No abstract

Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.

Unstable intermediates on the folding pathways of proteins can be stabilized sufficiently so that they accumulate to detectable extents by the addition of a suitable cosolute. Here, the effect of sodium sulfate (Na(2)SO(4)) on the folding of the SH3 domain of PI3 kinase was investigated in the presence of guanidine hydrochloride (GdnHCl) using intrinsic tyrosine fluorescence and 1-anilinonaphthalene-8-sulfonate (ANS) binding. The free energy of unfolding in water of the native state (N) increases linearly with Na(2)SO(4) concentration, indicating stabilization via the Hofmeister effect. The addition of 0.5 M Na(2)SO(4) causes accumulation of an early intermediate L, which manifests itself as (1) a sub-millisecond change in tyrosine and ANS fluorescence and (2) a curvature in the chevron plot. It is shown that L is a specific structural component of the initially collapsed ensemble. An intermediate, M, also accumulates in unfolding studies conducted in the presence of 0.5 M Na(2)SO(4) and manifests itself by causing a curvature in the unfolding arm of the chevron. M is shown to be a wet molten globule that binds to ANS under unfolding conditions and is stabilized to the same extent as N in the presence of Na(2)SO(4). A four-state U ↔ L ↔ M ↔ N scheme satisfactorily modeled the kinetic data. Thus, the folding of the PI3K SH3 domain in the presence of salt commences via the formation of a structured intermediate ensemble L, which accumulates before the rate-limiting step of folding. L subsequently proceeds to N via the late intermediate M that forms after the rate-limiting transition of folding.