Structure of bacteriophage T4 lysozyme refined at 1.7 Å resolution

Weaver, L.H.; Matthews, Brian W.

doi:10.1016/0022-2836(87)90636-x

Cited by 352 publications

(305 citation statements)

References 31 publications

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“…All of the mutants could be crystallized using conditions similar to those for wild-type lysozyme (Weaver & Matthews, 1987) and gave crystals large enough for high-resolution data collection. Prior to X-ray exposure the crystals were equilibrated with a solution of 1.05 M K2HP04, 1.26 M NaH2P04, 0.23 M NaCl, 1.4 mM 0-mercaptoethanol, pH 6.7.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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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“…For N163D, ASo is about two standard deviations above WT*, whereas for G113E it is about two standard deviations below. In the crystal structure of WT*, residues 162-164 at the C-terminus of the molecule are very mobile (Weaver & Matthews, 1987). In mutant N163D, however, the introduced aspartate apparently interacts favorably with the dipole of the C-terminal helix, and, as a result, in the crystal structure of this mutant residues 162-164 are better ordered than in WT* (data not shown).…”

Section: Mutants Isolatedmentioning

confidence: 94%

Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability

et al. 1993

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An M13 bacteriophage‐based in vivo screening system has been developed to identify T4 lysozyme mutants of enhanced thermal stability. This system takes advantage of easy mutagenesis in an M13 host, the production of functional T4 lysozyme during M13 growth, and the ability to detect lysozyme activity on agar plates. Of several mutagenesis procedures that were tested, the most efficient was based on misincorporation by avian myeloma virus reverse transcriptase. This one‐step mutagenesis and screening system has been used to find 18 random single‐site mutant lysozymes, of which 11 were heat resistant. Each of these had a melting temperature within 0.8–1.4°C of wild type, suggesting that the screening system is quite sensitive.

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“…The crystal structure of the wild-type protein (2LZM) was used as a template (Weaver & Matthews, 1987). Core residues for T4 lysozyme are 87, 91, 99, 102, 106, 111, 114, 118, 121, 126, 129, 133, 138, 149, 150, and 153.…”

Section: Modelsmentioning

confidence: 99%

Side-chain and backbone flexibility in protein core design 1 1Edited by A. R. Fersht

Desjarlais

Handel

1999

Journal of Molecular Biology

135

105

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We have developed a computational approach for the design and prediction of hydrophobic cores that includes explicit backbone¯exibility. The program consists of a two-stage combination of a genetic algorithm and monte carlo sampling using a torsional model of the protein. Backbone structures are evaluated either by a canonical force-®eld or a constraining potential that emphasizes the preservation of local geometry. The utility of the method for protein design and engineering is explored by designing three novel hydrophobic core variants of the protein 434 cro. We use the new method to evaluate these and previously designed 434 cro variants, as well as a series of phage T4 lysozyme variants. In order to properly evaluate the in¯uence of backbone¯exibility, we have also analyzed the effects of varying amounts of side-chain¯exibility on the performance of ®xed backbone methods. Comparison of results using a ®xed versus¯exible backbone reveals that, surprisingly, the two methods are almost equivalent in their abilities to predict relative experimental stabilities, but only when full side-chain¯exibility is allowed. The prediction of core side-chain structure can vary dramatically between methods. In some, but not all, cases the¯exible backbone method is a better predictor of structure. The development of a¯exible backbone approach to core design is particularly important for attempts at de novo protein design, where there is no prior knowledge of a precise backbone structure.

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Structure of bacteriophage T4 lysozyme refined at 1.7 Å resolution

Cited by 352 publications

References 31 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

Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability

Side-chain and backbone flexibility in protein core design 1 1Edited by A. R. Fersht

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