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R Wynn

Publications and source records attributed to R Wynn.

At least 37 records · Page 2Linked to original sources
PubMed1997

Comparison of straight chain and cyclic unnatural amino acids embedded in the core of staphylococcal nuclease.

We have determined by X-ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this behavior and have suggested that it may arise from the addition of side-chain vibrational and oscillational motions with each bond as a side chain grows away from the relatively rigid protein main chain and/or the population of multiple rotamers (Wynn R, Harkins P, Richards FM. Fox RO. 1996. Mobile unnatural amino acid side chains in the core of staphylococcal nuclease. Protein Sci 5:1026-1031). Reduction of the number of degrees of freedom by cyclization of a side chain would be expected to constrain these motions. These side chains are in fact well defined in the structures described here. Over-packing of the protein core results in a 1.0 A shift of helix 1 away from the site of mutation. Additionally, we have determined the structure of a side chain containing a single hydrogen to fluorine atom replacement on a methyl group. A fluorine atom is intermediate in size between methyl group and a hydrogen atom. The fluorine atom is observed in a single position indicating it does not rotate like methyl hydrogen atoms. This change also causes subtle differences in the packing interactions.

Amino Acids↗
PubMed1997

The number of words spoken by male doctors and male and female patients in a sample of British general practitioner-patient consultations.

OBJECTIVE: To investigate the relationship of the amount of words spoken by general practitioners and patients to the main topics of the consultations and the sex of the patients. DESIGN, SETTING AND SUBJECTS: Sample of 60 face-to-face doctor-patient consultations, with five different doctors and 60 different patients, randomly chosen from a database of British general practitioner-patient consultations. All the doctors were male; 33 patients were female, 27 male. MAIN OUTCOME MEASURES: Number of words spoken by doctors and patients, related to sex of patients and the consultations' main topics. RESULTS: There was no significant difference in the overall amount of words spoken by doctors and patients. However, doctors uttered significantly more words than their patients in those consultations in which the main topic was somatic, while patients spoke significantly more words than their doctors in those consultations in which the main topic was psychosocial. CONCLUSIONS: The main topics of the consultations were more important than the sex of the patients with regard to the proportion of words uttered by doctors and by patients.

Adolescent↗
PubMed1996

An observer study for direct comparison of clinical efficacy of electronic to film portal images.

PURPOSE: To directly compare clinical efficacy of electronic to film portal images. METHODS AND MATERIALS: An observer study was designed to compare clinical efficacy of electronic to film portal images acquired using a liquid matrix ion-chamber electronic portal imaging device and a conventional metal screen/film system. Both images were acquired simultaneously for each treatment port and the electronic portal images were printed on gray-level thermal paper. Four radiation oncologists served as observers and evaluated a total of 44 sets of images for four different treatment sites: lung, pelvis, brain, and head/neck. Each set of images included a simulation image, a double-exposure portal film, and video paper prints of electronic portal images. Eight to nine anatomical landmarks were selected from each treatment site. Each observer was asked to rate each landmark in terms of its clinical visibility and to rate the ease of making the pertinent verification decision in the corresponding electronic and film portal images with the aid of the simulation image. RESULTS: Ratings for the visibility of landmarks and for the verification decision of treatment ports were similar for electronic and film images for most landmarks. However, vertebral bodies and several landmarks in the pelvis such as the acetabulum and public symphysis were more visible in the portal film images than in the electronic portal images. CONCLUSION: The visibility of landmarks in electronic portal images is comparable to that in film portal images. Verification of treatment ports based only on electronic portal images acquired using an electronic portal imaging device is generally achievable.

Observer Variation↗
PubMed1996

Mobile unnatural amino acid side chains in the core of staphylococcal nuclease.

The structures of several variants of staphylococcal nuclease with long flexible unnatural amino acid side chains in the hydrophobic core have been determined by X-ray crystallography. The unnatural amino acids are disulfide moieties between the lone cysteine residue in V23C nuclease and methane, ethane, 1-n-propane, 1-n-butane, 1-n-pentane, and 2-hydroxyethyl thiols. We have examined changes in the core packing of these mutants. Side chains as large as the 1-n-propyl cysteine disulfide can be incorporated without perturbation of the structure. This is due, in part, to cavities present in the wild-type protein. The longest side chains are not well defined, even though they remain buried within the protein interior. These results suggest that the enthalpy-entropy balance that governs the rigidity of protein interiors favors tight packing only weakly. Additionally, the tight packing observed normally in protein interiors may reflect, in part, the limited numbers of rotamers available to the natural amino acids.

Amino Acids↗
PubMed1996

Polar day and polar night: month of year and time of day and the use of physical and pharmacological restraint in a north Norwegian university psychiatric hospital.

All episodes of restraint in a Norwegian university psychiatric hospital during a five and a half-year period have been studied retrospectively. An examination of the protocols in which the use of restraint is noted reveals that patients' aggression and the subsequent use of restraint follows daily and seasonal rhythms. There is a daily peak with most use of restraint in the afternoon and early evening and a seasonal peak- with the most use of restraint in autumn. Patients' age and sex also influence the daily and seasonal patterns of restraint. It is suggested that the 24-hour patterns as well as the seasonal patterns of aggression and restraint result from the combined influence, in susceptible patients, of light-dark cycles and variations in life-events and the ward environment.

Adolescent↗
PubMed1995

Ionizable P1 residues in serine proteinase inhibitors undergo large pK shifts on complex formation.

The burial of charged residues in proteins is rare as it is thermodynamically strongly disfavored. However, in "standard mechanism" protein inhibitors of serine proteinases, the P1 residue, which is highly exposed, becomes buried in the S1 specificity pocket of the enzyme. In many enzymes, such as Streptomyces griseus proteinase B (SGPB) the S1 pocket is hydrophobic. We measured the pH dependence of the association equilibrium constant for the interaction of SGPB with turkey ovomucoid third domain P1 mutants, Glu18 OMTKY3 and His18 OMTKY3. In order to eliminate the effects of other ionizable groups on the enzyme and the inhibitor, we divided these pH dependences by the pH dependence of the association equilibrium constant for the Gln18 OMTKY3 mutant. This yielded for Glu18, pKf (free inhibitor) of 4.46 +/- 0.05 and pKc (complex) of 8.74 +/- 0.06. For His18 the values are pKf 6.63 +/- 0.08 and pKc 4.31 +/- 0.07. At low pH values Glu18 variant is a relatively good inhibitor for SGPB. This may be biologically relevant.

Amino Acid Sequence↗
PubMed1995

Mixed disulfide intermediates during the reduction of disulfides by Escherichia coli thioredoxin.

The reduction of disulfides by thioredoxin involves a two-step mechanism. The first step features an intermolecular attack of Cys32 of thioredoxin on the disulfide with formation of a protein mixed disulfide and release of 1 equiv of thiol. The second step involves intramolecular breakdown of the mixed disulfide intermediate via attack of Cys35 with concomitant formation of the oxidized protein and release of a second equivalent of thiol. Study of mixed disulfide intermediates for Escherichia coli thioredoxin is exceedingly difficult because the second step is highly favorable. We have studied these intermediates via two approaches. First, Cys35 can be mutated to the similar but chemically nonreactive residue serine. This precludes breakdown of the intermediate. Second, "mass action trapping" techniques can be used because the second step of the mechanism is first-order in the forward direction and second-order in the reverse direction. This has yielded a thermodynamic breakdown of the reaction into its two component steps. Results for reaction of thioredoxin and 2-hydroxyethyl disulfide indicate that about half of the free energy change for the entire process is associated with the first step. Comparison with a small molecule cysteine analog suggests that significant interactions stabilize the mixed disulfide intermediate. Two-dimensional NMR analysis of the C35S thioredoxin 32C-beta-mercaptoethanol mixed disulfide shows packing interactions between the mixed disulfide moiety and Trp31 and Ile75. Additionally, studies with C35S thioredoxin show that substitution of the cysteine residue slightly perturbs the equilibrium for the first step in the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Substitution of charged residues into the hydrophobic core of Escherichia coli thioredoxin results in a change in heat capacity of the native protein.

Two site-directed mutants of Escherichia coli thioredoxin (L78K and L78R) were designed to study the effect of placing a charged residue in the hydrophobic core of the protein. Both mutants retain catalytic activity in the assembly of phage M13. Thermal denaturation of both these mutant proteins at pH 7.0 shows a reduction of stability of approximately 4 kcal.mol-1 with respect to the oxidized wild-type form. The thermal denaturation of the protein fits a dimeric state model. A significant reduction in the change in heat capacity (delta Cp) on unfolding is observed compared to oxidized wild-type thioredoxin. We present data to indicate that this reduction in delta Cp is attributable to structural perturbations resulting in localized unfolding of the native protein and exposure to solvent of residues that are buried in the wild-type protein.

Calorimetry, Differential Scanning↗
PubMed1995

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes.

Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1995

Flow cytometric determination of pre-transfusion red cell volume in fetuses and neonates requiring transfusion based on RhD+ dilution by transfused D- red cells.

We report a case of feto-maternal haemorrhage and describe a new flow-cytometric method of determining a fetus's or infant's pre-transfusion red cell volume (RCV). We validate this method against an established technique, employing fetal haemoglobin (HbF) dilution, for determining the RCV in fetuses and neonates requiring intravascular transfusion. We discuss advantages and other potential applications of this new method.

Blood Transfusion, Intrauterine↗
PubMed1994

Thermodynamic effects of reduction of the active-site disulfide of Escherichia coli thioredoxin explored by differential scanning calorimetry.

Intramolecular disulfide bonds in protein molecules, whether present in the wild-type protein or engineered via site-directed mutagenesis, are capable of significantly increasing the stability. Establishing thermodynamic parameters associated with the redox formation of this linkage is often encumbered by other global structural changes within the protein molecule. The active site of Escherichia coli thioredoxin possesses a disulfide/dithiol in a short loop, oxidation/reduction of which is accompanied by little structural alteration of the protein. Data for the thermal denaturation of the reduced protein are presented, which on comparison to the data obtained for the oxidized form [Ladbury, J.E., Wynn, R., Hellinga, H.W., & Sturtevant, J.M. (1993) Biochemistry 32, 7526-7530] are used to establish thermodynamic parameters for the redox reaction in this molecule. Data for an isosteric double mutation in the active site of thioredoxin (Cys32Ser/Cys35Ser) are also presented. Although the wild-type and mutated proteins show a similar reduction in free energy compared to the oxidized form (-3.0 +/- 0.4 and -3.1 +/- 0.3 kcal mol-1, respectively), the enthalpic and entropic contributions to this destabilization are different for the two proteins.

Binding Sites↗
PubMed1994

Psychiatric morbidity in primary health care in Santiago, Chile. Preliminary findings.

BACKGROUND: The aims were to determine the prevalence of psychiatric morbidity among primary care attenders in a poor suburb of Santiago and to study the relationship with health service use. METHOD: A cross-sectional survey was made of 163 consecutive attenders to a primary care clinic. RESULTS: Eleven per cent of the sample gave a psychological reason for consultation and the prevalence of psychiatric morbidity was 53%, defined using the revised Clinical Interview Schedule. Women and those of lower socio-economic status were at higher risk. Physicians recognised 14% of the psychiatric morbidity. Attenders with psychiatric morbidity consulted more frequently. CONCLUSIONS: There is a need to improve the recognition and management of psychiatric morbidity in primary care in Chile and other less developed countries. This could lead to the more efficient use of scarce health care resources in primary care.

Adult↗
PubMed1993

Partitioning the effects of changes in a protein to the folded or unfolded forms by using a thermodynamic cycle: a change in Escherichia coli thioredoxin does not affect the unfolded state.

Previously, we have introduced a method whereby novel disulfide side chains can be produced in the interior of a protein by modifying a cysteine residue after denaturant-induced unfolding [Wynn, R., & Richards, F. M. (1993) Proteins: Struct., Funct., Genet. 2, 395-403]. Here the disulfide exchange equilibrium constant, Kred, between the protein C32S,C35S-L78C thioredoxin and 2-hydroxyethyl disulfide is studied as a function of urea concentration. Since the disulfide exchange reaction and the stability of the proteins are thermodynamically linked, independent knowledge of the stabilities of the modified and unmodified proteins along with measurements of Kred allows us to assign Kred values for the folded and urea-unfolded forms of the protein. We find that the disulfide exchange reaction is more favorable for the folded protein, in agreement with the increased stability of the modified protein, and that the Kred values for both states are independent of the urea concentration. Finally, Kred values for the unfolded protein are the same, within experimental error, as that for N-acetylcysteine methylamide, an analog of cysteine in a peptide chain without the possibility of intramolecular interactions. Thus, we conclude that modification of position 78 of thioredoxin does not affect the unfolded state. The relevance of these results toward protein stability studies is discussed.

Cysteine↗
PubMed1993

Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position.

The effects of pH in the range 6.0-8.0 on the thermodynamics of the reversible thermal unfolding of Escherichia coli thioredoxin in the oxidized state have been determined over a range of concentrations using differential scanning calorimetry. The thermal denaturation indicated an inverse temperature dependence on concentration. The data were shown to fit a model based on dimerization of both the native and denatured states of the protein. The degree of dimerization of both states was found to be pH dependent. The previously described importance of protonation of the anomalously titrating aspartic acid 26 residue [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry 30 ,7603-7609] was apparently verified by the agreement between the experimentally determined delta delta Gzerod and the calculated delta delta GzeroH in the pH range 7.0-8.0.

Aspartic Acid↗
PubMed1993

Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques.

We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1-n-propane, 1-n-butane, and 1-n-pentane thiols. These mutants all show native-like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmCl]1/2, and the free energy of unfolding at zero denaturant concentration, delta G(H2O), give inverse orders of stability. This effect is due to changes in m, the dependence of delta G0 unfolding on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.

Amino Acids↗
PubMed1993

Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues.

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. beta-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of gamma-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6-18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19-56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive-site peptide bond X18-Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive-site-intact inhibitor variants.

Amino Acid Sequence↗