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H Nicholson

Publications and source records attributed to H Nicholson.

At least 19 recordsLinked to original sources
PubMed1992

Structure of a hinge-bending bacteriophage T4 lysozyme mutant, Ile3-->Pro.

The mutant T4 phage lysozyme in which isoleucine 3 is replaced by proline (I3P) crystallizes in an orthorhombic form with two independent molecules in the asymmetric unit. Relative to wild-type lysozyme, which crystallizes in a trigonal form, the two I3P molecules undergo large hinge-bending displacements with the alignments of the amino-terminal and carboxy-terminal domains changed by 28.9 degrees and 32.9 degrees, respectively. The introduction of the mutation, together with the hinge-bending displacement, is associated with repacking of the side-chains of Phe4, Phe67 and Phe104. These aromatic residues are clustered close to the site of the mutation and are at the junction between the amino and carboxyl-terminal domains. As a result of this structural rearrangement the side-chain of Phe4 moves from a relatively solvent-exposed conformation to one that is largely buried. Mutant I3P also crystallizes in the same trigonal form as wild-type and, in this case, the observed structural changes are restricted to the immediate vicinity of the replacement. The main change is a shift of 0.3 to 0.5 A in the backbone of residues 1 to 5. The ability to crystallize I3P under similar conditions but in substantially different conformations suggests that the molecule undergoes large-scale hinge-bending displacements in solution. It is also likely that these conformational excursions are associated with repacking at the junction of the N-terminal and C-terminal domains. On the other hand, the analysis is complicated by possible effects of crystal packing. The different I3P crystal structures show substantial differences in the binding of solvent, both at the site of the Ile3-->Pro replacement and at other internal sites.

Bacteriophage T4↗
PubMed1992

Folding kinetics of T4 lysozyme and nine mutants at 12 degrees C.

The kinetics of unfolding and refolding of T4 lysozyme and nine of its mutants have been investigated as a function of guanidinium chloride concentration at 12 degrees C. All show simple two-state, first-order kinetics. Two types of mutants were studied: proline-alanine interchanges and substitutions at position 3 with side chains of varying hydrophobicity. Crystal structures are available for seven of the ten proteins. The effect of mutations on the folding kinetics is more pronounced and complex than on equilibrium thermodynamics. The proteins fall into two broad kinetic classes with one class rather close to the wild type. P86A is a mutant with marked changes in kinetics but only a very small change in stability. Since the 86 position is in the middle of an alpha-helix, the indications are that the helix containing an A residue is more stable in the transition state than one containing a P residue. The other mutants are more complicated, with the refolding and unfolding rates unequally affected by the mutations. On the basis of comparisons with other investigations, we conclude that the rate-determining step in the presence of guanidinium chloride is not the same as in aqueous solution and that it most likely precedes it. The indications are that we are studying the formation of a transition intermediate which is destabilized by the denaturant and which resembles the A intermediate of the framework or molten globule models for protein folding.

Amino Acid Sequence↗
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↗
PubMed1992

Vasopressin biosynthesis in rodent Leydig cells.

Local biosynthesis of the peptide hormone vasopressin is demonstrated in vitro in Leydig cells derived from rat and mouse testis. Cycloheximide-sensitive production of the nonapeptide was shown for rat Leydig cells in primary culture. A polymerase chain reaction technique demonstrated the presence of functionally constituted vasopressin mRNA in rat and mouse testis, primary mouse Leydig cells and in rat and mouse Leydig tumour cell lines (MA10, R2C). Stimulation of cells with gonadotropins, however, had no effect either on peptide production or on levels of specific mRNA. Similarly, treatment of the MA10 cell line with a phorbol ester, or with rat atriopeptin, which activate other second messenger pathways, had no influence on vasopressin mRNA levels. The results are discussed in terms of an autocrine regulatory system which would provide the cell with information about its microenvironment.

Animals↗
PubMed1991

Analysis of the interaction between charged side chains and the alpha-helix dipole using designed thermostable mutants of phage T4 lysozyme.

It was shown previously that the introduction of a negatively charged amino acid at the N-terminus of an alpha-helix could increase the thermostability of phage T4 lysozyme via an electrostatic interaction with the "helix dipole" [Nicholson, H., Becktel, W. J., & Matthews, B. W. (1988) Nature 336, 651-656]. The prior report focused on the two stabilizing substitutions Ser 38----Asp (S38D) and Asn 144----Asp (N144D). Two additional examples of stabilizing mutants, T109D and N116D, are presented here. Both show the pH-dependent increase in thermal stability expected for the interaction of an aspartic acid with an alpha-helix dipole. Control mutants were also constructed to further characterize the nature of the interaction with the alpha-helix dipole. High-resolution crystal structure analysis was used to determine the nature of the interaction of the substituted amino acids with the end of the alpha-helix in both the primary and the control mutants. Control mutant S38N has stability essentially the same as that of wild-type lysozyme but hydrogen bonding similar to that of the stabilizing mutant S38D. This confirms that it is the electrostatic interaction between Asp 38 and the helix dipole, rather than a change in hydrogen-bonding geometry, that gives enhanced stability. Structural and thermodynamic analysis of mutant T109N provide a similar control for the stabilizing replacement T109D. In the case of mutant N116D, there was concern that the enhanced stability might be due to a favorable salt-bridge interaction between the introduced aspartate and Arg 119, rather than an interaction with the alpha-helix dipole. The additivity of the stabilities of N116D and R119M seen in the double mutant N116D/R119M indicates that favorable interactions are largely independent of residue 119. As a further control, Asp 92, a presumed helix-stabilizing residue in wild-type lysozyme, was replaced with Asn. This decreased the stability of the protein in the manner expected for the loss of a favorable helix dipole interaction. In total, five mutations have been identified that increase the thermostability of T4 lysozyme and appear to do so by favorable interactions with alpha-helix dipoles. As measured by the pH dependence of stability, the strength of the electrostatic interaction between the charged groups studied here and the helix dipole ranges from 0.6 to 1.3 kcal/mol in 150 mM KCl. In the case of mutants S38D and N144H, NMR titration was used to measure the pKa's of Asp 38 and His 144 in the folded structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis.

Six designed mutants of T4 lysozyme were created in an attempt to create putative salt bridges on the surface of the protein. The first three of the mutants, T115E (Thr 115 to Glu), Q123E, and N144E, were designed to introduce a new charged side chain close to one or more existing charged groups of the opposite sign on the surface of the protein. In each of these cases the putative electrostatic interactions introduced by the mutation include possible salt bridges between residues within consecutive turns of an alpha-helix. Effects of the mutations ranged from no change in stability to a 1.5 degrees C (0.5 kcal/mol) increase in melting temperature. In two cases, secondary (double) mutants were constructed as controls in which the charge partner was removed from the primary mutant structure. These controls proteins indicate that the contributions to stability from each of the engineered salt bridges is very small (about 0.1-0.25 kcal/mol in 0.15 M KCl). The structures of the three primary mutants were determined by X-ray crystallography and shown to be essentially the same as the wild-type structure except at the site of the mutation. Although the introduced charges in the T115E and Q123E structures are within 3-5 A of their intended partner, the introduced side chains and their intended partners were observed to be quite mobile. It has been shown that the salt bridge between His 31 and Asp 70 in T4 lysozyme stabilizes the protein by 3-5 kcal/mol [Anderson, D. E., Becktel, W. J., & Dahlquist, F. W. (1990) Biochemistry 29, 2403-2408]. To test the effectiveness of His...Asp interactions in general, three additional double mutants, K60H/L13D, K83H/A112D, and S90H/Q122D, were created in order to introduce histidine-aspartate charge pairs on the surface of the protein. Each of these mutants destabilizes the protein by 1-3 kcal/mol in 0.15 M KCl at pH values from 2 to 6.5. The X-ray crystallographic structure of the mutant K83H/A112D has been determined and shows that there are backbone conformational changes of 0.3-0.6 A extending over several residues. The introduction of the histidine and aspartate presumably introduces strain into the folded protein that destabilizes this variant. It is concluded that pairs of oppositely charged residues that are on the surface of a protein and have freedom to adopt different conformations do not tend to come together to form structurally localized salt bridges. Rather, such residues tend to remain mobile, interact weakly if at all, and do not contribute significantly to protein stability. It is argued that the entropic cost of localizing a pair of solvent-exposed charged groups on the surface of a protein largely offsets the interaction energy expected from the formation of a defined salt bridge. There are examples of strong salt bridges in proteins, but such interactions require that the folding of the protein provides the requisite driving energy to hold the interacting partners in the correct rigid alignment.

Electrochemistry↗
PubMed1991

Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft.

Substitution of Thr26 by Gln in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type. Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization. One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp. The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified. The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant. Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant. Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp). Model building suggests that the side chain of Gln26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity. In contrast, the crystal structures of the revertants reveal that the double substitutions (Gln26 and His18, or Gln26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone. This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.

Base Sequence↗
PubMed1991

Structural and genetic analysis of electrostatic and other interactions in bacteriophage T4 lysozyme.

The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein. Such studies can provide quantitative information on the contributions made by different types of interactions including hydrogen bonds, hydrophobic interactions, salt bridges and disulphide bridges. To determine the contribution of long-range electrostatic interactions a combination of charge-change mutations was used to reduce the overall formal charge on T4 lysozyme at neutral pH from +9 to +1 units. Such changes in charge were found to have little effect on the stability of the molecule. Salt bridges engineered on the surface of the protein also were found to contribute little to stability. In contrast, the introduction of acidic groups designed to interact with the partial positive charges at the N-termini of alpha-helices consistently increased the stability of the protein. It is argued that this difference between electrostatic salt-bridge interactions and electrostatic 'helix-dipole' interactions lies in the entropic cost of bringing together the interacting partners. In an attempt to simplify the folding problem, and also to further investigate the helix propensity of different amino acids, a series of alanines was introduced within an alpha-helix of T4 lysozyme. The resultant protein not only folds normally but is also more stable than the wild-type enzyme, adding further support to recent evidence that alanine is a helix-favouring amino acid.

Electricity↗
PubMed1991

Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths.

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.

Crystallization↗
PubMed1991

Children's freely produced synonyms for seven key emotions.

74 children from Grades Kindergarten, 4, and 8 were interviewed and asked to produce synonyms for seven key emotions (happy, sad, afraid, mad, comfortable, proud, guilty). Children produced more synonyms in Grades 4 and 8 than in Kindergarten, and more children from Kindergarten were unable to produce synonyms than those from the higher grades. The words guilty and proud were associated with the smallest number of synonyms while the words happy and sad were associated with the largest. At the Kindergarten level only, boys produced more synonyms than girls.

Age Factors↗
PubMed1989

Contributions of left-handed helical residues to the structure and stability of bacteriophage T4 lysozyme.

Non-glycine residues in proteins are rarely observed to have "left-handed helical" conformations. For glycine, however, this conformation is common. To determine the contributions of left-handed helical residues to the stability of a protein, two such residues in phage T4 lysozyme, Asn55 and Lys124, were replaced with glycine. The mutant proteins fold normally and are fully active, showing that left-handed non-glycine residues, although rare, do not have an indispensable role in the folding of the protein or in its activity. The thermodynamic stability of the Lys124 to Gly variant is essentially identical with that of wild-type lysozyme. The Asn55 to Gly mutant protein is marginally less stable (0.5 kcal/mol). These results indicate that the conformational energy of a glycine and a non-glycine residue in the left-handed helical conformation are very similar. This is consistent with some theoretical energy distributions, but is inconsistent with others, which suggest that replacements of the sort described here might increase the stability of the protein by up to 5 kcal/mol. Crystallographic analysis of the mutant proteins shows that the backbone conformation of the Lys124 to Gly variant is essentially identical with that of the wild-type structure. In the case of the Asn55 to Gly replacement, however, the (phi, psi) values of residue 55 change by about 20 degrees. This suggests that the energy minimum for left-handed glycine residues is not the same as that for non-glycine residues. This is strongly indicated also by a survey of accurately determined protein crystal structures, which suggests that the energy minimum for left-handed glycine residues is near (phi = 90 degrees, psi = 0 degrees), whereas that for non-glycine residues is close to (phi = 60 degrees, psi = 30 degrees). This apparent energy minimum for glycine is not clearly predicted by any of the theoretical (phi, psi) energy contour maps.

Amino Acid Sequence↗
PubMed1989

Autistic children's ability to interpret faces: a research note.

Ten autistic children were compared with 10 non-autistic children matched for chronological age and performance IQ on two tests of finding the odd face out of a set of photographs of faces, two tests of labelling photographs of faces, and a test of labelling photographs of common objects. The autistic children were significantly worse than the non-autistic children at finding the odd person out and the odd facial expression of emotion out, and at labelling facial expressions of emotion. They did no worse than the non-autistic children at labelling upside down faces or at labelling objects. The results, which replicate the findings of Hobson (J. Child Psychol, Psychiat. 27, 321-342, 671-680, 1986; Communication, 20, 12-17, 1986) are consistent with other evidence for a specific perceptual abnormality in at least some children with autism, the nature of which is discussed.

Adolescent↗
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

Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability.

To investigate the relation between protein stability and the predicted stabilities of individual secondary structural elements, residue Pro86 in an alpha-helix in phage T4 lysozyme was replaced by ten different amino acids. The x-ray crystal structures of seven of the mutant lysozymes were determined at high resolution. In each case, replacement of the proline resulted in the formation of an extended alpha-helix. This involves a large conformational change in residues 81 to 83 and smaller shifts that extend 20 angstroms across the protein surface. Unexpectedly, all ten amino acid substitutions marginally reduce protein thermostability. This insensitivity of stability to the amino acid at position 86 is not simply explained by statistical and thermodynamic criteria for helical propensity. The observed conformational changes illustrate a general mechanism by which proteins can tolerate mutations.

Enzyme Stability↗
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↗
PubMed1979

Diabetes and thyroid disease during pregnancy.

In a prospective study of 100 consecutive diabetic pregnancies, 1 was complicated by hyperthyroidism and 4 by hypothyroidism. The association of hypothyroidism with diabetes mellitus merits special attention as this combination of diseases affected 4 of 20 (20%) White's class D and F diabetics. Hydramnios and/or spontaneous premature labor were features of diabetic pregnancies complicated by compensated hypothyroidism. Delayed diagnosis of hypothyroidism may have contributed to the fatal congenital malformations in one of these fetuses, but the remaining infants survived, including 1 infant suffering from severe respiratory distress syndrome (RDS).

Congenital Abnormalities↗