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

Zhang, X.-J.; Baase, W.A.; Matthews, Brian W.

doi:10.1002/pro.5560010608

Cited by 64 publications

(34 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutations at these sites presumably increase protein stability by relieving the putative strain present in the native structure and by interacting with residues defining the binding-site groove. Both mutants S117A (7) and N132A (8) (33,34) which, in turn, seem less active than their psychrophilic counterparts (35 …”

Section: Discussionmentioning

confidence: 99%

A relationship between protein stability and protein function.

Shoichet

Baase

Kuroki

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

663

461

View full text Add to dashboard Cite

Enzymes are thought to use their ordered structures to facilitate catalysis. A corollary of this theory suggests that enzyme residues involved in function are not optimized for stability. We tested this hypothesis by mutating functionally important residues in the active site of T4 lysozyme. Six mutations at two catalytic residues, Glu-11 and Asp-20, abolished or reduced enzymatic activity but increased thermal stability by 0.7-1.7 kcal mol-1. Nine mutations at two substrate-binding residues, Ser-117 and Asn-132, increased stability by 1.2-2.0 kcal-mol'1, again at the cost of reduced activity. X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme. In two of these structures the enzyme undergoes a general conformational change, similar to that seen in an enzyme-product complex. These results support a relationship between stability and function for T4 lysozyme. Other evidence suggests that the relationship is general.The ordered, functional structures of proteins reflect two tendencies that are often opposed. On one hand, proteins fold to minimize their free energy. On the other hand, they organize themselves to recognize a ligand or a transition state (1). Minimizing free energy leads to well-packed hydrophobic interiors and hydrophilic exteriors (2). Maximizing function leads to active-site clefts where charged and polar groups are sequestered from water (3, 4) and where hydrophobic patches are exposed to solvent.The hypothesis that there is a balance between stability and function can be stated most strongly as follows: protein residues that contribute to catalysis or ligand binding are not optimal for protein stability. This "stability-function" hypothesis predicts that it usually should be possible to replace residues known to be important for function, reducing protein activity but concomitantly increasing stability of the folded protein.Here we describe experiments that directly test the stabilityfunction hypothesis in T4 lysozyme, an enzyme well characterized for the effects of mutation on structure and stability (5). Five residues were replaced (Table 1 and Fig. 1). These included two residues implicated in chemical catalysis, 11,12), as well as thyee others thought to have a role in substrate binding, Gly-30, Ser-117, and Asp-132. We measured the thermodynamic stability and kinetic activity of the mutant lysozymes. To determine the structural consequences of the substitutions, we determined x-ray crystal structures for several of these proteins. MATERIALS AND METHODSMutagenesis and Protein Purification. Mutations were introduced into the T4 lysozyme gene borne by M13 phage derivative M13mpl8 T4e by mismatched oligonucleotides using the method of Kunkel et al (13) as detailed (6, 14).

show abstract

Section: Discussionmentioning

confidence: 99%

A relationship between protein stability and protein function.

Shoichet

Baase

Kuroki

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

663

461

View full text Add to dashboard Cite

show abstract

“…g The measured ÁÁGs are taken from Serrano et al (1992a,b), Matouschek et al (1989), , Jackson & Fersht (1994), Itzhaki et al (1995), , Zhang et al (1992Zhang et al ( , 1995 and .…”

Section: Mutations Of Residues With a Solvent Accessibility Of Less Tmentioning

confidence: 99%

Predicting protein stability changes upon mutation using database-derived potentials: solvent accessibility determines the importance of local versus non-local interactions along the sequence

Gilis

Rooman

1997

Journal of Molecular Biology

206

129

View full text Add to dashboard Cite

For 238 mutations of residues totally or partially buried in the protein core, we estimate the folding free energy changes upon mutation using database-derived potentials and correlate them with the experimentally measured ones. Several potentials are tested, representing different kinds of interactions. Local interactions along the chain are described by torsion potentials, based on propensities of amino acids to be associated with backbone torsion angle domains. Non-local interactions along the sequence are represented by distance potentials, derived from propensities of amino acid pairs or triplets to be at a given spatial distance. We ®nd that for the set of totally buried residues, the best performing potential is a combination of a distance potential and a torsion potential weighted by a factor of 0.4; it yields a correlation coef®cient between computed and measured changes in folding free energy of 0.80. For mutations of partially buried residues, the best potential is a combination of a torsion potential and a distance potential weighted by a factor of 0.7, and for the previously analysed mutations of solvent accessible residues, it is a torsion potential taken individually; the respective correlation coef®cients reach 0.82 and 0.87. These results show that distance potentials, dominated by hydrophobic interactions, represent best the main interactions stabilizing the protein core, whereas torsion potentials, describing local interactions along the chain, represent best the interactions at the protein surface. The prediction accuracy reached by the distance potentials is, however, lower than that of the torsion potentials. A possible reason for this is that distance potentials would not describe correctly the effect on protein stability due to cavity formation upon mutating a large into a small amino acid. Last but not least, our results indicate that although local interactions, responsible for secondary structure formation, do not dominate in the protein core, they are not negligible for all that. They have a signi®cant weight in the delicate balance between all the interactions that ensure protein stability.# 1997 Academic Press Limited

show abstract

“…33 In other words, the replacement of either Ser117 or Asn132 with a non-H-bonding substitute results in a significant increase in stability. [N132A has not been constructed as a single mutant but based on its beneficial effect on other variants, 36 we anticipate that it also would be stabilizing (by about 1.5-2.0 C)]. Why is an apparent destabilizing interaction between Ser117 and Asn132 retained in the native lysozyme structure?…”

mentioning

confidence: 99%

Lessons from the lysozyme of phage T4

et al. 2010

View full text Add to dashboard Cite

An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (T m ) for any point mutant is 5.1°C for the mutant Ser 117 fi Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 Å that apparently has an unfavorable van der Waals contact. Increases in T m of more than 3-4°C for point mutants are rare, whereas several different types of destabilizing substitutions decrease T m by 20°C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of ''large-tosmall'' mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C-terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically-engineered dimers. Among the 43 space groups, P2 1 2 1 2 1 and P2 1 were observed most frequently, consistent with the prediction of Wukovitz and Yeates.

show abstract

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

Cited by 64 publications

References 30 publications

A relationship between protein stability and protein function.

A relationship between protein stability and protein function.

Predicting protein stability changes upon mutation using database-derived potentials: solvent accessibility determines the importance of local versus non-local interactions along the sequence

Lessons from the lysozyme of phage T4

Contact Info

Product

Resources

About