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 degreesC. 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 degreesC and possibly more than 145 degreesC 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.