Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar

Abstract

Osmolytes stabilize proteins against denaturation, but little is known about how their stabilizing effect might affect a protein folding pathway. Here, we report the effects of the osmolytes, trimethylamine-N-oxide, and sarcosine on the stability of the native state of barstar as well as on the structural heterogeneity of an early intermediate ensemble, I E , on its folding pathway. Both osmolytes increase the stability of the native protein to a similar extent, with stability increasing linearly with osmolyte concentration. Both osmolytes also increase the stability of I E but to different extents. Such stabilization leads to an acceleration in the folding rate. Both osmolytes also alter the structure of I E but do so differentially; the fluorescence and circular dichroism properties of I E differ in the presence of the different osmolytes. Because these properties also differ from those of the unfolded form in refolding conditions, different burst phase changes in the optical signals are seen for folding in the presence of the different osmolytes. An analysis of the urea dependence of the burst phase changes in fluorescence and circular dichroism demonstrates that the formation of I E is itself a multistep process during folding and that the two osmolytes act by stabilizing, differentially, different structural components present in the I E ensemble. Thus, osmolytes can alter the basic nature of a protein folding pathway by discriminating, through differential stabilization, between different members of an early intermediate ensemble, and in doing so, they thereby appear to channel folding along one route when many routes are available.

Effects of denaturants and osmolytes on proteins are accurately predicted by the molecular transfer model

“…The extracted m values for all these osmolytes vary only moderately for protein L (m ϭ Ϫ(0.1 to 0.2) kcal mol Ϫ1 M Ϫ1 ) and for CspTm (m ϭ Ϫ(0.5 to 0.3) kcal mol Ϫ1 M Ϫ1 ; see Table 1). The nearly constant m values for the osmolytes is consistent with experiments that have found that m values for TMAO and sarcosine are roughly the same for barstar (39). As a result of the small m values the osmolytes increase the stability of the small proteins only modestly (Ϸ1 kcal/mol).…”

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

“…3 a and b), indicating that the polypeptide chain does not encounter a sizeable free energy barrier while folding to I E . This interpretation gains credence from several earlier observations: (i) the structure of I E is different in the presence of different osmolytes and different salts (25,26); (ii) different intramolecular distances in I E contract in a gradual and asynchronous manner, upon a reduction in denaturant concentration (9); (iii) the free energy difference between U and I E turns out to be less than k B T (25,26) if the U-to-I E transition is assumed to be barrier-limited; (iv) I E is loosely packed throughout its structure (33) and hence is expected to form with a lack of cooperativity (20,34); and (v) most importantly, gradual structural changes in the folded as well as unfolded forms of barstar have been detected in equilibrium unfolding studies (12,13).…”

“…Far-UV CD and fluorescence measurements in the millisecond time domain were done by using a Biologic SFM-4 module, as described previously (8,9,26).…”

“…Because the sub-ms folding reaction is well separated in time from subsequent folding reactions that occur in the 100-ms time domain (Fig. 1 a Inset and b Inset), the observed FRET-measured ultrafast rate as well as the ANSfluorescence-measured very fast rate represent the rates of events leading to the formation of the early intermediate I E identified previously on the folding pathway of barstar (25,26). In the previous studies, I E was found to be fully populated at a few milliseconds of folding, and the ANS-f luorescencemeasured rate is likely to be the direct rate of formation of I E .…”

“…Much is known about the folding mechanism of barstar (24)(25)(26)(27)(28)(29)(30), and earlier studies with millisecond time resolution had indicated, albeit indirectly, that the initial, sub-ms folding reaction of barstar is a gradual process (8,9). A very early collapsed structure-less globule, U C , gets transformed into a structured early intermediate, I E (9,25,26), at a few milliseconds of folding. I E then transforms to a late intermediate and native protein in the subsequent major folding reaction in the 100-ms time domain (24,25).…”