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, Philip; Matthews, Brian W.

doi:10.1002/pro.5560021222

Cited by 26 publications

(17 citation statements)

References 14 publications

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“…The strain relief may similarly explain the other thermostabilizing substitutions at position 101, but the requisite structures are unsolved. The enhancement of protein thermostability by the relaxation of strain has been observed in a mutant of T4 lysozyme as well (Pjura & Matthews, 1993).…”

Section: Alleviation Of a Destabilizing Steric Contact At Residue 101mentioning

confidence: 92%

Design and structural analysis of an engineered thermostable chicken lysozyme

Shih

Kirsch

1995

Protein Science

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A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (AT, = + 10.5 "C) and chemical (AC,n for guanidine hydrochloride denaturation = + 1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 "C) to 4.0 (differential scanning calorimetry, 74 "C) kcal mol" greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The 155L/S91T core construct with AT, = 3.3 "C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062. (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 3 1 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The DlOlS variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.Keywords: a-helix propensity; avian lysozyme sequences; chicken lysozyme; human lysozyme; interior packing; protein design; structures; thermodynamic stability A challenging goal of protein engineering is the design of proteins with significantly enhanced thermo-and chemical stabilities. A variety of successful routes to this objective has been undertaken. These include: (1) introducing disulfide bridges (Matsumura et al., 1989b); (2) increasing internal hydrophobic packing (Malcolm et al., 1990); (3) reducing solvent-exposed nonpolar surface area (Pakula & Sauer, 1990); (4) incorporating a metal association site (Kuroki et al., 1989); and (5) adding charged side chains to interact with the a-helix dipoles (Nicholson et al., 1988). However, the above strategies are not always successful and their pursuit has often yielded proteins that are less stable than the WT progenitors for reasons that are not fully understood (e.g., Karpusas et al., 1989;Matsumura et al., 1989a;Eijsink et al., 1992;Leontiev et al., 1993 Abbreviations: hs, a hyperstable variant of chicken lysozyme; GdnHCI, guanidine hydrochloride; T,, the midpoint temperature of the thermal denaturation transition; C,, the midpoint concentration of the GdnHCl denaturation profile; DSC, differential scanning calorimetry; AH,,,, calorimetrically determined enthalpy of denaturation; AC,, the difference between the heat capacities of the native and the denatured states; WT or wt, wild type.

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Section: Alleviation Of a Destabilizing Steric Contact At Residue 101mentioning

confidence: 92%

Design and structural analysis of an engineered thermostable chicken lysozyme

Shih

Kirsch

1995

Protein Science

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“…The structures of three representative mutants are described in the companion paper (Pjura & Matthews, 1993).…”

Section: Rimentioning

confidence: 99%

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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“…Two variants were found, which exhibited increased reversible melting temperatures with respect to the wild-type protein. The results also illustrate the power of random mutagenesis in obtaining variants with a desired phenotype (Pjura and Matthews 1993 ). UV irradiation is one of the most popular physically induced methods of mutagenesis.…”

Section: Random Mutagenesis Of Bacteriophagesmentioning

confidence: 74%

Molecular and Chemical Engineering of Bacteriophages for Potential Medical Applications

Hodyra

Dąbrowska

2014

Arch. Immunol. Ther. Exp.

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Recent progress in molecular engineering has contributed to the great progress of medicine. However, there are still difficult problems constituting a challenge for molecular biology and biotechnology, e.g. new generation of anticancer agents, alternative biosensors or vaccines. As a biotechnological tool, bacteriophages (phages) offer a promising alternative to traditional approaches. They can be applied as anticancer agents, novel platforms in vaccine design, or as target carriers in drug discovery. Phages also offer solutions for modern cell imaging, biosensor construction or food pathogen detection. Here we present a review of bacteriophage research as a dynamically developing field with promising prospects for further development of medicine and biotechnology.

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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

Cited by 26 publications

References 14 publications

Design and structural analysis of an engineered thermostable chicken lysozyme

Design and structural analysis of an engineered thermostable chicken lysozyme

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

Molecular and Chemical Engineering of Bacteriophages for Potential Medical Applications

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