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Biomedical subjects

N D Clarke

Publications and source records attributed to N D Clarke.

At least 19 recordsLinked to original sources
PubMed1998

A hybrid sequence approach to the paracelsus challenge.

Inspired by the Paracelsus Challenge of Rose and Creamer (Proteins 19: 1-3, 1994), we have designed a protein sequence that is 50% identical to an all-helical protein but is intended to fold into a largely beta-sheet structure. Rather than attempt a de novo design, our strategy was to construct a hybrid sequence based on a helical "parent" protein (434 Cro) and a "target" protein with the desired fold (the B1 domain of protein G). The hybrid sequence (Crotein-G) is 50% identical to 434 Cro but is also 62% identical to the B1 domain of protein G. We also created a variant of Crotein-G (ZCrotein-G) that contains a potential His3Cys1 zinc binding site. At low protein concentrations and in the presence of 20% 2,2,2-trifluoroethanol (TFE) (v/v), the circular dichroism spectra of the designed proteins are distinct from that of 434 Cro and similar to that of the B1 domain of protein G. However, the proteins fail to denature in a cooperative manner. Furthermore, aggregation occurs at moderate protein concentrations or in the absence of TFE. Addition of zinc to ZCrotein-G does not promote structure formation. In summary, 434 Cro has been altered to something that may resemble the B1 domain of protein G, but the protein does not adopt a native structure.

Amino Acid Sequence↗
PubMed1997

Specificity of DNA repair methyltransferases determined by competitive inactivation with oligonucleotide substrates: evidence that Escherichia coli Ada repairs O6-methylguanine and O4-methylthymine with similar efficiency.

DNA repair methyltransferases (MTases) are stoichiometric acceptor molecules that are irreversibly inactivated in the course of removing a methyl group from O6-methylguanine (meG)-DNA or O4-methylthymine (meT)-DNA. A new assay has been developed to determine the relative efficiency of repair of meG and meT. The assay is based on the deprotection of methylated restriction sites in synthetic oligonucleotides and can be used to measure meG repair or meT repair directly. More importantly, relative repair efficiencies can be measured in competition experiments, using each of the methylated oligomers in turn as an inhibitor of repair for the other. Relative repair rates are determined by numerical solution of the coupled rate equations that describe this competition to the experimental data. We find that the human MTase repairs meT about 35-fold less well than meG, qualitatively similar to earlier studies. Contrary to previous reports, however, we find that Escherichia coli Ada repairs meG and meT with nearly equal efficiency. This finding, in conjunction with other recent reports, may indicate that low meT repair is a relatively unusual characteristic of the human homolog.

Bacterial Proteins↗
PubMed1997

Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.

DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.

Amino Acid Sequence↗
PubMed1997

Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase: renaturation and rapid purification of the protein.

A synthetic gene was constructed that encodes human DNA methylguanine methyltransferase (hMGMT). The synthetic gene was designed with a number of unique restriction sites to facilitate cassette mutagenesis and to reflect the preferences found among genes in Escherichia coli. Both the full-length gene and a gene for a functional variant (hMGMT delta C) that lacks the C-terminal 28 codons were constructed, and the genes were overexpressed using a T7 RNA polymerase promoter. The proteins are made in the form of insoluble aggregates but the truncated form of the protein (hMGMT delta C) has been successfully denatured, renatured, and purified to near homogeneity by ion exchange. Methyltransferase activity assays of hMGMT delta C demonstrate that the reconstituted protein has substantial DNA repair activity, though somewhat less than full-length hMGMT that had been expressed and purified in a soluble form. Mass spectrometry of a mixture of proteolytic fragments confirmed the protein sequence and indicated no detectable oxidation of the active site cysteine. The protein was determined to be monomeric by gel filtration chromatography, and circular dichroism spectra for renatured hMGMT delta C and fully soluble hMGMT are consistent with the renatured protein preparation being fully folded. Refolded hMGMT delta C had a curious propensity to form large aggregates in a time-dependent manner when injected into a dynamic light scattering instrument; this aggregation behavior was not observed for hMGMT purified in a soluble form. Differences in susceptibility to aggregation may account for differences in methyltransfer activity. Yields of purified protein were approximately 5 mg/liter of culture.

Amino Acid Sequence↗
PubMed1996

Specific recognition of A/G and A/7,8-dihydro-8-oxoguanine (8-oxoG) mismatches by Escherichia coli MutY: removal of the C-terminal domain preferentially affects A/8-oxoG recognition.

Escherichia coli MutY is a 39 kDa adenine DNA glycosylase and 3' apurinic/apyrimidinic (AP) lyase that is active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. 8-oxoG (7,8-dihydro-8-oxoguanine or GO) is a major stable product of oxidative damage, and A/GO mismatches may be particularly important biological substrates for MutY. Proteolytic digestion of MutY using thermolysin was found to produce two relatively stable fragments of 25 and 12 kDa. The 25 kDa fragment begins at the N terminus of MutY and spans the region homologous with E. coli endonuclease III, a DNA glycosylase/AP lyase that repairs oxidatively damaged pyrimidines. The 12 kDa fragment, which consists of much of the rest of MutY, had no detectable activity. The purified 25 kDa fragment (M25) had nearly wild-type binding and cleavage activities with A/G-mismatched substrates. Binding to A/GO-mismatched DNA, however, was dramatically reduced in M25 compared to that in intact protein. Borohydride-dependent enzyme-DNA cross-linking, which is a hallmark of the reaction of several DNA glycosylases that possess concomitant AP lyase activity, was also substantially reduced when M25 was allowed to react with A/GO-mismatched DNA. The significant differences in M25 recognition and reactivity with A/G and A/GO mismatches suggest that the C-terminal region of MutY, a region with no homologous counterpart in E. coli endonuclease III, plays an important role in the repair of mismatched DNA arising from oxidation damage.

Adenine↗
PubMed1996

Innervation of the chick cornea analyzed in vitro.

PURPOSE: During the early stages (embryonic day 3 [E3]) of avian corneal development, nerve fibers extend from the trigeminal ganglion to the corneal limbus. On E11, these nerve fibers enter the cornea and extend through the secondary stroma to begin innervation of the epithelium on E13. This process of innervation is concomitant with the cornea's dehydration and transition from opacity to transparency; thus, suggesting a link between innervation and the attainment of corneal function. This investigation attempts to ascertain whether the developing cornea can support its innervation in vitro and whether there is a possible developmental interrelationship between corneal innervation and dehydration, with the associated transition from opacity to transparency. METHODS: Isolated corneas from either E8 or E14 chicks were co-cultured with E8 dorsal root ganglia. After 4 days of culture, innervation was visualized by silver staining and immunohistochemistry. Changes in corneal composition and organization associated with this innervation in vitro were analyzed by measuring changes in specific hydration, thickness and compaction, and incorporation of [35S]sulfate into glycosaminoglycans during co-culture. RESULTS: The E8 and E14 corneas support extensive innervation in vitro. Developing nerve fibers extend through the secondary stroma to innervate the epithelium. In vitro innervation of E8, but not E14, corneas was associated with a decrease in corneal specific hydration, whereas control corneas (without dorsal root ganglia) failed to show any such changes. E8 corneas also showed a significant increase in compaction when innervated in vitro. Corneal innervation in vitro did not significantly change the overall incorporation of [35S]sulfate into glycosaminoglycans. Furthermore, incorporation of [35S]sulfate into corneal sulfated glycosaminoglycans (sGAG) is not influenced by either the number of nerve fibers innervating the cornea or nerve growth factor (NGF). In addition, the distribution of staining of the corneal glycosaminoglycans, chondroitin sulfate and keratan sulfate, and peanut agglutinin-binding epitopes, suggests that these molecules are not associated with inhibition of axonal development. CONCLUSIONS: The in vitro system described here is a useful model to understand the process of corneal development. Co-culture has shown that corneal innervation promotes the process of dehydration, which is dependent on the age of the cornea. However, other functionally related refinements necessary for transparency-notably proteoglycan synthesis-may not be linked to innervation or NGF production. The authors conclude that the development of transparency is dependent on corneal innervation, though not exclusively, and that other controlling factors also are required.

Animals↗
PubMed1995

Covariation of residues in the homeodomain sequence family.

Homeodomains are 60 amino acid DNA binding domains found in numerous eukaryotic transcription factors. The homeodomain family is a useful system for studying sequence-structure relationships because several hundred sequences are known and the structures of several homeodomains have been determined. Covariation of amino acid residues in the homeodomain family has been investigated to see whether strongly covariant residue pairs can be understood in terms of the structure and function of these domains. Among 16 strongly covariant pairs examined, 2 are explained by the ability to form salt bridges, and 9 appear related to the DNA binding function of the proteins. For the remaining 5 pairs, the rationale for covariance remains unclear and the likelihood of artifactual correlations is discussed in the context of experimental and evolutionary biases in the selection of sequences. No significant correlation was found between covariance and structural proximity in the hydrophobic core.

Binding Sites↗
PubMed1995

Metal search: a computer program that helps design tetrahedral metal-binding sites.

We describe a computer program (Metal Search) that helps design tetrahedrally coordinated metal binding sites in proteins of known structure. The program takes as input the backbone coordinates of a protein and outputs lists of four residues that might form tetrahedral sites if wild-type amino acids were replaced by cysteine or histidine. The program also outputs the side chain dihedral angles of the amino acids and the coordinates of the predicted metal ion. The only function evaluated by Metal Search is the ability of side chains to meet simple geometric criteria for formation of a tetrahedral site, but these criteria are sufficient to produce a manageably small list that can then be evaluated by other means. The program has been used in the introduction of zinc binding sites in the designed four-helix bundle protein alpha 4 and in the B1 domain of streptococcal protein G, and in both cases the tetrahedral coordination of a bound metal ion has been confirmed (Klemba, M., Gardner, K. H., Marino, S., Clarke, N.D., and Regan, L., Nature: Structural Biology 2:368-373, 1995).

Amino Acids↗
PubMed1995

Sequence 'minimization': exploring the sequence landscape with simplified sequences.

The challenges of protein engineering arise, in part, from the enormous number of possible sequences and the almost unimaginably small fraction of such sequences that can be studied experimentally or computationally. Fortunately, not all possibilities need to be considered because many different sequences can adopt the same structure. Of the vast number of sequences that fold into a given conformation, some are 'simpler' than the sequences of typical proteins. Studying protein sequences that are simpler helps focus attention on the principal determinants of structure. Recent examples of this strategy are the simplification of protein surfaces and cores, the use of a binary 'code' for protein design and the structural analysis of random simple sequences.

Alanine↗
PubMed1995

Novel metal-binding proteins by design.

We describe the successful design of a tetrahedral His3Cys Zn(II)-binding site in a small protein of known structure: the B1 domain of Streptococcal protein G. The B1 variants containing the novel metal-binding site were characterized using a combination of optical absorption, circular dichroism and NMR spectroscopies. The results indicate that the designed proteins bind Zn(II) with high affinity and tetrahedral coordination geometry, and that the overall secondary and tertiary structure of the B1 domain is maintained.

Amino Acid Sequence↗
PubMed1994

A proposed mechanism for the self-splicing of proteins.

Intervening protein sequences, called inteins, are intronlike elements that are removed posttranslationally, apparently by self-splicing. The conserved and essential residues of precursor proteins consist of an asparagine as the last residue of the intein and a hydroxyl- or thiol-containing residue immediately following both splice junctions. Evidence for a branched intermediate has been reported [Xu, M.-Q., Southworth, M., Mersha, F., Hornstra, L. & Perler, F. (1993) Cell 75, 1371-1377]; however, the chemical nature of the branched structure is unclear. I propose a mechanism that includes the formation of a branched structure, provides an explanation for the reversal of branch formation observed at high pH, and accounts for each of the essential amino acids. The branched structure is formed by nucleophilic attack of the asparagine side chain on the N-terminal splice junction. The nature of this branched structure is a distinguishing feature of the model and can be experimentally tested.

Asparagine↗
PubMed1994

Structural studies of the engrailed homeodomain.

The structure of the Drosophila engrailed homeodomain has been solved by molecular replacement and refined to an R-factor of 19.7% at a resolution of 2.1 A. This structure offers a high-resolution view of an important family of DNA-binding proteins and allows comparison to the structure of the same protein bound to DNA. The most significant difference between the current structure and that of the 2.8-A engrailed-DNA complex is the close packing of an extended strand against the rest of the protein in the unbound protein. Structural features of the protein not previously noted include a "herringbone" packing of 4 aromatic residues in the core of the protein and an extensive network of salt bridges that covers much of the helix 1-helix 2 surface. Other features that may play a role in stabilizing the native state include the interaction of buried carbonyl oxygen atoms with the edge of Phe 49 and a bias toward statistically preferred side-chain dihedral angles. There is substantial disorder at both ends of the 61 amino acid protein. A 51-amino acid variant of engrailed (residues 6-56) was synthesized and shown by CD and thermal denaturation studies to be structurally and thermodynamically similar to the full-length domain.

Amino Acid Sequence↗
PubMed1991

The DNA binding arm of lambda repressor: critical contacts from a flexible region.

Segments of protein that do not adopt a well-ordered conformation in the absence of DNA can still contribute to site-specific recognition of DNA. The first six residues (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6-) of phage lambda repressor are flexible but are important for site-specific binding. Low-temperature x-ray crystallography and codondirected saturation mutagenesis were used to study the role of this segment. All of the functional sequences have the form [X]1-[X]2-[Lys or Arg]3-[Lys]4-[Lys or Arg]5-[X]6. A high-resolution (1.8 angstrom) crystal structure shows that Lys3 and Lys4 each make multiple hydrogen bonds with guanines and that Lys5 interacts with the phosphate backbone. The symmetry of the complex breaks down near the center of the site, and these results suggest a revision in the traditional alignment of the six lambda operator sites.

Amino Acid Sequence↗
PubMed1990

A tetrahedral zinc(II)-binding site introduced into a designed protein.

The ultimate goal of protein engineering is to create novel proteins which will adopt predetermined structures, bind specified ligands, and catalyze new reactions. Here we describe the successful introduction of metal-binding activity into a model four helix bundle protein. The designed binding site is tetrahedral and is formed by two Cys and two His ligands on adjacent helices. We have introduced this site into the protein and characterized the binding activity. Using 65Zn(II), we have shown that the protein binds Zn(II), that the sulfhydryls are essential for binding, and that binding occurs to the protein monomer. The designed protein binds metals with high affinity: we estimate the dissociation constants as 2.5 X 10(-8) M for Zn(II) and 1.6 X 10(-5) M for Co(II). The characteristic absorption spectrum of the Co(II)-substituted protein fully supports the model of a tetrahedral binding site comprised of two Cys and two His ligands. Circular dichroism studies indicate that no significant changes in secondary structure occur between the metal-bound and metal-free forms of the protein. However, the metal-bound form is substantially stabilized toward denaturation by GuHCl compared to the metal-free form.

Amino Acid Sequence↗
PubMed1990

Identification of protein folds: matching hydrophobicity patterns of sequence sets with solvent accessibility patterns of known structures.

Hydrophobic side chains often are buried in the interior of a protein, and evolutionarily related proteins usually maintain the hydrophobic character of buried positions. In this paper we show that a pattern of hydrophobicity values derived from a set of related protein sequences is well correlated with the linear pattern of side-chain solvent accessibility values, calculated from a known protein structure representative of the sequences. In several cases, information from aligned sequences can be used to select the correct tertiary fold from a large data base of protein structures.

Algorithms↗
PubMed1988

Evidence from cassette mutagenesis for a structure-function motif in a protein of unknown structure.

The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes. Evidence is presented that an enzyme of unknown structure [Ile-transfer RNA (tRNA) synthetase] may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase). This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis. Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine. Few amino acid substitutions appear to be tolerated in this region. However, one substitution (independently isolated twice) increased the Michaelis constant Km for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the Km for adenosine triphosphate, the apparent Km for tRNA, or the rate constant kcat.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II.

We have constructed two recombinant plasmids which harbour functions involved in DNA repair of alkylation damage in Escherichia coli. One plasmid carries the tag+ gene encoding 3-methyladenine DNA glycosylase I while the other carries alkA+ encoding 3-methyladenine DNA glycosylase II. The plasmids were isolated from plasmid stocks carrying total cellular DNA by selection for their ability to complement the methylmethanesulphonate(MMS)-sensitive phenotype of an E. coli mutant (tag ada) deficient in both 3-methyladenine DNA glycosylases I and II. Both plasmids increase the plating efficiency of such a mutant on methylmethanesulphonate plates by a factor of more than 10(5). The tag gene is located on a 6 (kbp) HindIII fragment, and the presence of the tag plasmid in the cells results in 15-fold overproduction of 3-methyladenine DNA glycosylase I. The other plasmid restores 3-methyladenine DNA glycosylase II deficiency in alkA mutant cells, and results in 3-fold overproduction of this enzyme after alkylation induction. The induction is ada+-dependent and we conclude that this plasmid contains the structural gene for 3-methyladenine DNA glycosylase II, including its control region responding to alkylation induction. However, the plasmid does not complement fully the MMS-sensitive phenotype of alkA mutants which suggests that the plasmid may not include the entire alkA operon.

Cloning, Molecular↗