Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein.
Explore the source record for details and available documents.
Cytosine Nucleotides↗
SEARCH · PubMed Health
Results for “S-protein”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7.
Explore the source record for details and available documents.
Hydrogen-Ion Concentration↗
Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins.
The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group.
Animals↗
Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A.
1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.
Deuterium↗
Conformation of ribonuclease S-protein.
Ribonuclease S-protein exhibits a pH-dependent conformational transition between folded and unfolded states, and some unfolded S-protein persists up to pH 8. The histidine C2 proton resonance of the unfolded species was erroneously assigned by Bradbury et al. to histidine residue 119 of the folded species.
Histidine↗
Studies on polypeptides. XXVI. Synthesis of the N-terminal 1--23 peptide sequence of rat pancreatic ribonuclease; enzymatic activity of the hybrid complex with bovine S-protein.
A description is given of the synthesis by fragment condensation of the peptide Gly-Glu-Ser-Arg-Glu-Ser-Ser-Ala-Asp-Lys-Phe-Lys-Arg-Gln-His-Met-Asp-Thr-Glu-Gly-Pro-Ser-Lys corresponding to the 1--23 amino acid sequence of rat pancreatic ribonuclease. This rat peptide combined with bovine S-protein yields a fully active ribonuclease S' analogue.
Amino Acid Sequence↗
Isolation of human S-protein, an inhibitor of the membrane attack complex of complement.
Explore the source record for details and available documents.
Amino Acids↗
The C5b-6 complex: formation, isolation, and inhibition of its activity by lipoprotein and the S-protein of human serum.
Explore the source record for details and available documents.
Alum Compounds↗
Binding of desoxycholate, phosphatidylcholine vesicles, lipoprotein and of the S-protein to complexes of terminal complement components.
Explore the source record for details and available documents.
Binding Sites↗
Relation between structure and function in some partially synthetic ribonucleases S'. Enzymic and spectroscopic investigation on [Orn10, Asn14]-RNase S' and 1epsilon, 7epsilon, 10delta-triguanidino-[Orn10, Asn14]-RNase S'.
Some analogues have been prepared of S-peptide, the peptide obtained together with S-protein from subtilisn-modified beef pancreatic R Nase A. The syntheses are described of [Orn10, Asn14]-S-peptide and 1epsilon, 7epsilon, 10delta-triguanidino-[Orn10, Asn14]-S-peptide. The S-peptide analogues are able to activate S-protein at the level of the parent [Orn10]-S-peptide and 1epsilon, 7epsilon-diguanidino-S-peptide respectively, although at high peptide-to-protein molar ratios. After their recombination with S-protein the buried character of Tyr-25 was restored, as judged from difference absorption and circular dichroism spectra in the near-ultraviolet region. These findings indicate that the asparaginyl residue is a possible naturally occurring substituent in the R Nase A sequences whose state of amidation in position 14 has not yet been defined.
Amino Acid Sequence↗
The SC5b-7 complex: formation, isolation, properties, and subunit composition.
Activation of C in C8-depleted serum results in the formation of a soluble complex containing C5, C6, and C7. The complex has an electrophoretic mobility of an alpha-globulin, an s-rate of 18.5S, and a m.w. of 668,000 daltons. This complex was isolated and upon SDS polyacrylamide gel electrophoresis it was found to contain, in addition to C5b, C6 and C7, an 88,000 dalton glycoprotein. The protein was identified as the band V protein of the soluble C5b-9 complex. It is referred to as SIIIs-protein, or S-protein. Since the S-protein does not bind to C5b-6, it is concluded that it is incorporated during the fusion of C5b-6 with C7. The SC5b-7 complex exhibits the same neoantigen as the SC5b-9 complex, but compared to the C5b-6 complex it appears to contain an additionally qualitatively distinct neoantigen.
Animals↗
Optimization of a Semi-nested PCR Protocol for Amplifying the Entire Spike Protein Region to Identify SARS-CoV-2 Variants in Wastewater.
Wastewater-based monitoring of SARS-CoV-2 and other pathogens is a widely adopted tool for assessing epidemic dynamics. While quantitative assays are commonly used to estimate infection levels in catchment populations, phylogenetic information-such as identifying circulating variants-is also crucial for public health. However, applying the widely used ARTIC protocol for full-genome sequencing to wastewater samples has proven challenging, likely due to the limited specificity and sensitivity of multiplex RT-PCR in such complex matrices. In this study, we developed and optimized a semi-nested RT-PCR assay targeting the full S-protein coding region (~4000 bases) for phylogenetic characterization of SARS-CoV-2 in wastewater. By reducing multiplexing and using single-plex reactions for both RT and PCR steps, we successfully amplified ~2000 bp fragments. Amplicons were sequenced using the Flongle Flow Cell platform. The optimized method-consisting of reverse transcription with specific primers followed by three parallel single-plex semi-nested PCRs-yielded over 1,000 SARS-CoV-2-like reads per primer set in 30 out of 39 wastewater samples in treatment plants in Japan, including those with <10 copies per analyte. Variant proportions were estimated using a newly developed approach based on single-nucleotide variant pattern matrix, revealing the presence of multiple co-circulating variants, including XBB lineages, JN.1, and notably BA.2.75, which was undetected in domestic clinical surveillance. These results highlight the effectiveness of our approach for detecting temporal shifts in SARS-CoV-2 variants, even at low RNA concentrations.
Polymerase Chain Reaction↗
Assignment of the imidazole ring nitrogen protons of histidine 48 in the proton NMR spectrum of ribonuclease A in water solution.
Several exchangeable resonances, designated a, b, c and d are observed in the 11-14 ppm (from 2,2-dimethyl-2-silapentane-5-sulfonate) region of the proton spectrum of ribonuclease A in water solution. We describe a number of lines of evidence suggesting the assignment of peaks b and c to the N1 and N3 protons of His 48, which occupies an interior position in the protein remote from the active site. This evidence includes the observation that the binding of Cu(II) and 3'-CMP (cytidine 3'-monophosphate) has no effect on these resonances. Further evidence includes pH titration data showing a pKa of approx. 2 for these protons, solvent exchange rates in the native state and with disulfide bridges IV-V and III-VIII cleaved, the observation of the carboxymethylated enzymes CM-His12-RNAase A and CM-His119-RNAase A, and of the modified enzymes Des(1-21)-RNAase A (S-protein) and Des(119-124)-RNAase A.
Amino Acid Sequence↗
Ramu stunt virus genome reveals previously unreported segments and nucleocapsid domain duplication in Mechlorovirus.
Ramu stunt virus (RmSV), a member of the genus Mechlorovirus within the family Phenuiviridae, was previously described as a six-segmented RNA virus infecting sugarcane. In this study, we re-examined type material and additional isolates using high-throughput sequencing and RT-PCR validation, revealing that RmSV possesses a nine-segmented genome, making it the largest reported in the Phenuiviridae. This expanded architecture includes duplicated RNA segments (RNA 2a and RNA 2b) encoding nucleocapsid-like proteins and two novel segments (RNA 7 and RNA 8). Comparative analysis showed that RNA 2a and 2b share about 84% amino acid identity, while RNA 5 encodes a third nucleocapsid homolog, indicating unprecedented domain redundancy. Structural modeling confirmed that all three nucleocapsid proteins maintain a conserved fold despite low sequence identity, with electrostatic mapping suggesting differential RNA-binding potential. Additionally, RNA 6 encodes a hypothetical protein structurally similar to the rice stripe virus disease-specific S-protein, implicating a role in symptom development. Transcript abundance analysis revealed RNA 6 as the most highly expressed segment across isolates. These findings revise the genomic composition of RmSV, highlight mechanisms of genome plasticity and adaptive evolution in plant-infecting bunyaviruses, and underscore practical implications for diagnostic assay design, resistance breeding, and biosecurity surveillance.
Genome, Viral↗
Location of the antigenic determinants of bovine pancreatic ribonuclease.
Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least three antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data, however, for each purified specific antibody preparation, carried out by the method of Berzofsky et al. [Berzofsky, J. A., Curd, J. G., & Schechter, A. N. (1976) Biochemistry, 15, 2113], leads to an estimate of four for the number of antigenic determinants in ribonuclease; this estimate had also been made earlier by Stelos et al. [Stelos, P., Fothergill, J. E., & Singer, S. J. (1960) J. Am. Chem. Soc. 82, 6034]. We find that one determinant is associated with each of segments 1--13 and 80--124 and two with segment 31--79. No antigenic activity could be detected for segment 14--29 either in native ribonuclease or in the free fragment. These conclusions are based on (1) the use of specific peptides to isolate purified antibodies by affinity chromatography, (2) immunoprecipitation of an antigenic peptide from the peptic digest of ribonuclease, (3) competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1--13, 31--79, and 80--124, the tryptic peptides 40--61 and 105--224, S-peptide, S-protein, and des(121--124)-RNase], and (4) comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the four antigenic determinants are localized around the alpha-helical portion of segment 1--10, somewhere in segment 40--61, at the beta bend in segment 63--75, and either at the beta bend or beta sheet in segment 87--104 of native ribonuclease.
Amino Acids↗
[Nature of the heterogeneity of 30S ribosomal subparticles in vitro. I. Effect of large centrifugal fields during 30S subparticle isolation on their capacity for codon-dependent tRNA binding].
Measurements of association constants (Ka) of specific [14C]Phe-tRNAPhe with a 30S..poly(U) complex revealed that values of these constants vary from 0.5.10(7) up to 1.5.10(8) M--1 when different 30S subunit preparations were used at the same medium conditions (20 mM Mg2+, 200 mM NH4, 0 degrees C). Analysis of these data showed that the higher the rotor speeds were used during separation of 70S ribosomes into subunits, the less Ka values were measured. In special experiments on sedimentation of pure 30S subunits at different rotor speeds it was found that the decrease of Ka values was caused due to the additional reversible dissocation of ribosomal proteins from 30S subunits at high (the order of 100 000.g) centrifugal fields. As a possible mechanism of such dissociation we suggest the influence of high hydrostatic pressure on the association constants of S-proteins with 30S subunits. Data presented in this paper demonstrate that at least one of the reasons for the physical and functional heterogeneity of 30S subunits in vitro derives from the application of high centrifugal fields during isolation of ribosomal subunits.
Cell Fractionation↗
[Nature of the heterogeneity of the 30S ribosomal subunits in vitro. II. Two types of inactivation of the 30S subunits of Escherichia coli ribosomes].
The influence of concentration of monovalent cations on the binding constant of Phe-tRNAPhe to 30S.poly(U) complex was studied. Two types of inactivation of the 30S subunits by ammonium ions at the low magnesium concentration (1 mM) were found. The first type of inactivation was observed at high concentrations of NH4+ ions (from 0.5 to 1.5 M) and due to the dissociation of ribosomal proteins from 30S subunits. This inactivation only decreased the binding constant of Phe-tRNAPhe to 30S.poly(U) complex up to 50 times but all 30S subunits were equally achieved in Phe-tRNAPhe binding. This type of inactivation was reversible, addition of S-proteins restored the association constant to the original value. At low concentration of NH4+ ions (below 100 mM) about half of the 30S subunits is irreversibly inactivated (the binding constant of Phe-tRNAPhe decreased below detectable level) probably as a result of conformational changes in ribosomal RNA. Both types of inactivation of the 30S subunits can take place during the preparation of isolated subunits of ribosomes.
Escherichia coli↗
Studies on polypeptides. XXIX. Synthesis of the N-terminal tetradecapeptide sequence of ribonuclease A, incorporating beta-(3-pyridyl)-L-alanine at position 12.
A description is given of the synthesis by fragment condensation of 12-(3-Pyd) Ala S-peptide (1--14), the 1--14 peptide sequence of ribonuclease A in which the histidine-12 residue is replaced by beta-(3-pyridyl)-L-alanine. The peptide binds strongly and stoichiometrically to S-protein; the resulting complex is enzymatically inactive.
Alanine↗