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D S Auld

Publications and source records attributed to D S Auld.

At least 37 records · Page 2Linked to original sources
PubMed1995

Switching recognition of two tRNA synthetases with an amino acid swap in a designed peptide.

The genetic code is based on specific interactions between transfer RNA (tRNA) synthetases and their cognate tRNAs. The anticodons for methionine and isoleucine tRNAs differ by a single nucleotide, and changing this nucleotide in an isoleucine tRNA is sufficient to change aminoacylation specificity to methionine. Results of combinatorial mutagenesis of an anticodon-binding-helix loop peptide were used to design a hybrid sequence composed of amino acid residues from methionyl- and isoleucyl-tRNA synthetases. When the hybrid sequence was transplanted into isoleucyl-tRNA synthetase, active enzyme was generated in vivo and in vitro. The transplanted peptide did not confer function to methionyl-tRNA synthetase, but the substitution of a single amino acid within the transplanted peptide conferred methionylation and prevented isoleucylation. Thus, the swap of a single amino acid in the transplanted peptide switches specificity between anticodons that differ by one nucleotide.

Amino Acid Sequence↗
PubMed1995

Protein thermal denaturation, side-chain models, and evolution: amino acid substitutions at a conserved helix-helix interface.

Random mutant libraries with substitutions at the interface between the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c were screened. All residue combinations that have been identified in naturally occurring cytochrome c sequences are found in the libraries. Mutants with these combinations are biologically functional. Enthalpies, heat capacities, and midpoint temperatures of denaturation are used to determine the entropy and Gibbs free energy of denaturation (delta GD) for the ferri form of the wild-type protein and 13 interface variants. Changes in delta GD cannot be allocated solely to enthalpic or entropic effects, but there is no evidence of enthalpy-entropy compensation. The lack of additivity of delta GD values for single versus multiple amino acid substitutions indicates that the helices interact thermodynamically. Changes in delta GD are not in accord with helix propensities, indicating that interactions between the helices and the rest of the protein outweigh helix propensity. Comparison of delta GD values for the interface variants and nearly 90 non-cytochrome c variants to side-chain model data leads to several conclusions. First, hydrocarbon side chains react to burial-like transfer from water to cyclohexane, but even weakly polar side chains respond differently. Second, despite octanol being a poor model for protein interiors, octanol-to-water transfer free energies are useful stability predictors for changing large hydrocarbon side chains to smaller ones. Third, unlike cyclohexane and octanol, the Dayhoff mutation matrix predicts stability changes for a variety of substitutions, even at interacting sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Zinc metallochemistry in biochemistry.

The chemically stable but stereochemically flexible, non-toxic nature of zinc combined with its amphoteric properties has permitted it to orchestrate a number of zinc-binding motifs critical to life processes. For zinc enzymes, catalytic, cocatalytic, and structural zinc sites exist. DNA-binding proteins have zinc fingers, twists, and clusters exist.

Amino Acid Sequence↗
PubMed1995

Matrilysin: expression, purification, and characterization.

The expression vector pGEX-2T under the control of the IPTG-inducible tac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin, HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.

Base Sequence↗
PubMed1994

Zinc content of promatrilysin, matrilysin and the stromelysin catalytic domain.

Promatrilysin expressed in Escherichia coli and Chinese hamster ovary cells contains 2.36 +/- 0.19 and 2.13 +/- 0.39 moles of zinc per mole of protein, respectively, while the activated enzyme contains 2.22 +/- 0.21. The catalytic domain of stromelysin-1 expressed in E. coli contains 2.22 +/- 0.11. Thus these matrix metalloproteinases contain two metal binding sites at which zinc is bound firmly and possibly a third site at which it is bound weakly. Promatrilysin and matrilysin do not contain significant amounts of Fe, Cu, Mn, or Ni. All known matrix metalloproteinases have a sequence homologous to the zinc binding site of astacin, HExxHxxGxxH, suggesting that one of the zinc sites is catalytic in agreement with the known inhibition of these enzymes by chelators.

Amino Acid Sequence↗
PubMed1993

XAFS studies of carboxypeptidase A: detection of a structural alteration in the zinc coordination sphere coupled to the catalytically important alkaline pKa.

X-ray absorption fine structure (XAFS) spectra of carboxypeptidase A (ZnCPD) show progressive spectral changes particularly in the near edge region when the pH is changed from neutral to alkaline values. Both least square fitting and radial distribution function (RDF) data analysis yield two distributions of atoms in the first coordination shell of ZnCPD at all pH values and at both 150 and 297 K. Direct comparison of the first and higher coordination shells of ZnCPD reveals structural differences between pH 7.0 and pH 9.9. At pH 7.0, the zinc ion has four ligand atoms (N or O) at an average distance of 2.024 +/- 0.006 A, and a smaller distribution of 1.3 atoms (N or O) at 2.54 +/- 0.05 A from the zinc ion (from the least square fitting analysis). At pH 9.9, the larger distribution contains four atoms at a 0.022 A shorter distance (2.002 A) from the zinc, while the smaller distribution contains 0.7 atoms at 2.52 +/- 0.06 A. The smaller distribution can be attributed mainly to the contribution of the epsilon 2-oxygen of Glu 72 and to the atoms farther away for which the contribution cannot be fully separated from the first shell peak. The structural changes of ZnCPD at intermediate pHs are consistent with the changes observed at pH 7.0 and pH 9.9. The XAFS Debye-Waller factor shows an increased structural disorder for the four atom distribution at the alkaline pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Changes in global stability and local structure of cytochrome c upon substituting phenylalanine-82 with tyrosine.

We have examined the F82Y;C102T variant of Saccharomyces cerevisiae iso-1-cytochrome c using high-resolution proton nuclear magnetic resonance spectroscopy, chemical denaturation, and differential scanning calorimetry. Comparison of proton chemical shifts, paramagnetic shifts, and nuclear Overhauser effects indicates structural changes are localized to the vicinity of position 82. One alteration involves the rearrangement of the side chain of leucine-85. Using many more proton assignments than were available in the initial report [G. J. Pielak, R. A. Atkinson, J. Boyd, and R. J. P. Williams, Eur. J. Biochem. 177, 179-185 (1988)], a second alteration involving an interaction between arginine-13 and tyrosine-82 is observed. The interaction appears to involve a hydrogen bond with the eta-protons of arginine's guanido group acting as donor and tyrosine's phenolic eta-oxygen as acceptor. In spite of this potentially-stabilizing interaction, the free energy of denaturation decreases by approximately 2.4 kcal mol-1. Results are discussed with respect to alterations in the native and denatured states.

Cytochrome c Group↗
PubMed1993

Cocatalytic zinc motifs in enzyme catalysis.

Cocatalytic zinc binding sites are characteristic of enzyme molecules which contain two or more zinc and/or other metal atoms. In each site an aspartate, glutamate, or histidine residue simultaneously binds to two zinc atoms or a zinc and a different metal atom. In the resultant amino acid bridge, two of the cocatalytic metal atoms bind to the same amino acid. Consequently the participating metal atoms are in close proximity and function as a catalytic unit, typical of this motif. In these functional units aspartate seems to be preferred over glutamate. Serine, threonine, tryptophan, and lysine residues are encountered as zinc ligands, although they have not so far been identified as ligands in monozinc enzymes or DNA-binding zinc proteins. The resultant coordination spheres and their mechanistic implications raise interesting questions for further study.

Alkaline Phosphatase↗
PubMed1993

Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c.

Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in omega-loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, delta Gd, at 298 K to the free energy change for local unfolding, delta Gop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in delta Gop. Comparisons of delta Gd to delta Gop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high-energy conformations of the reduced form appears to involve the axially coordinated heme.

Amides↗
PubMed1993

Probing weakly polar interactions in cytochrome c.

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur-containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c genes at positions that form a weakly polar aromatic-aromatic interaction, the wild-type protein. To determine if a weakly polar sulfur-aromatic interaction replaced the aromatic-aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cysteine analog, S-methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur-containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur-containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2-3 kcal mol-1 less stable than iso-1-cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 +/- 0.3 kcal mol-1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur-containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.

Cysteine↗
PubMed1993

Low-temperature stopped-flow rapid-scanning spectroscopy: performance tests and use of aqueous salt cryosolvents.

An ultimate goal of enzymology is to determine the structure of intermediates in catalysis in solution. Metalloenzymes provide a unique opportunity in this regard because the metal atom is critical to catalysis. In the case of zinc enzymes, replacement of the zinc by the chromophoric cobalt atom can result in the identification of intermediates in catalysis. This is most easily accomplished by examining the enzyme-catalyzed reaction under rapid-mixing and rapid-scanning subzero conditions. Aqueous salt solutions are particularly useful as cryosolvents whenever hydrophobic forces are important for substrate binding. Rapid-freeze or chemical quenching of these metallointermediates can allow further structural characterization by such approaches as EPR spectroscopy and X-ray absorption fine structure. Such a combination of approaches can bridge the information gained by examining only the structure of the enzyme in the crystalline state by X-ray diffraction and that obtained by examining only steady-state enzymatic activities.

Amino Acid Sequence↗
PubMed1992

pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

X-ray absorption fine structure study of the active site of zinc and cobalt carboxypeptidase A in their solution and crystalline forms.

A comparative study on the metal environment of Zn(II)-carboxypeptidase A (ZnCPD) and Co(II)-carboxypeptidase A (CoCPD) in their solution and crystalline forms using the X-ray absorption fine structure (XAFS) technique has been conducted. The first coordination sphere of Zn for ZnCPD in its solution state is found to consist of two distributions of atoms, with four atoms (N or O) located at an average distance of 2.03 +/- 0.01 A and one atom (N or O) located at 2.57 +/- 0.04 A. The four-atom distribution remains the same for ZnCPD in its crystalline state, but the fifth atom is found at 2.36 +/- 0.04 A. Examination of the higher coordination shell, between 2.7 and 4.2 A, reveals the presence of two imidazoles. Combined with X-ray crystallographic results, a structural model is proposed. The four atoms at an average distance of 2.03 A are assigned to the two delta 1 nitrogens of His-69 and His-196, one epsilon 1 oxygen of Glu-72, and the oxygen of a coordinated water molecule. The atom at 2.57 A for ZnCPD in solution is assigned to the epsilon 2 oxygen of Glu-72. The results for CoCPD in solution are similar with the four atoms at an average distance of 2.08 +/- 0.01 A and one atom at 2.50 +/- 0.04 A, which moves to 2.34 +/- 0.04 A in the crystalline enzyme. The intensity of the 3d "pip" peak for CoCPD is consistent with a distorted tetragonal metal geometry for the solution form of the enzyme which is converted to a more pentacoordinated metal site for the crystalline enzyme. The first shell distribution of crystalline CoCPD is quite disordered, which may be largely due to the disorder of His-69 and His-196 as indicated by higher shell analysis. Thus, the XAFS studies show that the metal coordination spheres in the zinc and cobalt enzymes are quite similar in the solution state but differ from their crystalline counterparts. The XAFS studies provide the necessary background for measurement of substrate- and inhibitor-promoted structural changes in the metal coordination sphere of the zinc and other metal-substituted carboxypeptidases in the solution state.

Animals↗
PubMed1992

Functional zinc-binding motifs in enzymes and DNA-binding proteins.

Zinc is now known to be an integral component of a large number and variety of enzymes and proteins involved in virtually all aspects of metabolism, thus accounting for the fact that this element is essential for growth and development. The chemistry of zinc, superficially bland, in reality has turned out to be ideally appropriate and versatile for the unexpected development of multiple and unique chemical structures which biology has used for specific life processes. The present discussion will centre on those distinctive zinc-binding motifs that are critical both to enzyme function and the expression of the genetic message. X-Ray diffraction structure determination of 15 zinc enzymes belonging to IUB classes I-IV provide absolute standards of reference for the identity and nature of zinc ligands in their families. Three types of zinc enzyme binding motifs emerge through analysis of these: catalytic, coactive or cocatalytic, and structural. In contrast to zinc enzymes virtually all DNA-binding proteins contain multiple zinc atoms. With the availability of NMR and X-ray structure analyses three distinct motifs now emerge for those: zinc fingers, twists and clusters.

Amino Acid Sequence↗
PubMed1992

Active zinc binding sites of zinc metalloenzymes.

The participation of zinc in widely diversified biological reactions focuses attention on its chemistry. A number of its properties relate to its biological utilization and versatility. Its amphoteric properties allow the zinc-coordinated water to exist as a "hydronium" or hydroxide ion even at neutrality. Its coordination sphere is flexible and adapts to a wide variety of ligands, allowing for a multiplicity of types and numbers of coordination complex geometries. Its stable d shell signifies that it is neither oxidized nor reduced; yet it participates in enzymatic oxidoreduction reactions in coordination with an organic cofactor. X-ray crystallographic analyses of twelve zinc enzymes now show that catalytic zinc is bound by three protein ligands, whereas structural zinc atoms are fully coordinated by four ligands. Water is always a ligand to the catalytic zinc while the protein ligands occur in an order of frequency of His >> Glu > Asp = Cys. The zinc-bound water is the critical component of the active site; it is activated for enzymatic catalysis by the identity and arrangement of the ligands coordinated to zinc. Thus, ultimately, it is this water molecule which, upon entering the zinc coordination sphere, is activated either by ionization, polarization or displacement. As a result of the properties of this metal, zinc metalloenzymes and zinc proteins participate in a wide variety of metabolic processes including carbohydrate, lipid, protein and nucleic acid synthesis, regulation and degradation.

Amino Acid Sequence↗
PubMed1991

Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.

The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.

Amino Acid Sequence↗