Design and structural analysis of an engineered thermostable chicken lysozyme.
A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (delta Tm approximately +10.5 degrees C) and chemical (delta Cm 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 degrees C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mol-1 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 I55L/S91T core construct with delta Tm = 3.3 degrees 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 31 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 D101S 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.
Shih
P
P
Department of Chemistry, University of California, Berkeley 94720, USA.
Kirsch
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds; D002152:Calorimetry, Differential Scanning; D002645:Chickens; D002942:Circular Dichroism; D003370:Coturnix; D018360:Crystallography, X-Ray / Q000379:methods; D004795:Enzyme Stability; D005260:Female; D014644:Genetic Variation; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D008961:Models, Structural; D009113:Muramidase / Q000096:biosynthesis* / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011784:Quail; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013816:Thermodynamics; D014422:Turkeys
D006146:Guanidines; D011994:Recombinant Proteins; D009113:Muramidase; D019791:Guanidine