A mutant T4 lysozyme displays five different crystal conformations

Faber, H.R.; Matthews, Brian W.

doi:10.1038/348263a0

Cited by 230 publications

(173 citation statements)

References 19 publications

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“…results). It is presumed in the case of these variants that it requires very little energy to change the hinge-bending angle (Faber & Matthews, 1990), and the present results tend to suggest that this is true in general, as the mutations described here are well away from the hinge-bending region (Fig. 1).…”

Section: Large-scale Changes In Con Formationsupporting

confidence: 63%

“…Rather, we suggest that the structural changes in the vicinity of the polyalanine helix favor an alternative crystal contact, and the hingebending angle adjusts to facilitate this new contact. Substantial variability in hinge-bending has been observed in two other T4 lysozyme variants, Met 6 --t Ile (Faber & Matthews, 1990) and Ile 3 -+ Pro (unpubl. results).…”

Section: Large-scale Changes In Con Formationmentioning

confidence: 97%

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Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within a-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the a-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions.All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest ( 5 0 . 4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133 +Ala made a-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino-and carboxyterminal domains changed by 3.6". This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.Keywords: alanine; lysozyme; protein folding; protein structure; thermostability In a systematic attempt to identify residues important in the folding and stability of phage T4 lysozyme we previously substituted four alanines within the a-helix that includes residues 126-134 . The helix is amphipathic, located on the surface of the carboxy-terminal domain, and is remote from the active site ( Fig. 1; Kinemage 1). In wild-type lysozyme this a-helix already includes three alanines. It was found that substitution of three solvent-exposed residues, Glu 128, Val 131, and Asn

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Section: Large-scale Changes In Con Formationsupporting

confidence: 63%

Section: Large-scale Changes In Con Formationmentioning

confidence: 97%

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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“…The structure of M6L has many similarities to the previously determined structure of M61 (Faber & Matthews, 1990). Both structures have cavities proximal to the site of mutation of about 40 A3, and both are destabilized equally relative to WT*.…”

Section: Methionine 6 + Leucinementioning

confidence: 56%

“…Within two days to two weeks, crystals appeared that belonged to space group P3221 and were isomorphous with those of WT* T4 lysozyme. Similarly to M61 (Faber & Matthews, 1990), a second crystal form was obtained for M6L (space group P212121).…”

Section: Crystallization and Structure Determinationmentioning

confidence: 99%

“…Refinement of atomic coordinates was via standard protocols with TNT (Tromd et al, 1987). Starting models for refinement were M61 in the P3221 space group for M6L (Faber & Matthews, 1990), and WT* (Eriksson et al, 1993) for the other four mutants. Water and other solvent molecules were not included in the starting model.…”

Section: Crystallization and Structure Determinationmentioning

confidence: 99%

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Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

et al. 1998

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The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met + Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.

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A new method for modeling large-scale rearrangements of protein domains

Maiorov

Abagyan

1997

Proteins

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A method for modeling large-scale rearrangements of protein domains connected by a single- or a double-stranded linker is proposed. Multidomain proteins may undergo substantial domain displacements, while their intradomain structure remains essentially unchanged. The method allows automatic identification of an interdomain linker and builds an all-atom model of a protein structure in internal coordinates. Torsion angles belonging to the interdomain linkers and side chains potentially able to form domain interfaces are set free while remaining torsions, bond lengths, and bond angles are fixed. Large-scale sampling of the reduced torsions conformational subspace is effected with the "biased probability Monte Carlo-minimization" method [Abagyan, R.A., Totrov, M.M. (1994): J. Mol. Biol. 235, 983-1002]. Solvation and side-chain entropic contributions are added to the energy function. A special procedure has been developed to generate concerted deformations of a double-stranded interdomain linker in such a way that the polypeptide chain continuity is preserved. The method was tested on Bence-Jones protein with a single-stranded linker and lysine/arginine/ornithine-binding (LAO) protein with a double-stranded linker. For each protein, structurally diverse low-energy conformations with ideal covalent geometry were generated, and an overlap between two sets of conformations generated starting from the crystallographically determined "closed" and "open" forms was found. One of the low-energy conformations generated in a run starting from the LAO "closed" form was only 2.2 A away from the structure of the "open" form. The method can be useful in predicting the scope of possible domain rearrangements of a multidomain protein.

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A mutant T4 lysozyme displays five different crystal conformations

Cited by 230 publications

References 19 publications

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

A new method for modeling large-scale rearrangements of protein domains

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