Highly nonexponential kinetics in the early-phase refolding of proteins at low temperatures.
Theory and simulations predict that the folding kinetics of protein-like heteropolymers become nonexponential and glassy (i.e., controlled by escape from different low-energy misfolded states) at low temperatures, but there was little experimental evidence for such behavior of proteins. We have developed a stopped-flow instrument working reliably down to -40 degrees C with high mixing capability and applied it to study the refolding kinetics of horse cytochrome c (cyt c) and hen egg white lysozyme at temperatures below 0 degrees C in the presence of antifreeze NaCl, LiCl, or ethylene glycol and above 0 degrees C in the presence and absence of antifreeze. The refolding was initiated by rapid dilution of the guanidine hydrochloride unfolded proteins, and the kinetics were monitored by intrinsic tryptophan fluorescence. Highly nonexponential kinetics extended over 3 decades in time (0.01-10 s) were observed in the early phases of the refolding of cyt c and lysozyme in the temperature range of -35 to 5 degrees C. These results are in agreement with the theoretical prediction, suggesting that the folding energy landscapes of these proteins are rugged in the upper portions.
Saigo
Satoshi
S
Division of Biophysics, Department of Physiology, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan. saigos@jichi.ac.jp
Shibayama
Naoya
N
D000818:Animals; D002645:Chickens; D003080:Cold Temperature; D003574:Cytochrome c Group / Q000737:chemistry*; D004527:Egg Proteins / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D009206:Myocardium / Q000201:enzymology; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013057:Spectrum Analysis / Q000379:methods; D014364:Tryptophan / Q000737:chemistry
D003574:Cytochrome c Group; D004527:Egg Proteins; D014364:Tryptophan; D009113:Muramidase; D019791:Guanidine