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K S Wilson

Publications and source records attributed to K S Wilson.

At least 145 records · Page 8Linked to original sources
PubMed1995

Structure of inhibited trypsin from Fusarium oxysporum at 1.55 A.

The structure of trypsin from the fungus Fusarium oxysporum has been refined at 1.55 A resolution by restrained least-squares minimization to an R-factor of 14.4%. The data were recorded from a single-crystal on the X31 beamline at EMBL, Hamburg, using a locally developed image-plate scanner. The final model consists of 1557 protein atoms, 400 water molecules, one molecule of isopropanol and one monoisopropyl phosphoryl inhibitor group covalently bound to the catalytic Ser195. Comparison of the structure with bovine trypsin reveals significant differences in the active site and suggests a possible explanation for the difference in substrate specificity between the two enzymes. In F. oxysporum trypsin the specificity pocket is larger than in bovine trypsin. This explains the preference of F. oxysporum trypsin for the bulkier arginine over lysine and the reverse preference in bovine trypsin. The binding cavity on the C-terminal side of the substrate is more restricted in F. oxysporum trypsin than in mammalian and Streptomyces griseus trypsins, which explains the relative inactivity of F. oxysporum trypsin towards peptide-pNA substrate analogues as an unfavourable steric interaction between the side of the binding cavity and the para-nitroanilino group of peptide-pNA. The observed restriction of the binding cavity does not lead to a reduced catalytic activity compared to other trypsins.

Journal Article↗
PubMed1995

Proteins at atomic resolution.

Experimental advances in data collection, including bright sources, cryogenic cooling and two-dimensional detectors, have made it tractable to record data to beyond 1.2 A for several proteins, yielding high-accuracy models and fine details of structure. For small metalloproteins, atomic-resolution data have enabled ab initio solution of the phase problem.

Computers↗
PubMed1995

How nature deals with stereoisomers.

All natural proteins are composed of L-amino acids and are inherently chiral. The properties of both L- and chemically synthesized D-amino acids are identical except in optically asymmetric interactions. Structural studies of D-I racemic mixtures of crystallographic interest are discussed. The review also gives some recent examples of stereospecificity: how L-proteins deal with L- or D-substrates and how enzymes can function as racemases. Two particular examples of stereoselectivity are then discussed.

Amino Acids↗
PubMed1995

Renal cell carcinoma with translocation (X;1). Further evidence for a cytogenetically defined subtype.

A renal cell carcinoma from a 15-year-old male had a 49,Y,t(X;1)(p11.2;q21), +der(X)t(X;1) (p11.2;q21), +5, -16, +17, +18 karyotype. This is the third report of a translocation involving a breakpoint at Xp11.2 in a renal cell carcinoma in a child. A total of nine cases of renal cell carcinoma involving Xp11, including this case, have been reported. Of the eight cases for which there are genetics reports, all are male. Patients with renal cell carcinoma with abnormalities at Xp11 appear to be younger than renal cell carcinoma patients overall.

Adolescent↗
PubMed1995

A common protein fold and similar active site in two distinct families of beta-glycanases.

The structure of Clostridium thermocellum endoglucanase CelC, a member of the largest cellulase family (family A), has been determined at 2.15 A resolution. The protein folds into an (alpha/beta)8 barrel, with a deep active-site cleft generated by the insertion of a helical subdomain. The structure of the catalytic core of xylanase XynZ, which belongs to xylanase family F, has been determined at 1.4 A resolution. In spite of significant differences in substrate specificity and structure (including the absence of the helical subdomain), the general polypeptide folding pattern, architecture of the active site and catalytic mechanism of XynZ and CelC are similar, suggesting a common evolutionary origin.

Amino Acid Sequence↗
PubMed1995

The family pet as an unlikely source of group A beta-hemolytic streptococcal infection in humans.

This study examines the possibility of the family pet serving as a reservoir for group A beta-hemolytic streptococcal infections in humans. We obtained oropharyngeal cultures from children with acute pharyngitis and concurrent oropharyngeal cultures from their household pets. Children with culture-proved group A beta-hemolytic streptococcal pharyngitis were detected in 26 of 42 households surveyed. Oropharyngeal cultures were also collected from a group of children without pharyngitis and their pets. Additionally 149 dogs and cats from a local veterinary hospital were cultured from 371 body sites including the oropharynx, axilla and vagina. All beta-hemolytic bacterial isolates were identified by colonial and microscopic morphology, catalase and pyrrolidonylarylamidase production, bacitracin susceptibility and serogrouping. No group A beta-hemolytic streptococci were recovered from any of the body sites surveyed from a total of 230 animals. Based on these findings, the family pet seems to be an unlikely reservoir for group A beta-hemolytic streptococci.

Acute Disease↗
PubMed1995

Stability of subtilisins and related proteinases (subtilases).

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN', Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2 x 10(-3)-10(-1) M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca2+ in the second (third in the case of thermitase) binding site increases the Tm values by 11-21 degrees C and the delta GD(H2O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca2+ concentration of 2 x 10(-2) M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the alpha-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca2+. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.

Binding Sites↗
PubMed1995

Randomized trial of interferon maintenance in multiple myeloma: a study of the National Cancer Institute of Canada Clinical Trials Group.

PURPOSE: To determine whether interferon maintenance therapy improves overall survival and response duration in patients with multiple myeloma who have responded to induction therapy with melphalan and prednisone. PATIENTS AND METHODS: In a multicenter trial, patients with symptomatic clinical stage I and stage II and III multiple myeloma were registered at diagnosis and those who responded to melphalan-prednisone (MP) were randomized either to receive interferon (2 mU/m2) subcutaneously three times per week or no maintenance. MP was discontinued in both groups once a stable response plateau of the monoclonal protein was reached. Interferon was continued until relapse, and then was restarted on subsequent response to MP. Interferon toxicity was recorded using a self-report diary. Survival and response duration were calculated using life-table methods, and were adjusted in the analysis for imbalances in baseline prognostic factors. RESULTS: Four hundred two patients were registered and 176 responders were randomized (85 to interferon and 91 to control). At a median follow-up time of 43 months, the median survival duration was 43 months for interferon and 35 months for control (P = .16), but when adjusted for chance imbalances in baseline prognostic factors (mainly performance status), the median survival duration was 44 months and 33 months for interferon and control, respectively (P = .049). Progression-free survival from randomization to first relapse also favored interferon (unadjusted P < .002; adjusted P < .003). Interferon toxicity caused 58% of patients to reduce their dose, of which 84% were able to return to the initial dose; 14% had to discontinue interferon treatment. CONCLUSION: Interferon maintenance therapy improves progression-free and overall survival of patients with multiple myeloma who respond to melphalan and prednisone. Toxicity is substantial and must be weighed by patients against the potential benefits in response duration and survival.

Aged↗
PubMed1995

RNA interactions in the regulation of transcription.

In this overview we describe interactions between RNA, DNA, and proteins that are involved in the regulation of the various functional cycles of E. coli transcription. The role of the RNA terminator hairpin in destabilizing the transcription elongation complex and controlling the efficiency of termination at various template positions near intrinsic terminators are discussed, as are the mechanisms whereby rho protein triggers RNA release at rho-dependent terminators. Mechanisms used by phage 1-coded antitermination factor N in "fine-tuning" termination efficiencies are also considered, as are the roles of kinetically different elongation complex conformations in controlling transcriptional fidelity. General principles of the control of transcription by such RNA-protein interaction mechanisms are discussed.

Bacteriophage lambda↗
PubMed1994

Crystal structure of a bacterial chitinase at 2.3 A resolution.

BACKGROUND: Chitinases cleave the beta-1-4-glycosidic bond between the N-acetyl-D-glucosamine units of which chitin is comprised. Chitinases are present in plants, bacteria and fungi, but whereas structures are available for two prototypic plant enzymes, no structure is available for a bacterial or fungal chitinase. RESULTS: To redress this imbalance, the structure of native chitinase A from Serratia marcescens has been solved by multiple isomorphous replacement and refined at 2.3 A resolution, resulting in a crystallographic R-factor of 16.2%. The enzyme comprises three domains: an all beta-strand amino-terminal domain, a catalytic alpha/beta-barrel domain, and a small alpha+beta-fold domain. There are several residues with unusual geometries in the structure. Structure determination of chitinase A in complex with N,N',N",N"'-tetra-acetylo-chitotetraose, together with biochemical and sequence analysis data, enabled the positions of the active-site and catalytic residues to be proposed. CONCLUSIONS: The reaction mechanism seems to be similar to that of lysozyme and most other glycosylhydrolases, i.e. general acid-base catalysis. The role of the amino-terminal domain could not be identified, but it has similarities to the fibronectin III domain. This domain may possibly facilitate the interaction of chitinase A with chitin.

Amino Acid Sequence↗
PubMed1994

1H, 13C, and 15N resonance assignments and secondary structure analysis of the HU protein from Bacillus stearothermophilus using two- and three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.

Nearly complete 1H, 13C, and 15N resonance assignments have been obtained for the protein HU from Bacillus stearothermophilus (dimer, 19.5 kDa) using double- and triple-resonance 2D and 3D NMR experiments. This has resulted in assignments of 91% of the observable protons, 98% of all 13C, and 92% of all 15N nuclei. NOEs obtained from a 3D time-shared NOESY-(13C,15N)-HSQC spectrum, exchange data of amide protons, and chemical shifts of the 1H alpha, 1HN, 13C beta, 13C alpha, 13CO, and 15N nuclei have been used to identify the secondary structure elements. Three alpha-helices (residues 3-13, 18-37, and 83-90) and three extended strands (residues 40-45, 48-62, and 67-82) have been found in HU. The arrangement of these elements of secondary structure is very similar to the X-ray structure [Tanaka et al. (1984) Nature 310, 376-381; White et al. (1989) Proteins 5, 281-288]. The conformation of the proposed DNA-binding region of HU, i.e., an antiparallel beta-hairpin, was not observed previously in the X-ray structure. In the NMR structure long range NOEs in the beta-arm region (residues 53-76) suggest a distortion between residue Pro-72 and Ala-73 and between Pro-63 and Gln-64 with concomitant distortions in the opposite strand. The NOE data indicate further that the loop region in the DNA-binding arms of HU is arranged as a type I beta-turn from Pro-63 to Gly-66.

Amino Acid Sequence↗
PubMed1994

11-fold symmetry of the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis determined by X-ray analysis.

The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis has been crystallized and examined by crystallography using X-ray synchrotron radiation diffraction data. Crystals of TRAP complexed with L-tryptophan belong to space group C2 with a = 156.8 A, b = 114.05 A, c = 105.9 A, beta = 118.2 degrees. Crystals of a potential heavy-atom derivative of TRAP complexed with 5-bromo-L-tryptophan grow in the same space group with similar cell dimensions. X-ray data for the native crystals and for the derivative have been collected to 2.9 A and 2.2 A resolution, respectively. Peaks in the self-rotation function and in the Patterson synthesis could only be explained by two 11-subunit oligomers (each formed by an 11-fold axis of symmetry) in the asymmetric unit lying with the 11-fold rotation axes parallel to each other. The consequence is that the TRAP molecule has 11-fold symmetry and contains 11 subunits.

Bacillus subtilis↗
PubMed1994

Stability of Escherichia coli transcription complexes near an intrinsic terminator.

Transcription complexes containing Escherichia coli RNA polymerase terminate RNA synthesis in response to intrinsic (rho-independent) terminators encoded in the DNA template. This type of termination involves the formation of a hairpin structure in the nascent RNA, leading to release of the RNA and polymerase from the DNA template. In this paper we show that RNA release occurs only within a zone extending over six template positions of the tR2 terminator of phage lambda. The upstream end of this zone is defined by a large and abrupt decrease in the stability of the transcription complex, and this decrease is reversed at the downstream end. Except within this termination zone, RNA chain elongation is completely processive at all template positions, over a wide range of temperatures (20 to 70 degrees). The stability of ternary complexes halted at each template position by the incorporation of 3'-deoxynucleotide substrate analogues was determined by means of an ultrafiltration RNA release assay. This assay shows that the nascent RNA is released from halted ternary complexes within the same zone in which termination occurs during active transcription. The termination efficiency at each position within the tR2 termination zone is a sensitive function of the concentration of the next nucleotide substrate to be incorporated into the nascent RNA. These results suggest that termination positions are defined by the thermodynamic stability of the polymerase complex, while the efficiencies of termination are determined by a kinetic competition between RNA elongation and release at each position. Based on a kinetic competition model, the temperature dependence of the efficiency indicates that the elongation pathway is favored enthalpically, while the termination pathway is favored entropically.

Bacteriophage lambda↗
PubMed1994

Crystal structure of domain A of Thermus flavus 5S rRNA and the contribution of water molecules to its structure.

This is the first high resolution crystal structure of an RNA molecule made by solid phase chemical synthesis and representing a natural RNA. The structure of the domain A of Thermus flavus ribosomal 5S RNA is refined to R = 18% at 2.4 A including 159 solvent molecules. Most of the 2'-hydroxyl groups as well as the phosphate oxygens are involved either in specific hydrogen bonds in intermolecular contacts or to solvent molecules. The two U-G and G-U base-pairs are stabilized by H-bonds supplied via three water molecules to compensate for the lack of base-pair hydrogen bonds. The structure shows for the first time in detail the importance of highly ordered internal water in stabilizing an RNA structure.

Base Sequence↗
PubMed1994

High-resolution structures of single-metal-substituted concanavalin A: the Co,Ca-protein at 1.6 A and the Ni,Ca-protein at 2.0 A.

The molecular structures of cobalt- and nickel-substituted concanavalin A have been refined at 1.6 and 2.0 A resolution, respectively. Both metal derivatives crystallize in space group I222 with approximate cell dimensions a = 89, b = 87 and c = 63 A and one monomer in the asymmetric unit. The final R factor for Co-substituted concanavalin A is 17.8% for 29 211 reflections with F > 1.0sigma(F) between 8.0 and 1.6 A. For Ni-substituted concanavalin A the final R factor is 15.9% for 16 128 reflections with F > 1.0sigma(F) between 8.0 and 2.0 A resolution. Both structures contain a transition-metal binding site and a calcium-binding site but, unlike Cd-substituted concanavalin A, do not have a third metal-binding site. The Co-substituted concanavalin A structure diffracts to the highest resolution of any concanavalin A structure reported to date. A comparison of the structures of Ni-, Co-, Cd-substituted and native concanavalin A gives an indication of coordinate errors, which is a useful baseline for comparisons with saccharide complexes of concanavalin A described in other work. We also give a detailed account of multiple conformations which were found for five side-chain residues.

Journal Article↗
PubMed1994

Crystallographic studies of Savinase, a subtilisin-like proteinase, at pH 10.5.

The effect of high pH (pH 10.5) on Savinase, a subtilisin-like serine proteinase, has been investigated using X-ray crystallography. The structures of two Savinase mutants were determined at two different pH values, namely pH 6.0, where the enzyme is inactive (this is the pH at which most of the structural work has been carried out on other serine proteinases), and pH 10.5, where Savinase is active. Comparison of these high resolution (0.16 nm) structures showed four related sets of changes between the two pH values. First, the difference in protonation state of the active-site histidine leads to a change in conformation of the active-site triad. Secondly, there are resulting changes in the water structure around this histidine residue with much increased mobility of the water in the active-site at pH 10.5. Thirdly, the two substrate-binding loops on either side of the binding site are less well linked by ordered water molecules at pH 10.5 and show substantially increased flexibility. Finally, the first substrate-binding loop, residues at positions 99-104, moves slightly so as to widen the substrate channel.

Amino Acid Sequence↗
PubMed1994

X-ray, NMR and molecular dynamics studies on reduced bovine superoxide dismutase: implications for the mechanism.

Single crystals of the reduced form of Cu, Zn superoxide dismutase (space group P2(1)2(1)2(1), one dimer per asymmetric unit) have been obtained and their X-ray structure refined at 1.9 A resolution. The structure shows that the imidazolate bridge is maintained in the present crystalline form. It is confirmed that in solution the bridge is broken and the involved histidine is protonated on the side of copper. Based on the NOE constraints, and with the aid of molecular dynamics calculations, a structural model is proposed for the molecule in solution. Both structures are considered significant as far as the enzymatic mechanism is concerned.

Animals↗