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N D Clarke

Publications and source records attributed to N D Clarke.

28 records · Page 2Linked to original sources
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↗
PubMed1977

Regulation of newly evolved enzymes. III Evolution of the ebg repressor during selection for enhanced lactase activity.

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.

Biological Evolution↗