Nonglassy kinetics in the folding of a simple single-domain protein.

Theory suggests that the otherwise rapid folding of simple heteropolymer models becomes "glassy"-dominated by multiple kinetically trapped misfolded states-at low temperatures or when the overall bias toward the native state is reduced relative to the depth of local minima. Experimental observations of nonsingle-exponential protein-folding kinetics have been taken as evidence that the protein-folding free energy landscape is similarly rough. No equivalent analysis, however, has been reported for a simple single-domain protein lacking prolines, disulfide bonds, prosthetic groups, or other gross structural features that might complicate folding. In an effort to characterize the glassiness of a folding free energy landscape in the absence of these potentially complicating factors, we have monitored the folding of a kinetically simple protein, peptostreptococcal protein L (protein L). We observe no statistically significant deviation from homogeneous single-exponential relaxation kinetics across temperatures ranging from near the protein's melting temperature to as low as -15 degrees C. On the basis of these observations, we estimate that, if there is a glass transition in the folding of protein L, it occurs at least 45 degrees C and possibly more than 145 degrees C below the freezing point of water. Apparently the folding free energy landscape of protein L is extremely smooth, which may be indicative of a rate-limiting step in folding that is, effectively, a nonglassy process.