence↗
PubMed1999

Location of translational initiation factor IF3 on the small ribosomal subunit.

The location of translational initiation factor IF3 bound to the 30S subunit of the Thermus thermophilus ribosome has been determined by cryoelectron microscopy. Both the 30S.IF3 complex and control 30S subunit structures were determined to 27-A resolution. The difference map calculated from the two reconstructions reveals three prominent lobes of positive density. The previously solved crystal structure of IF3 fits very well into two of these lobes, whereas the third lobe probably arises from conformational changes induced in the 30S subunit as a result of IF3 binding. Our placement of IF3 on the 30S subunit allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation.

Cell Fractionation↗
PubMed1999

Sexual orientation and suicidality: a co-twin control study in adult men.

BACKGROUND: Several recent studies have found a higher lifetime prevalence of suicide attempts in homosexual males compared with heterosexual control subjects or population rates. These studies used either convenience samples, most without controls, or population-based samples in which confounding factors such as depression and substance abuse were not measured. METHODS: This study used twins from the population-based Vietnam Era Twin Registry, Hines, Ill. An analytic sample of 103 middle-aged male-male twin pairs from the registry was identified in which one member of the pair reported male sex partners after age 18 years while the other did not. Four lifetime symptoms of suicidality as measured by the Diagnostic Interview Schedule were analyzed: thoughts about death, wanting to die, thoughts about committing suicide, and attempted suicide. A composite measure of reporting at least one suicidality symptom was also assessed. RESULTS: Same-gender sexual orientation is significantly associated with each of the suicidality measures. Unadjusted matched-pair odds ratios follow: 2.4 (95% confidence interval [CI], 1.2 - 4.6) for thoughts about death; 4.4 (95% CI, 1.7 - 11.6) for wanted to die; 4.1 (95% CI, 2.1 - 8.2) for suicidal ideation; 6.5 (95% CI, 1.5 - 28.8) for attempted suicide; and 5.1 (95% CI, 2.4 - 10.9) for any of the suicidal symptoms. After adjustment for substance abuse and depressive symptoms (other than suicidality), all of the suicidality measures remain significantly associated with same-gender sexual orientation except for wanting to die (odds ratio, 2.5 [95% CI, 0.7 - 8.81). CONCLUSIONS: The substantially increased lifetime risk of suicidal behaviors in homosexual men is unlikely to be due solely to substance abuse or other psychiatric comorbidity. While the underlying causes of the suicidal behaviors remain unclear, future research needs to address the inadequacies in the measurement of both sexual orientation and suicidality in population-based samples.

Adult↗
PubMed1999

Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.

Amino Acid Sequence↗
PubMed1999

The role of alpha and beta platelet-derived growth factor receptor in the vascular response to injury in nonhuman primates.

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.

Animals↗
PubMed1999

Chicago community-based asthma intervention trial: feasibility of delivering peer education in an inner-city population.

The most effective means of educating children with asthma and their families has not been clearly demonstrated in previous studies. Peer education is uniquely suited to the complex problems encountered in underserved populations. The purpose of this study was to show the feasibility of delivering a peer education program for children with asthma and the effect of the program on indoor allergen levels in an inner-city population in Chicago. Overall, the program was well received. Baseline allergen levels were consistent with some previous studies in showing low levels of mite allergens and high levels of cockroach allergens, with 79.6% of samples having levels > 8 U/g. A total of 28.2% of samples had cat allergen levels > 2 microg/g, although only 9.7% of homes had cats, confirming previous reports that cat allergen is ubiquitous. Mold levels were seasonal, with the highest levels in the summer. Results from this study suggest that intervention programs should focus more on elimination of cockroaches than was previously appreciated, while minimizing the use of pesticides, and on identification of the sources of cat allergen. Structural and psychosocial issues in homes need to be addressed in future studies. This study has demonstrated the feasibility of delivering peer education in a inner-city population and highlighted the need for comprehensive intervention strategies addressing complex issues facing underserved neighborhoods.

Adolescent↗
PubMed1998

Position and orientation of the globular domain of linker histone H5 on the nucleosome.

It is essential to identify the exact location of the linker histone within nucleosomes, the fundamental packing units of chromatin, in order to understand how condensed, transcriptionally inactive chromatin forms. Here, using a site-specific protein-DNA photo-crosslinking method, we map the binding site and the orientation of the globular domain of linker histone H5 on mixed-sequence chicken nucleosomes. We show, in contrast to an earlier model, that the globular domain forms a bridge between one terminus of chromatosomal DNA and the DNA in the vicinity of the dyad axis of symmetry of the core particle. Helix III of the globular domain binds in the major groove of the first helical turn of the chromatosomal DNA, whereas the secondary DNA-binding site on the opposite face of the globular domain of histone H5 makes contact with the nucleosomal DNA close to its midpoint. We also infer that helix I and helix II of the globular domain of histone H5 probably face, respectively, the solvent and the nucleosome. This location places the basic carboxy-terminal region of the globular domain in a position from which it could simultaneously bind the nucleosome-linking DNA strands that exit and enter the nucleosome.

Animals↗
PubMed1998

Structure of the histone acetyltransferase Hat1: a paradigm for the GCN5-related N-acetyltransferase superfamily.

We have solved the crystal structure of the yeast histone acetyltransferase Hat1-acetyl coenzyme A (AcCoA) complex at 2.3 A resolution. Hat1 has an elongated, curved structure, and the AcCoA molecule is bound in a cleft on the concave surface of the protein, marking the active site of the enzyme. A channel of variable width and depth that runs across the protein is probably the binding site for the histone substrate. A model for histone H4 binding by Hat1 is discussed in terms of possible sources of specific lysine recognition by the enzyme. The structure of Hat1 provides a model for the structures of the catalytic domains of a protein superfamily that includes other histone acetyltransferases such as Gcn5 and CBP.

Acetyl Coenzyme A↗
PubMed1998

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif.

We report the 1.7 A crystal structure of ribosomal protein S4 from Bacillus stearothermophilus. To facilitate the crystallization, 41 apparently flexible residues at the N-terminus of the protein have been deleted (S4Delta41). S4Delta41 has two domains; domain 1 is completely alpha-helical and domain 2 comprises a five-stranded antiparallel beta-sheet with three alpha-helices packed on one side. Domain 2 is an insertion within domain 1, and it shows significant structural homology to the ETS domain of eukaryotic transcription factors. A phylogenetic analysis of the S4 primary structure shows that the likely RNA interaction surface is predominantly on one side of the protein. The surface is extensive and highly positively charged, and is centered on a distinctive canyon at the domain interface. The latter feature contains two arginines that are totally conserved in all known species of S4 including eukaryotes, and are probably crucial in binding RNA. As has been shown for other ribosomal proteins, mutations within S4 that affect ribosome function appear to disrupt the RNA-binding sites. The structure provides a framework with which to probe the RNA-binding properties of S4 by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1998

Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance.

Antibiotic resistance is rapidly becoming a major medical problem. Many antibiotics are directed against bacterial ribosomes, and mutations within both the RNA and protein components can render them ineffective. It is well known that the majority of these antibiotics act by binding to the ribosomal RNA, and it is of interest to understand how mutations in the ribosomal proteins can produce resistance. Translational accuracy is one important target of antibiotics, and a number of ribosomal protein mutations in Escherichia coli are known to modulate the proofreading mechanism of the ribosome. Here we describe the high-resolution structures of two such ribosomal proteins and characterize these mutations. The S5 protein, from the small ribosomal unit, is associated with two types of mutations: those that reduce translational fidelity and others that produce resistance to the antibiotic spectinomycin. The L6 protein, from the large subunit, has mutations that cause resistance to several aminoglycoside antibiotics, notably gentamicin. In both proteins, the mutations occur within their putative RNA-binding sites. The L6 mutations are particularly drastic because they result in large deletions of an RNA-binding region. These results support the hypothesis that the mutations create local distortions of the catalytic RNA component.When combined with a variety of structural and biochemical data, these mutations also become important probes of the architecture and function of the translational machinery. We propose that the C-terminal half of S5, which contains the accuracy mutations, organizes RNA structures associated with the decoding region, and the N-terminal half, which contains the spectinomycin-resistance mutations, directly interacts with an RNA helix that binds this antibiotic. As regards L6, we suggest that the mutations indirectly affect proofreading by locally distorting the EF-Tu.GTP.aminoacyl tRNA binding site on the large subunit.

Amino Acid Sequence↗
PubMed1998

Conformational variability of the N-terminal helix in the structure of ribosomal protein S15.

BACKGROUND: Ribosomal protein S15 is a primary RNA-binding protein that binds to the central domain of 16S rRNA. S15 also regulates its own synthesis by binding to its own mRNA. The binding sites for S15 on both mRNA and rRNA have been narrowed down to less than a hundred nucleotides each, making the protein an attractive candidate for the study of protein-RNA interactions. RESULTS: The crystal structure of S15 from Bacillus stearothermophilus has been solved to 2.1 A resolution. The structure consists of four alpha helices. Three of these helices form the core of the protein, while the N-terminal helix protrudes out from the body of the molecule to make contacts with a neighboring molecule in the crystal lattice. S15 contains a large conserved patch of basic residues which could provide a site for binding 16S rRNA. CONCLUSIONS: The conformation of the N-terminal alpha helix is quite different from that reported in a recent NMR structure of S15 from Thermus thermophilus. The intermolecular contacts that this alpha helix makes with a neighboring molecule in the crystal, however, closely resemble the intramolecular contacts that occur in the NMR structure. This conformational variability of the N-terminal helix has implications for the range of possible S15-RNA interactions. A large, conserved basic patch at one end of S15 and a cluster of conserved but exposed aromatic residues at the other end provide two possible RNA-binding sites on S15.

Amino Acid Sequence↗