Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Abstract

To determine whether a protein folding reaction can occur in the absence of a dominant barrier is crucial for understanding its complexity. Here direct ultrafast kinetic measurements have been used to study the initial submillisecond (sub-ms) folding reaction of the small protein barstar. The cooperativity of the initial folding reaction has been explored by using two probes: fluorescence resonance energy transfer, through which the contraction of two intramolecular distances is measured, and the binding of 8-anilino-1-naphthalene sulfonic acid, through which the formation of hydrophobic clusters is monitored. A fast chain contraction is shown to precede the formation of hydrophobic clusters, indicating that the sub-ms folding reaction is not cooperative. The observed rate constant of the sub-ms folding reaction monitored by 8-anilino-1-naphthalene sulfonic acid fluorescence has been found to be the same in stabilizing conditions (low urea concentrations), in which specific structure is formed, and in marginally stabilizing conditions (higher urea concentrations), where virtually no structure is formed in the product of the sub-ms folding reaction. The observation that the folding rate is independent of the folding conditions suggests that the initial folding reaction occurs in the absence of a dominant free energy barrier. These results provide kinetic evidence that the formation of specific structure need not be slowed down by any significant free energy barrier during the course of a very fast protein folding reaction.noncooperative ͉ submillisecond protein folding

Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

“…2 and 3). Exponential kinetics are usually associated with an all-or-none process, but can also be produced by a gradual diffusive process occurring over many small distributed barriers (39). Indeed, a recent time-resolved FRET study of the unfolding of monellin strongly supports the latter possibility (42).…”

“…2 C and D Insets) in which tight packing interactions are cooperatively disrupted. However, a sigmoidal transition does not necessarily indicate cooperative unfolding, but can instead arise from gradual unfolding (8,13,39). Hence, the possibility that the N to I transition is a continuous transition cannot be ruled out at the present time.…”

“…2 A and B Insets), and the N to I reaction may appear to be continuous when studied by FRET, only because the swelling of N distorts the appearance of the transition. If the transition from the native to the dry molten globule state is indeed a continuous one, then the 2 states would be separated by many small distributed free-energy barriers (Յ3 k BT ), instead of 1 single dominant barrier (17,39). Indeed, computer simulations have suggested that the initial fast transition during the unfolding of a protein may be a gradual structural transition (40).…”

Continuous dissolution of structure during the unfolding of a small protein

“…Recent equilibrium unfolding studies using high-resolution structural probes or single-molecule fluorescence detection, have also indicated that unfolding may occur in several steps (11) and even gradually via a continuum of intermediate forms (12)(13)(14)(15)(16)(17). Nevertheless, definitive kinetic evidence for a gradual protein unfolding or folding reaction has been scarce, and only indirect (18)(19)(20).…”

Structural and dynamic aspects of protein clocks: how can they be so slow and stable?

“…Approximate time scales of motions were taken from Refs. [52][53][54] Cyanobacterial Kai protein oscillator 2155 barrierless transition is often discussed with respect to the early phases of protein folding [55], but such reactions [large A(T) and E a = 0] should be fast ([*10 4 s -1 ) and are considered to be limited by chain diffusion (i.e., to be temperature dependent, Eq. 1).…”