Cold denaturation and 2H2O stabilization of a staphylococcal nuclease mutant.
Cold denaturation is now recognized as a general property of proteins but has been observed only under destabilizing conditions, such as moderate denaturant concentration or low pH. By destabilizing the protein using site-directed mutagenesis, we have observed cold denaturation at pH 7.0 in the absence of denaturants in a mutant of staphylococcal nuclease, which we call NCA S28G for a hybrid protein between staphylococcal nuclease and concanavalin A in which there is the point mutation Ser-28----Gly. The temperature of maximum stability (tmax) as determined by circular dichroism (CD) was 18.1 degrees C, and the midpoints of the thermal unfolding transitions (tm) were 0.6 degrees C and 30.0 degrees C. These values may be compared with the tm of 52.5 degrees C for wild-type staphylococcal nuclease, for which no cold denaturation was observed under these conditions. When the stability of the mutant was examined in 2H2O by NMR, CD, or fluorescence, a substantial increase in the amount of folded protein at the tmax was noted as well as a decrease in tmax, reflecting increased stability.
Antonino
L C
LC
Department of Chemistry, University of California, Santa Cruz 95064.
Kautz
R A
RA
Nakano
T
T
Fox
R O
RO
Fink
A L
AL
D002151:Calorimetry; D002942:Circular Dichroism; D003080:Cold Temperature; D003208:Concanavalin A / Q000235:genetics / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism*; D017666:Deuterium Oxide; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013861:Thiocyanates / Q000494:pharmacology; D014867:Water / Q000378:metabolism*
D006146:Guanidines; D011994:Recombinant Proteins; D013861:Thiocyanates; D014867:Water; D003208:Concanavalin A; C054436:guanidine thiocyanate; D003903:Deuterium; D008836:Micrococcal Nuclease; D017666:Deuterium Oxide; D019791:Guanidine