Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein

Alber, Tom; Sun, Dao Pin; Nye, Julie A.; Muchmore, David C.; Matthews, Brian W.

doi:10.1021/bi00387a002

Cited by 263 publications

(158 citation statements)

References 34 publications

Supporting

Mentioning

142

Contrasting

Order By: Relevance

“…Other evidence in support of this idea in- cludes the demonstration by Sauer and coworkers that amino acids in a given protein can have a low "information content" (Bowie et al, 1990). Also, amino acid substitutions of mobile solvent-exposed residues on the surface of a protein generally have little effect on stability (Hecht et al, 1986;Alber et al, 1987). In experiments parallel to those reported here, Heinz et al (1992) have constructed a mutant lysozyme with 10 consecutive alanines (residues 40-49) in a different a-helix and have shown that that variant also folds normally.…”

Section: Tolerance Of a Polyalanine Helixmentioning

confidence: 70%

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

1992

View full text Add to dashboard Cite

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

show abstract

Section: Tolerance Of a Polyalanine Helixmentioning

confidence: 70%

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

1992

View full text Add to dashboard Cite

show abstract

“…Mutations that substantially destabilize T4 lysozyme tend to be restricted to those amino acids that have the lowest thermal motion (Alber et al, 1987;Matthews, 1993). Takano et al (1995) have also suggested that there might be an inverse correlation between mobility and destabilization for Ile Val substitutions in human lysozyme.…”

Section: Packing Density and Thermal Motionmentioning

confidence: 99%

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

et al. 1998

Self Cite

View full text Add to dashboard Cite

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts 5 0.3 A); in other cases mainchain atoms moved up to 2 A.In the case of Ile 29 -+ Ala the structure col!apsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 4 Ala (V149A) and Met 6 -+ Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the. cavity. The Val 149 -+ Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 -+ Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu + Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol" k 3 . Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu + Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).

show abstract

“…These include T59S (Bell et al, 1992), T152S (Alber et al, 1987a;Dao-pin et al, 1991), T157S (Alber et al, 1987b), A98T (Alber et al, 1987a), A146T (Alber et al, 19861, and A160T (Alber et al, 1987a). All appear to be temperaturesensitive to a greater or lesser degree, showing that there is no reason to believe that threonine to serine (or alanine to threonine) substitutions are intrinsically stabilizing.…”

Section: Discussionmentioning

confidence: 99%

Structures of randomly generated mutants of T4 lysozyme show that protein stability can be enhanced by relaxation of strain and by improved hydrogen bonding via bound solvent

Pjura

Matthews

1993

Protein Science

Self Cite

View full text Add to dashboard Cite

The structures of three mutants of bacteriophage T4 lysozyme selected using a screen designed to identify thermostable variants are described. Each of the mutants has a substitution involving threonine. Two of the variants, Thr 26 + Ser (T26S) and Thr 151 -+ Ser (T151S), have increased reversible melting temperatures with respect to the wild-type protein. The third, Ala 93 -+ Thr (A93T), has essentially the same stability as wild type. Thr 26 is in the wall of the active-site cleft. Its replacement with serine results in the rearrangement of nearby residues, most notably Tyr 18, suggesting that the increase in stability may result from the removal of strain. Thr 151 in the wildtype structure is far from the active site and appears to sterically prevent the access of solvent to a preformed binding site. In the mutant, the removal of the methyl group allows access to the solvent binding site and, in addition, the Ser 151 hydroxyl rotates to a new position so that it also contributes to solvent binding. Residue 93 is in a highly exposed site on the surface of the molecule, and presumably is equally solvent exposed in the unfolded protein.It is, therefore, not surprising that the substitution Ala 93 -, Thr does not change stability. The mutant structures show how chemically similar mutations can have different effects on both the structure and stability of the protein, depending on the structural context. The results also illustrate the power of random mutagenesis in obtaining variants with a desired phenotype. Although knowledge of the mutant structures makes it possible to rationalize their behavior, it would have been very difficult to predict in advance that these mutants would be stabilizing.

show abstract

Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein

Cited by 263 publications

References 34 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

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

Structures of randomly generated mutants of T4 lysozyme show that protein stability can be enhanced by relaxation of strain and by improved hydrogen bonding via bound solvent

Contact Info

Product

Resources

About