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Biomedical subjects

W Furey

Publications and source records attributed to W Furey.

At least 37 records · Page 2Linked to original sources
PubMed1993

Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.

Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗
PubMed1992

Crystal structure of staphylococcal enterotoxin B, a superantigen.

The three-dimensional structure of staphylococcal enterotoxin B, which is both a toxin and a super-antigen, has been determined to a resolution of 2.5 A. The unusual main-chain fold containing two domains may represent a general motif adopted by all staphylococcal enterotoxins. The T-cell receptor binding site encompasses a shallow cavity formed by both domains. The MHCII molecule binds to an adjacent site. Another cavity with possible biological activity was also identified.

Animals↗
PubMed1992

Refined structure of rat Clara cell 17 kDa protein at 3.0 A resolution.

The rat Clara cell 17 kDa protein (previously referred to as the rat Clara cell 10 kDa protein) has been reported to inhibit phospholipase A2 and papain, and to also bind progesterone. It has been isolated from rat lung lavage fluid and crystallized in the space group P6(5)22. The structure has been determined to 3.0 A resolution using the molecular replacement method. Uteroglobin, whose amino acid sequence is 55.7% identical, was used as the search model. The structure was then refined using restrained least-squares and simulated annealing methods. The R-factor is 22.5%. The protein is a covalently bound dimer. Two disulfide bonds join the monomers together in an antiparallel manner such that the dimer encloses a large internal hydrophobic cavity. The hydrophobic cavity is large enough to serve as the progesterone binding site, but access to the cavity is limited. Each monomer is composed of four alpha-helices. The main-chain structure of the Clara cell protein closely resembles that of uteroglobin, but the nature of many of the exposed side-chains differ. This is true, particularly in a hypervariable region between residues 23 and 36, and in the H1H4 pocket.

Amino Acid Sequence↗
PubMed1990

Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast.

Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.

Macromolecular Substances↗
PubMed1990

Direct methods with single isomorphous replacement data. I. Reduction of systematic errors.

The direct-methods procedure for single isomorphous replacement (SIR) data [Hauptman (1982). Acta Cryst. A38, 289-294], as modified by Fortier, Moore & Fraser [Acta Cryst. (1985), A41, 571-577] has been implemented and tested with a large number of known structures. It was found that the modified procedure greatly reduces the bias toward 'unresolved' SIR invariant values associated with estimates of 0 or pi, but does not remove it entirely. If the heavy atoms are not in a centrosymmetric array the centroid of the distribution of invariant estimates is not centered on true protein values, but is biased toward conventional SIR values by up to 15 degrees, thus errors in the estimates are not random but systematic. When the heavy atoms are in a centrosymmetric array (or single heavy-atom site in space group P21), the distribution of estimates is often sharply bimodal, with peaks centered at both true invariant values and pure 'unresolved' SIR values. Simple procedures are given which can be applied in both situations to reduce significantly the bias with no overall loss of accuracy. An additional correction factor is then described which can be used to remove nearly all of the bias, and improve the accuracy as well. The result is that errors in the corrected invariant estimates are small in magnitude, but are now also random instead of systematic. Since the number of estimates greatly exceeds the number of phases, the remaining random errors should have little impact in phasing processes.

Bence Jones Protein↗
PubMed1985

Novel arrangement of immunoglobulin variable domains: X-ray crystallographic analysis of the lambda-chain dimer Bence-Jones protein Loc.

We have characterized and crystallized a human lambda I light-chain dimer, Bence-Jones protein Loc, which has variable (V) region antigenic determinants characteristic for the lambda I subgroup and constant (C) region determinants of the C lambda I gene Mcg. The crystal structure was determined to 3-A resolution; the R factor is 0.27. The angle formed by the twofold axes of the V and C domains, the "elbow bend", is 97 degrees, the smallest found so far for an antibody fragment. The antigen-binding site formed by the two V domains of the Loc light chain differs significantly from those of other immunoglobulin molecules (light-chain dimers and Fab fragments) for which X-ray crystallographic data are available. Whereas, in other antibody fragments, the V domains are related by a local twofold axis, a local twofold screw axis with a translational component of 3.5 A relates the V domains in protein Loc. In contrast to the classic antigen binding "pocket" formed by V domain interactions in the previously characterized antibody structures, the V region associations in protein Loc result in a central protrusion in the binding site, with grooves on two sides of the protrusion. The structure of protein Loc indicates that immunoglobulins are physically capable of forming a more diverse spectrum of antigen-binding sites than has been heretofore apparent. Moreover, the unusual protruding nature of the binding site may be analogous to structures required for some anti-idiotypic antibodies. Further, the complementarity-determining residues form parts of two independent grooves.(ABSTRACT TRUNCATED AT 250 WORDS)

Bence Jones Protein↗
PubMed1984

Heart surgery in infants: a preliminary assessment of maternal adaptation.

The present investigation was undertaken to determine the effects on the mother of having a child undergo heart surgery. Three groups of subjects were recruited and included mothers of infants undergoing heart surgery (Heart Surgery group), mothers of infants admitted on an emergency basis for nonsurgical/nonterminal illness (Inpatient Control), and mothers of infants visiting the pediatrician for well baby checks (Outpatient Control). Data were collected at three times: PRE, the day of admission; POST, the 7th day of admission or day of discharge (whichever came first); and FOLLOW-UP, 2 months after discharge. Relative to the other two groups, a differential attrition rate was evidenced in the Inpatient Control group and this finding is discussed. Given the attrition rate, analyses after PRE involved the Heart Surgery and Outpatient Control. Findings indicate that, relative to the Outpatient Control group, the Heart Surgery group reported greater distress at FOLLOW-UP than at any other time. The distress that was reported involved disruption in the family environment. Results of the analyses are discussed as consistent with posttraumatic stress theory.

Cardiac Surgical Procedures↗
PubMed1983

Structure of a novel Bence-Jones protein (Rhe) fragment at 1.6 A resolution.

The crystal structure of Rhe, a lambda-type Bence-Jones protein fragment, has been solved and refined to a resolution of 1.6 A. A model fragment consisting of the complete variable domain and the first three residues of the constant domain yields a crystallographic residual RF value of 0.149. The protein exists as a dimer both in solution and in the crystals. Although the "immunoglobulin fold" is generally preserved in the structure, there are significant differences in both the monomer conformation and in the mode of association of monomers into dimers, when compared to other known Bence-Jones proteins or Fab fragments. The variations in conformation within monomers are particularly significant as they involve non-hypervariable residues, which previously were believed to be part of a "structurally invariant" framework common to all immunoglobulin variable domains. The novel mode of dimerization is equally important, as it can result in combining site shapes and sizes unobtainable with the conventional mode of dimerization. A comparison of the structure with other variable domain dimers reveals further that the variations within monomers and between domains in the dimer are coupled. Some possible functional implications revealed by this coupling are greater variability, induced fitting of the combining site to better accommodate antigenic determinants, and a mechanism for relaying binding information from one end of the variable domain dimer to the other. In addition to providing the most accurate atomic parameters for an immunoglobulin domain yet obtained, the high resolution and extensive refinement resulted in identification of several tightly bound water molecules in key structural positions. These water molecules may be regarded as integral components of the protein. Other water molecules appear to be required to stabilize the novel conformation.

Amino Acid Sequence↗
PubMed1981

Structure of a 7Fe ferredoxin from Azotobacter vinelandii.

The structure of the 7Fe ferredoxin from Azotobacter vinelandii has been solved from a 3.0-A multiple isomorphous replacement map. The crystals belong to space group P43212 with a = 55.22, c = 95.20 A, and Z = 1. Heavy-atom derivatives were prepared with K2PtCl4,K2[OsO2(OH)4], and Na3RhCl6. Anomalous scattering data were collected for native (Fe) and Pt derivative crystals. The figure of merit for 3,322 reflections to 3.0 A is 0.74. The structure consists of an NH2-terminal core of residues 1-50 which form the Fe-S cluster sites, and a COOH-terminal chain of residues 51-107 which wraps around this core. The [3Fe-3S] cluster is ligated by cysteines 8, 11, 16, 20, and 49 and a sixth ligand which is either glutamic acid 18 or an exogenous small molecule. The [4Fe-4S] cluster is ligated by cysteines 24, 39, 42, and 45. The coordination of both Fe-S centers has been confirmed by fitting of the cluster atoms and residues 1-50 to unbiased 2Fo-Fc Fourier maps at 2.5-A resolution. The structure of the 3Fe center has also been confirmed with anomalous scattering difference Fourier maps using both isomorphous replacement and refined phases. The partially refined structure at 2.5 A (3,490 reflections, 6.0 sigma(F)) has R = 35%.

Azotobacter↗