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M A Hefford

Publications and source records attributed to M A Hefford.

At least 19 recordsLinked to original sources
PubMed1998

Introduction of potential helix-capping residues into an engineered helical protein.

MB-1 is an engineered protein that was designed to incorporate high percentages of four amino acid residues and to fold into a four-alpha-helix bundle motif. Mutations were made in the putative loop I and III regions of this protein with the aim of increasing the stability of the helix ends. Four variants, MB-3, MB-5, MB-11 and MB-13, have replacements intended to promote formation of an 'N-capping box'. The loop I and III sequences of MB-3 (both GDLST) and MB-11 (GGDST) were designed to cause alphaL C-terminal 'capping' motifs to form in helices I and III. MB-5 has a sequence, GPDST, that places proline in a favourable position for forming beta-turns, whereas MB-13 (GLDST) has the potential to form Schellman C-capping motifs. Size-exclusion chromatography suggested that MB-1, MB-3, MB-5, MB-11 and MB-13 all form dimers, or possibly trimers. Free energies for the unfolding of each of these variants were determined by urea denaturation, with the loss of secondary structure followed by CD spectroscopy. Assuming an equilibrium between folded dimer and unfolded monomer, MB-13 had the highest apparent stability (40.5 kJ/mol, with +/-2.5 kJ/mol 95% confidence limits), followed by MB-11 (39.3+/-5.9 kJ/mol), MB-3 (36.4+/-1.7 kJ/mol), MB-5 (34.7+/-2.1 kJ/mol) and MB-1 (29.3+/-1.3 kJ/mol); the same relative stabilities of the variants were found when a folded trimer to unfolded monomer model was used to calculate stabilities. All of the variants were relatively unstable for dimeric proteins, but were significantly more stable than MB-1. These findings suggest that it might be possible to increase the stability of a protein for which the three-dimensional structure is unknown by placing amino acid residues in positions that have the potential to form helix- and turn-stabilizing motifs.

Amino Acid Sequence↗
PubMed1997

Prediction of folding stability and degradability of the de novo designed protein MB-1 in cow rumen.

The authors have recently reported on the design of a protein (MB-1) enriched in methionine, threonine, lysine, and leucine. The protein is intended to be produced by rumen bacteria, in a way that would provide high producing lactating cows with limiting amino acids. In this report, MB-1 stability in the rumen is assessed, i.e., where the protein might be found after cell lysis or after being secreted by rumen bacteria. Current in vitro methods used to predict proteolytic degradability in the rumen were used for MB-1, as well as other natural proteins for comparison. MB-1 was found to be more susceptible to degradation than cytochrome c and ribonuclease A. Data indicate that MB-1 will be rapidly degraded if exposed to the rumen environment without protection. The contribution of folding stability to proteolytic stability was also examined. Rumen liquor components were selected to formulate a solution compatible with constraints of thermal denaturation studies. Denaturation curves show that the natural proteins were folded at rumen temperature. The MB-1 denaturation curves indicated that MB-1 does not unfold in a cooperative transition when heated from 20 to 70 degrees C. This suggests that MB-1 structure may be progressively modified as temperature increases, and that a continuum of conformations are available to MB-1. At 39 degrees C, a significant (50%) portion of MB-1 molecules had their tertiary structure unfolded, contributing to proteolytic degradability. Despite the unusual constraints used in MB-1 design (i.e., a maximized content in selected essential amino acids), results show that MB-1 has structural properties similar to previously reported de novo designed proteins.

Amino Acids, Essential↗
PubMed1997

A consensus residue analysis of loop and helix-capping residues in four-alpha-helical-bundle proteins.

A statistical study was performed on a set of proteins which adopt the four-alpha-helical-bundle tertiary motif in order to determine amino acid occurrences at helix-capping and loop positions. Eight X-ray crystal structures from the Brookhaven Protein Data Bank (PDB) were examined and N", N', Ncap, Ccap, C' and C" residues were assigned. In addition, a set of 55 protein sequences for the analogous proteins from different strains and species was taken from the Protein Information Resource and Swiss-Prot databanks. The residues at the capping and loop positions in this expanded data set were deduced by aligning these sequences with those from the PDB files. Similar trends were observed in the two data sets. In general, polar residues were predominant in the loops, although aromatic residues were also fairly common. Glycine, a highly flexible residue with an excellent 'helix-breaking' ability, was very common at the Ccap, C' and C" residues. Proline, which can force sharp turns in the direction of a peptide backbone, was only common at the N" residue. Residues which can participate in the N-capping box motif were found with high frequency. Capping motifs at the helix C-termini (Schellman and alphaL motifs) were also somewhat common, while another helix N-terminal stabilizing motif, the hydrophobic stable, was not common. The data presented in this study should prove useful for applying the 'consensus residue' approach to the de novo design of loop regions in helical bundle proteins.

Animals↗
PubMed1997

Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios.

As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens. We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B. fibrisolvens Bu49. While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct. Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance. The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis. The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype. Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism. Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented. pOM1 has been used in the construction of a new Escherichia coli-B. fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B. fibrisolvens harboring the pRJF1 plasmid.

Amino Acid Sequence↗
PubMed1995

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: comparison with other Butyrivibrio plasmids and application in the development of cloning vector.

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.

Amino Acid Sequence↗
PubMed1995

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156.

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM beta 1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 micrograms erythromycin/ml. Maximum efficiency was 1.1 x 10(5) transformants per micrograms plasmid DNA (average 3 x 10(4)), and restriction mechanisms reduced efficiency by a factor of 2 x 10(2). Nonselective growth for 200 generations gave no measurable loss of plasmid.

Escherichia coli↗
PubMed1995

Design, expression, and initial characterization of MB1, a de novo protein enriched in essential amino acids.

Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, lysine and leucine. Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure. We therefore attempted to create a stable de novo protein, highly enriched in these essential amino acids, that would adopt the alpha-helical bundle fold. The design process was an iterative one. The consensus residues (based on the properties profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-termini. Using these data, the helices in our de novo protein were designed by inserting the residues M, T, K and L as often as possible at positions where their volume, hydrophobicity and charge match the consensus found in natural bundle helices. Short sequences of strong turn formers were used to join the helices and adjust the predicted p1 to 7.7, while a number of local and global factors were used to refine our design. Further, the sequence was checked to eliminate various known protease targets in E. coli. The sequence of our de novo protein, MB1, is: MAT-EDMTDMMTTLFKTMQLLTK-SEPTA-MDEATKTATTMKNHLQNLMQK-TKNKE DMTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA+ ++ , where dashes separate long helices from short, turn forming linkers. A gene coding for this protein was assembled from synthetic oligonucleotides, then fused to the maltose binding protein gene under the control of a tac promoter. The fusion protein was expressed in E. coli, purified and cleaved to yield maltose binding protein and our de novo protein, MB1. MB1 was found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K and L.

ATP-Binding Cassette Transporters↗
PubMed1993

The complete nucleotide sequence of a small cryptic plasmid from a rumen bacterium of the genus Butyrivibrio.

The complete nucleotide sequence of a plasmid, designated pRJF1, isolated from a rumen bacterium of the genus Butyrivibrio, has been determined. pRJF1 is a small plasmid (2631 bp) which shows the high AT content (64%) typical of plasmids from gram-positive organisms. Computer analysis of sequence data revealed two major open reading frames encoded on the same strand but in different frames. The smaller, ORF1 (435 bp), is preceded by Shine Dalgarno (SD) and Escherichia coli-10, -35 sequences and is predicted to encode an acidic polypeptide of 145 aa. The putative protein shows little homology to known bacterial proteins except in a small region similar to part of the Pre protein (plasmid recombination enzyme) of Bacillus plasmid pTB913. Pre proteins of the pTB913 family, however, show conservation of several amino acids in the amino terminal region of the protein that may be related to function. This pattern of amino acid conservation is not observed in ORF1. The predicted product of ORF2 (849 bp) is a basic protein of 283 as that shows significant similarity to repA gene products of plasmids from gram-negative (but not gram-positive) bacteria. No typical Shine Dalgarno sequence or -10, -35 sequences precede this coding region. While sequence homologous to nick sites of plasmids from gram-positive organisms that replicate via ssDNA intermediates were also detected in pRJF1, the order of the three consecutive inverted repeats typical of nick sites is not conserved. pRJF1, however, does contain an extended region of directly and inversely repeated sequence motifs reminiscent of theta replication origins.

Amino Acid Sequence↗
PubMed1992

Bipartite organization of the Bacillus subtilis endo-beta-1,4-glucanase revealed by C-terminal mutations.

The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-beta-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host. The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate. CD spectra indicated that the bulk of the alpha-helical secondary structure in EG470 was contained within EG300. However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and approximately 4- to 5-fold higher with carboxymethylcellulose (soluble cellulose). These results along with data which show that EG470 binding capacity to microcrystalline cellulose is approximately 11 times more than that of EG300, demonstrate the importance of residues 330-499 for non-catalytic binding of cellulose. A construct of the cel gene carrying a deletion of codons 330-499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine-326 and serine-321, respectively. Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids.

Bacillus subtilis↗
PubMed1990

The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-beta-1,4-glucanases is essential for enzymatic activity.

We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.

Amino Acid Sequence↗
PubMed1989

Chemical properties of the histidine residue of secretin: evidence for a specific intramolecular interaction.

Secretin has a single histidine residue located at the amino terminus which plays a crucial role in its biological activity. The chemical properties, viz. pK and reactivity, of the alpha-amino and imidazole groups of this residue were determined at a secretin concentration of 10(-6) M in 0.1 M KCl at 37 degrees C. Competitive labelling using tritiated 1-fluoro-2,4-dinitrobenzene (DNP-F) as the labelling reagent was the experimental approach employed. The alpha-amino group was found to have a pK value of 8.83 and a reactivity 5-times that of the alpha-amino group in the model compound, histidylglycine. For the imidazole function a pK value of 8.24 and a reactivity 26-times that of the imidazole function in histidylglycine was found. Both these groups in secretin had pK values which were shifted one pK unit higher than in histidylglycine, but like the model compound the reactivity of the imidazole function was still linked to the state of ionization of the alpha-amino group. These observations are interpreted as evidence for the existence of a major conformational state in dilute aqueous solution in which the amino-terminal histidine of secretion is interacting with a negatively charged carboxyl group.

Binding, Competitive↗
PubMed1987

Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages.

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single-stranded DNA and RNA phages.

Amino Acid Sequence↗
PubMed1987

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.

Bacillus thuringiensis↗
PubMed1986

Structure-function relationships in the free insulin monomer.

The chemical properties of the functional groups of insulin were determined at a concentration (0.5 microM) where the predominant species of insulin is the free (unassociated) monomeric unit. The glycine N-terminus and the four tyrosine phenolic groups had the same properties as in the associated forms of insulin. On the other hand the lysine epsilon-amino group and the two histidine imidazole groups had substantially altered properties. Some alteration in the properties of the phenylalanine N-terminus was also observed. The reactivity-pH profile for the imidazole groups showed a second ionization with a pKa of 10.1 in addition to an ionization with a pKa of 6.8. On the basis of the X-ray-crystallographic structure of hexameric insulin the observed changes can be accounted for by disruption of monomer-monomer or dimer-dimer interactions in the associated states of insulin. It is concluded that the conformation of the monomeric unit of insulin is essentially the same in its free and associated states in solution.

Amino Acid Sequence↗
PubMed1985

Unusual chemical properties of N-terminal histidine residues of glucagon and vasoactive intestinal peptide.

An N-terminal histidine residue of a protein or peptide has two functional groups, viz., an alpha-amino group and an imidazole group. A new procedure, based on the competitive labeling approach described by Duggleby and Kaplan [Duggleby, R. G., & Kaplan, H. (1975) Biochemistry 14, 5168-5175], has been developed by which the chemical reactivity of each functional group in such a residue can be determined as a function of pH. Only very small amounts of material are required, which makes it possible to determine the chemical properties in dilute solution or in proteins and polypeptides that can be obtained in only minute quantities. With this approach, the reactivity of the alpha-amino group of histidylglycine toward 1-fluoro-2,4-dinitrobenzene gave an apparent pKa value of 7.64 +/- 0.07 at 37 degrees C, in good agreement with a value of 7.69 +/- 0.02 obtained by acid-base titration. However, the reactivity of the imidazole function gave an apparent pKa value of 7.16 +/- 0.07 as compared to the pKa value of 5.85 +/- 0.01 obtained by acid-base titration. Similarly, in glucagon and vasoactive intestinal peptide (VIP), apparent pKa values of 7.60 +/- 0.04 and 7.88 +/- 0.18, respectively, were obtained for the alpha-amino of their N-terminal histidine, and pKa values of 7.43 +/- 0.09 and 7.59 +/- 0.18 were obtained for the imidazole function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.

Acetic Anhydrides↗