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W J Becktel

Publications and source records attributed to W J Becktel.

At least 19 recordsLinked to original sources
PubMed1997

Comparison of van der Waals and semiempirical calculations of the molecular volumes of small molecules and proteins.

Molecular volumes for hydrocarbons, amino acids, peptides, and 14 globular proteins were calculated by techniques using van der Waals radii and by semiempirical molecular orbital methods. The resulting values were compared to experimentally determined volumes. The values obtained by methods employing van der Waals radii were found to be up to three times smaller than the experimentally determined values in the case of proteins, 25% smaller than the experimental values for peptides, and up to 50% greater than experimental values for simple hydrocarbons. For the semiempirical calculations, neither the type nor precision of the calculation altered the percentage of the electron density required to reproduce the experimentally observed volumes for any of the different types of molecules tested. For molecules en vacuo, the amount of electron density included was approximately 98.5% of the total calculated value. For solvated molecules, the percentage was closer to 99.5%. From the results of our studies, we conclude that semiempirical techniques are more reliable, less arbitrary, and hence are more accurate for the determination of molecular volumes. The methods by which we employ semiempirical techniques for determination of molecular volume will be described in detail.

Algorithms↗
PubMed1995

Structure and stability of histone HMf from the hyperthermophilic archaeon Methanothermus fervidus.

The secondary and quaternary structures and stabilities of recombinant (r) forms of the HMfA and HMfB histones from Methanothermus fervidus have been investigated by CD spectroscopy and formaldehyde-mediated protein-protein cross-linking. Both proteins were shown to be dimers in solutions containing 5-1300 mM KCl, at pH 6-10 and 25-83 degrees C, and specifically in 1 M KCl, at pH 7.5 and 83 degrees C, conditions which approximate those in vivo in M. fervidus cells. Heat treatment of a mixture of rHMfA and rHMfB homodimers resulted in the formation of rHMfA.rHMfB heterodimers, as demonstrated by two-dimensional PAGE. Heterodimer formation did not result in a CD-detectable conformational change from the homodimer states, indicating that homogeneous (rHMfA)2 and (rHMfB)2 preparations may be considered as structural models of heterodimers. At pH 2, both rHMfA and rHMfB were denatured under low-salt (< 0.2 M KCl) conditions, and their conformations were stabilized in a cooperative manner by increasing KCl concentration, with cooperativity constants for KCl uptake of 2.7 and 3.1, respectively. The alpha-helical conformations of rHMfA and rHMfB were salt-dependent, at both pH 2 and pH 7.5, with maximal helicities in 1 M KCl of 84% and 63% at pH 2, and 72% and 65% at pH 7.5, respectively. The data obtained indicate that the structures of HMfA and HMfB, in 100-200 mM KCl at pH 7.5 and 25 degrees C, are likely to be very similar to their in vivo structures, even though these conditions are far removed from those found in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Molecular volume.

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Algorithms↗
PubMed1994

Osmolyte mediation of T7 DNA polymerase and plasmid DNA stability.

The thermal stability of T7 DNA polymerase and pGEM4Z plasmid DNA in the presence and absence of the osmolyte N-methylglycine (sarcosine) was determined by means of UV spectroscopy. The decrease in melting temperature observed upon addition of sarcosine to solutions containing the plasmid DNA is linear with the concentration of sarcosine present. The enthalpy of the transition is also linear in its relationship to the melting temperature, and the entropy of the transition is linear in the natural log of the melting temperature. The slopes of both the entropic and enthalpic plots are equal. Destabilization of the plasmid DNA is observed to be entropically driven. The melting temperature of the T7 DNA polymerase complex is increased from 41 degrees C by addition of sarcosine to the solution. The relationship between the amount of sarcosine added and the melting temperature is linear, with a temperature of 61 degrees C observed for a 6 M solution. No clear trend of the effect of sarcosine on the enthalpy or entropy of the transitions could be observed.

DNA↗
PubMed1994

pH-induced conformational transitions of Cry IA(a), Cry IA(c), and Cry IIIA delta-endotoxins in Bacillus thuringiensis.

Three protoxins and corresponding delta-endotoxins from Bacillus thuringiensis (BT) were studied by means of circular dichroism spectroscopy and size-exclusion HPLC. At neutral pH, the Cry IIIA toxin exists only as a 65-kDa monomer. The toxins of Cry IA(a) and Cry IA(c) exist both as 66-kDa monomers and as oligomers with apparent molecular masses greater than 220 kDa. At neutral pH, interconversion between monomer and oligomer is slow, and the two, separate forms exist for several days. Equilibration between the monomer and oligomer in both Cry IA(a) and Cry IA(c) toxins is facilitated by increasing the pH of the solutions to above 10. The relative amounts of monomer and oligomer depend upon temperature, pH, and buffer composition. CD spectra of the protoxins and toxins indicate a large helix content. The CD spectra of HPLC-isolated, monomeric Cry IA(a) and Cry IA(c) are quite similar, but are different from the spectrum of Cry IIIA. The Cry IA(a) and Cry IA(c) protoxins exhibit more helical CD spectra than the corresponding toxins. The CD spectra, with the pH titration of Cry IA(a), reveal that there is a significant increase in helical content as the pH is changed from neutral to alkaline values, but no decrease at low pH. A similar titration of Cry IIIA revealed no significant change in structure from pH 6 to pH 11. The CD spectrum of Cry IIIA at pH 2 indicates that the helical content of the toxin has significantly decreased. The magnitude of the 222-nm signal decreases from pH 7 to 2, with a midpoint of approximately pH 4.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Purification and N-terminal sequence analysis of pea chloroplast protein synthesis factor EF-G.

Chloroplast protein synthesis elongation factor G (chlEF-G) has been purified from whole-cell extracts of light-induced pea (Pisum sativum) seedlings. The first step in the purification scheme relies on the affinity of organellar EF-G for Escherichia coli ribosomes in the presence of the antibiotic, fusidic acid. A complex between organellar EF-G, E. coli ribosomes, GDP, and fusidic acid was isolated by high-speed centrifugation. The largest major protein eluted from this complex by high salt has an apparent molecular weight of 86,000 and is only a minor component of similar preparations from dark-grown seedlings. The same polypeptide copurifies with EF-G activity upon size exclusion HPLC on a Waters Protein-Pak 200SW column. The N-terminal amino acid sequence of chlEF-G has been determined by direct sequencing of gel-purified protein. Like many proteins that are processed upon import into chloroplasts, it has an N-terminal alanine residue. Part of the putative chlEF-G gene has been amplified using oligonucleotides corresponding to the N-terminal amino acid sequence of the purified protein and to highly conserved sequences within the GTP-binding domains of other elongation factors. The deduced amino acid sequence displays high sequence identity to the corresponding region of the chloroplast EF-G gene product from soybean, somewhat less similarity to bacterial EF-Gs, and only low homology to mitochondrial EF-G and to eukaryotic cytoplasmic EF-2 genes. The chlEF-G gene appears to be encoded by a two-copy gene family in pea and a single-copy gene in Arabidopsis thaliana.

Amino Acid Sequence↗
PubMed1992

Dissection of helix capping in T4 lysozyme by structural and thermodynamic analysis of six amino acid substitutions at Thr 59.

Threonine 59, a helix-capping residue at the amino terminus of the longest helix in T4 phage lysozyme, was substituted with valine, alanine, glycine, serine, asparagine, and aspartic acid. The valine, alanine, and glycine replacements were observed to be somewhat more destabilizing than serine, asparagine, and aspartic acid. The crystal structures of the different variants showed that changes in conformation occurred at the site of substitution, including Asp 61, which is nearby, as well as displacement of a solvent molecule that is hydrogen-bonded to the gamma-oxygen of Thr 59 in wild-type lysozyme. Neither the structures nor the stabilities of the mutant proteins support the hypothesis of Serrano and Fersht (1989) that glycine and alanine are better helix-capping residues than valine because a smaller-sized residue allows better hydration at the end of the helix. In the aspartic acid and asparagine replacements the substituted side chains form hydrogen bonds with the end of the helix, as does threonine and serine at this position. In contrast, however, the Asp and Asn side chains also make unusually close contacts with carbon atoms in Asp 61. This suggests a structural basis for the heretofore puzzling observations that asparagine is more frequently observed as a helix-capping residue than threonine [Richardson, J. S., & Richardson, D. C. (1988) Science 240, 1648-1652] yet Thr----Asn replacements at N-cap positions in barnase were found to be destabilizing [Serrano, L., & Fersht, A. R. (1989) Nature 342, 296-299].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme.

It was previously shown that the two replacements Gly 77-->Ala (G77A) and Ala 82-->Pro (A82P) increase the thermostability of phage T4 lysozyme at pH 6.5. Such replacements are presumed to restrict the degrees of freedom of the unfolded protein and so decrease the entropy of unfolding [B. W. Matthews, H. Nicholson, and W. J. Becktel (1987) Proceedings of the National Academy of Science USA Vol. 84, pp. 6663-6667]. To further test this approach, three additional replacements--G113A, K60P and A93P--have been constructed. On the basis of model building, each of these three replacements was judged to be less than optimal because it would tend to introduce unfavorable van der Waals contacts with neighboring parts of the protein. The presence of such contacts was verified for G113A and K60P by conformational adjustments seen in the crystal structures of these mutant proteins. In the case of G113A there are backbone conformational changes of 0.5-1.0 A in the short alpha-helix, 108-113, that includes the site of substitution. In the case of K60P the pyrrolidine ring shows evidence of strain. The thermal stability of each of the three variants at both pH 2.0 and pH 6.5 was found to be very close to that of wild-type lysozyme. The results suggest that the procedure used to predict sites for both Xaa-->Pro and Gly-->Ala is, in principle, correct. At the same time, the increase in stability expected from substitutions of this type is modest, and can easily be offset by strain associated with introduction of the alanine or proline. This means that the criteria used to select substitutions that will increase thermostability have to be stringent at least. In the case of T4 lysozyme this severely limits the number of sites. The analysis reveals a significant discrepancy between the conformational energy surface predicted for the residue preceding a proline and the conformations observed in crystal structures.

Bacteriophage T4↗
PubMed1990

pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme.

The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pKa values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pKa value of 9.1 in the native state and 6.8 in the unfolded state at 10 degrees C in moderate salt. Similarly, the aspartate pKa is shifted to a value of about 0.5 in the native state from its value of 3.5-4.0 in the unfolded state. These shifts in pKa show that the salt bridge is stabilized 3-5 kcal/mol. This implies that the salt bridge stabilizes the native state by 3-5 kcal/mol as compared to the unfolded state. This is reflected in the thermodynamic stability of mutants of the protein in which Asp70, His31, or both are replaced by asparagine. These observations and consideration of the thermodynamic coupling of protonation state to folding of proteins suggest a mechanism of acid denaturation in which the unfolded state is progressively stabilized by protonation of its acid residues as pH is lowered below pH 4. The unfolded state is stabilized only if acidic groups in the folded state have lower pKa values than in the unfolded state. When the pH is sufficiently low, the acid groups of both the native and unfolded states are fully protonated, and the apparent unfolding equilibrium constant becomes pH independent. Similar arguments apply to base-induced unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗
PubMed1989

Stabilization of phage T4 lysozyme by engineered disulfide bonds.

Four different disulfide bridges (linking positions 9-164, 21-142, 90-122, and 127-154) were introduced into a cysteine-free phage T4 lysozyme at sites suggested by theoretical calculations and computer modeling. The new cysteines spontaneously formed disulfide bonds on exposure to air in vitro. In all cases the oxidized (crosslinked) lysozyme was more stable than the corresponding reduced (noncrosslinked) enzyme toward thermal denaturation. Relative to wild-type lysozyme, the melting temperatures of the 9-164 and 21-142 disulfide mutants were increased by 6.4 degrees C and 11.0 degrees C, whereas the other two mutants were either less stable or equally stable. Measurement of the equilibrium constants for the reduction of the engineered disulfide bonds by dithiothreitol indicates that the less thermostable mutants tend to have a less favorable crosslink in the native structure. The two disulfide bridges that are most effective in increasing the stability of T4 lysozyme have, in common, a large loop size and a location that includes a flexible part of the molecule. The results suggest that stabilization due to the effect of the crosslink on the entropy of the unfolded polypeptide is offset by the strain energy associated with formation of the disulfide bond in the folded protein. The design of disulfide bridges is discussed in terms of protein flexibility.

Disulfides↗
PubMed1988

Enhanced protein thermostability from designed mutations that interact with alpha-helix dipoles.

Two different genetically engineered amino-acid substitutions designed to interact with alpha-helix dipoles in T4 lysozyme are shown to increase the thermal stability of the protein. Crystallographic analyses of the mutant lysozyme structures suggest that the stabilization is due to electrostatic interaction and does not require precise hydrogen bonding between the substituted amino acid and the end of the alpha-helix.

Aspartic Acid↗
PubMed1988

Disulfide bonds and thermal stability in T4 lysozyme.

Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.

Disulfides↗
PubMed1987

Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding.

It is proposed that the stability of a protein can be increased by selected amino acid substitutions that decrease the configurational entropy of unfolding. Two such substitutions, one of the form Xaa----Pro and the other of the form Gly----Xaa, were constructed in bacteriophage T4 lysozyme at sites consistent with the known three-dimensional structure. Both substitutions stabilize the protein toward reversible and irreversible thermal denaturation at physiological pH. The substitutions have no effect on enzymatic activity. High-resolution crystallographic analysis of the proline-containing mutant protein (Ala-82----Pro) shows that its three-dimensional structure is essentially identical with the wild-type enzyme. The overall structure of the other mutant enzyme (Gly-77----Ala) is also very similar to wild-type lysozyme, although there are localized conformational adjustments in the vicinity of the altered amino acid. The combination of a number of such amino acid replacements, each of which is expected to contribute approximately 1 kcal/mol (1 cal = 4.184 J) to the free energy of folding, may provide a general strategy for substantial improvement in the stability of a protein.

Calorimetry↗
PubMed1985

A lysoplate assay for Escherichia coli cell wall-active enzymes.

A benchtop assay based upon digestion of purified Escherichia coli peptidoglycan suspended in an agarose gel matrix is described. Enzymes for which these cell walls are substrates are applied to wells in the gel and diffuse into the gel. Activity is measured visually by the size of clear disks formed around the wells as the peptidoglycan is digested. Using this assay, it is possible to screen large numbers of cell wall-active enzymes for sensitivity to pH, ionic strength, denaturant, temperature, or other factors without interference from endogenous autolytic enzymes. Data are presented to show the limits of detection and linearity of the assay. For an assay time of 14 h, as little as 1 nmol per liter of bacteriophage T4 lysozyme and 200 nmol per liter of hen egg white lysozyme were detected. Longer assay times decrease these limits by as much as an order of magnitude. The salt dependence of T4 lysozyme and several of its temperature-sensitive mutants was also determined. Finally, an example of the use of the assay during lysozyme purification to determine active column fractions is presented.

Biological Assay↗