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W M Westler

Publications and source records attributed to W M Westler.

At least 19 recordsLinked to original sources
PubMed1997

Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site.

The binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

Anilino Naphthalenesulfonates↗
PubMed1996

Protonation-state dependence of hydrogen bond strengths and exchange rates in a serine protease catalytic triad: bovine chymotrypsinogen A.

Hydrogen-1 nuclear magnetic resonance spectroscopy was used to measure D/H fractionation factors and the temperature dependence of the rate of hydrogen exchange at two sites in the catalytic triad of chymotrypsinogen (hydrogen bond between aspartate-102 and histidine-57 and hydrogen bond between histidine-57 and serine-195) as a function of the protonation states of the constituent residues. Connectivities in one-dimensional spectra used to assign NMR data were collected at three pH values: pH 9, at which His-57 is neutral and Asp-102 is negatively charged; pH 3.5, at which His-57 is positively charged and Asp-102 is negatively charged; and pH 1, at which His-57 is positively charged and Asp-102 is neutral. The signal from H epsilon 2 of histidine-57 was assigned by reference to 1H-1H NOE connectivities at pH 3.5 to the previously assigned signals from the H epsilon 1 and H delta 2 of the same residue. The D/H fractionation factor, phi, for the hydrogen bond between Asp-102 and His-57 changed from phi = 2 at pH 9 to phi = 0.4 at pH 3.5. From studies of model systems, it may be concluded that a change of phi of this magnitude corresponds to a large increase in hydrogen bond strength. A signal from the hydrogen bond between Ser-195 and His-57 was detected only at the lower pH values studied. The D/H fractionation factor for this hydrogen bond was phi = 0.7 at pH 3.5, indicative of a moderately strong interaction. Data obtained at pH 1 indicate that the hydrogen bond between Asp-102 and His-57 is weakened but that the hydrogen bond between His-57 and Ser-195 persists. The results are consistent with the hypothesis that changes in hydrogen bonding strength serve to lower barriers along the reaction coordinate in the catalytic mechanism. Large pH-dependent changes were found in the activation enthalpy (delta H ++) for exchange with protons from the solvent at the hydrogen bond between aspartate-102 and histidine-57: delta H ++ was approximately 10-12 kcal.mol-1 higher at pH 3.5 than at pH 1 or 9.

Animals↗
PubMed1996

Continuous probability distribution (CUPID) analysis of potentials for internal rotations.

The continuous probability distribution (CUPID) approach for analyzing the rotamer populations from NMR spin-spin couplings and nuclear Overhauser enhancements [Z. Dzakula, W.M. Westler, A.S. Edison, and J.L. Markley, J. Amer. Chem. Soc. 114, 6195 (1992)] can be expanded to allow computation of the rotational potential from the Fourier coefficients of the angular probability distribution. This approach provides a general solution to the nonnegativity problem, which appears when lack of data causes a serious truncation in the Fourier series that defines the probability distribution. In favorable cases, this approach also allows thermodynamic characterization of internal rotation. Use of this extension of the CUPID method is illustrated by the analysis of internal rotations in an amino acid, two peptides, and an oligosaccharide from published experimental data. Three strategies have been devised for dealing with cases where the experimental input data do not provide enough information for complete reconstruction of the potential: (1) two-dimensional grid search for the undetermined third-order Fourier coefficients of the potential, (2) transfer of these coefficients from related model compounds, and (3) restriction of the magnitudes of the Fourier coefficients as required by the assumption of fast-exchange averaging of the input parameters. In addition, equations for translating uncertainties in experimental NMR input data into errors in calculated continuous probability distributions of rotamers are presented. The dependence of errors on various features of the distributions has been studied systematically from simulations. The results show that, typically, the confidence intervals are +/- 30-40 degrees for dihedral angles and +/- 0.2 for rotamer populations. For chi 1 rotamers of amino acids, the analysis is most sensitive to the uncertainties in C'-H beta couplings. A critical reexamination of the use of Gaussian functions to reconstruct a probability distribution is presented. In particular, the simplifying assumption of identical widths for all Gaussian probability peaks has been justified by showing that it does not lead to large errors in other CUPID parameters. finally, the angular dependencies of cross-relaxation rates, their uncertainties, and the potential for their use in studying chi 1 internal rotations in amino acids are discussed.

Electron Spin Resonance Spectroscopy↗
PubMed1996

NMR studies of Pi-containing extracellular and cytoplasmic compartments in brain.

The inorganic phosphate (Pi) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of Pi. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated Pi spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from infinity to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of Pi buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the Pi peaks arising from the three compartments with different pH values if each peak made up 10-35% of total Pi area. In vivo, we identified the plasma compartment by intraarterial infusion of Pi. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that Pi compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger Pi peaks represented intracellular spaces.

Animals↗
PubMed1995

Protein binding chiral discrimination of HPLC stationary phases made with whole, fragmented, and third domain turkey ovomucoid.

Individual protein domains and two domains in combination were prepared by enzymatic and chemical cleavage of turkey ovomucoid followed by isolation and purification by size-exclusion and ion-exchange chromatography. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers among a wide range of racemates. The columns made from whole turkey ovomucoid displayed chiral activity toward many racemates, where as a combination of the first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exhibited enantioselective protein binding for fused-ring aromatic weak acids. Glycosylation of the third domain did not affect chiral recognition. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids responsible for binding. Molecular modeling of the ligand-protein complexation provided insights into the ability of a protein surface to discriminate enantiomers on the basis of multiple intermolecular interactions.

Amino Acid Sequence↗
PubMed1995

Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative [2Fe-2S]ferredoxin.

Two alternative T7 RNA promoter/polymerase systems have been employed for the heterologous expression of a plant-type [2Fe-2S]ferredoxin, Anabaena 7120 vegetative ferredoxin, in Escherichia coli at high levels (approximately 20 mg/liter of culture). One system was used when 15N-labeling the ferredoxin uniformly by growing E. coli with 15NH4Cl as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating 2H-, 13C-, and 15N-labeled amino acids. The labeled ferredoxin samples were studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Results from 1H and 2H NMR studies of samples containing [2H alpha]Cys, [2H beta 2, beta 3]Cys, [13 C beta]-Cys, and [15N]Cys have confirmed previous cysteinyl proton resonance assignments (L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M. Holden, B. L. Jacobson, I. Rayment, and J. L. Markley (1991) Biochemistry 30, 7363-7368). All four 13C NMR peaks arising from the four cysteinyl beta-carbons and all four 15N NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys46, which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of 13Ci-15Ni+1 coupling in a ferredoxin sample with incorporated [13C']Ala and [15N]Cys. The nitrogen signal of Cys 41 was assigned tentatively on the basis of its chemical shift and T1 relaxation time. The cysteinyl beta-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) beta-protons. The beta-carbons resonance from Cys46 in the oxidized state has been assigned by its correlation with the corresponding resonance in the reduced state; this was accomplished by following the progressive air oxidation of a protein sample reduced by dithionite in the presence of methyl viologen. The spin-lattice relaxation times of the beta-carbons of the two cysteines coordinated to Fe)III) were similar in the oxidized and reduced states. This suggests that the antiferromagnetic coupling present in the reduced cluster has little influence on the electronic relaxation time of the Fe(III). Studies of the temperature dependence of the 1H, 13C, and 15N signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (delta delta/delta T-1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although delta delta/delta T-1 is positive for Cys49 1H beta 2 and 1H beta 3, it is negative for Cys49 13C beta. Although delta delta/delta T-1 is negative for protons of cysteines ligated to Fe(II) and positive for protons of cysteines ligated to Fe(III), it is positive for all the cysteinyl nitrogens.(ABSTRACT TRUNCATED AT 400 WORDS)

Anabaena↗
PubMed1995

Comparison of the accuracy of protein solution structures derived from conventional and network-edited NOESY data.

Network-editing experiments are variants of the basic NOESY experiment that allow more accurate direct measurement of interproton distances in macromolecules by defeating specific spin-diffusion pathways. Two network-editing approaches, block-decoupled NOESY and complementary-block-decoupled-NOESY, were applied as three-dimensional, heteronuclear-edited experiments to distance measurement in a small protein, turkey ovomucoid third domain (OMTKY3). Two-hundred and twelve of the original 655 distance constraints observed in this molecule (Krezel AM et al., 1994, J Mol Biol 242:203-214) were improved by their replacement by distances derived from network-edited spectra, and distance geometry/simulated annealing solution structure calculations were performed from both the unimproved and improved distance sets. The resulting two families of structures were found to differ significantly, the most important differences being the hinge angle of a beta-turn and an expansion of the sampled conformation space in the region of the reactive-site loop. The structures calculated from network-editing data are interpreted as a more accurate model of the solution conformation of OMTKY3.

Animals↗
PubMed1994

Estimates of phi and psi torsion angles in proteins from one-, two- and three-bond nuclear spin-spin couplings: application to staphylococcal nuclease.

Calculated coupling constants (3JHNH alpha, 1JC alpha H alpha, 2JC'H alpha, 1JC alpha N and 2JC alpha N) from our accompanying paper [Edison, A.S. et al. (1994) J. Biomol. NMR, 4, 519-542] have been used to generate error surfaces that can provide estimates of the phi and psi angles in proteins. We have used experimental coupling data [3JHNH alpha: Kay, L.E. et al. (1989) J. Am. Chem. Soc., 111, 5488-5490; 1JC alpha H alpha: Vuister, G. W. et al. (1993) J. Biomol. NMR, 3, 67-80; 2JC'H alpha: Vuister, G.W. and Bax, A. (1992) J. Biomol. NMR, 2, 401-405; 1JC alpha N and 2JC alpha N: Delaglio, F. et al. (1991) J. Biomol. NMR, 1, 439-446] to create error surfaces for selected residues of the protein staphylococcal nuclease. The residues were chosen to include all those with five experimental couplings, as well as some with four experimental couplings, to demonstrate the relative importance of 3JHNH alpha and 1JC alpha H alpha. For most of the cases, we obtained good agreement between the X-ray structure [Loll, P.J. and Lattman, E.E. (1989) Protein Struct. Funct. Genet., 5, 183-201] and the NMR data.

Magnetic Resonance Spectroscopy↗
PubMed1993

Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.

The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.

Amino Acid Sequence↗
PubMed1993

A versatile perifusion system for the NMR spectroscopy of bovine retina. Assignment of resonances and effect of ischemia.

A perifusion system has been designed for metabolic nuclear magnetic resonance (NMR) spectroscopy studies of the bovine retina. This system, which allows the use of unmodified commercial probes for multinuclear studies, is shown here to maintain the perifused retina in a metabolic steady state at 25 degrees C, as deduced from its 31P and 1H NMR spectra. The 31P NMR ex vivo spectrum of the retina has been characterized, and its major resonances have been assigned in tissue extracts. We have found that excised retinal tissue subjected to 2 hr of ischemia is capable, upon perifusion with oxygenated medium, of recovering its resting spectral pattern. Thus, this system offers a versatile model for studies of physiological events related with ischemia in a neural tissue, without associated systemic effects.

Animals↗
PubMed1993

Overexpression and purification of avian ovomucoid third domains in Escherichia coli.

Genetic engineering studies of ovomucoid domains have been hindered by the lack of an efficient procedure for overproducing this protein. The novel scheme presented here has led to the isolation of chicken ovomucoid third domain (OMCHI3) at a level of 22 mg pure protein/l Escherichia coli culture medium. The gene coding for OMCHI3 was fused to the 3' end of the gene encoding staphylococcal nuclease (SNase). Expression of the chimeric gene was placed under control of the strong transcription and translation signals of the phage T7 promoter. Upon isopropyl-beta-D-galactopyranoside induction, the cells harboring the target plasmid efficiently overproduced the protein (30% of the total soluble protein). The 56-residue fragment corresponding to OMCHI3 was then liberated by cyanogen bromide (CNBr) cleavage at a genetically engineered methionine residue located at the nuclease--OMCHI3 junction (OMCHI3 lacks an internal methionine). SDS--PAGE, enzyme inhibition studies and NMR spectroscopy all indicated that the recombinant OMCHI3 has properties identical to those of OMCHI3 isolated from its natural source. The expression system was easily adapted for the production of [98% U 15N] OMCHI3. The expression vector was mutated for overexpression of turkey ovomucoid third domain (OMTKY3), which differs from OMCHI3 by three amino acid substitutions. Since many other avian ovomucoid domains also lack methionine residues, this approach should be suitable for large-scale production and isotope labeling of homologous proteinase inhibitors with a variety of inhibitory specificities.

Amino Acid Sequence↗
PubMed1992

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.

Animals↗
PubMed1991

Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the 2Fe-2S* cluster.

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1991

Refinement of the NMR solution structure of a protein to remove distortions arising from neglect of internal motion.

The effect of internal motion on the quality of a protein structure derived from nuclear magnetic resonance (NMR) cross relaxation has been investigated experimentally. Internal rotation of the tyrosine-31 ring of turkey ovomucoid third domain was found to mediate magnetization transfer; the effect led to underestimation of proton-proton distances in its immediate neighborhood. Experimental methods that distinguish pure cross relaxation from chemical exchange mediated cross relaxation were used to separate true distances from distorted ones. Uncorrected and corrected sets of distances, where the corrections took internal motion into account, each were used as input to a distance geometry program for structural modeling. Each set of distances yielded a family of similar (converged) structures. The two families of structures differed considerably (2 A) in the region of tyrosine-31. In addition, differences as large as 1 A were observed at other positions throughout the structure. These results emphasize the importance of analyzing the effects of internal motions in order to obtain more accurate NMR solution structures.

Amino Acid Sequence↗
PubMed1991

A relational database for sequence-specific protein NMR data.

A protein NMR database has been designed and is being implemented. The database is intended to contain solution NMR results from proteins and peptides (larger than 12 residues). A relational database format has been chosen that indexes data by: primary journal citation, molecular species, sequence-related and atom-specific assignments, and experimental conditions. At present, all data are entered from the primary refereed literature. Examples are given of sample queries to the database. Possible distribution formats are discussed.

Animals↗