Revealing what gets buried first in protein folding.

After decades of research, the nature of the rate-limiting step in the folding of globular proteins still elicits a range of opinions (1). The properties of the associated transition state (TS) provide critical insights into possible folding mechanisms. However, the characterization of the TS is challenging because atomic level methods cannot be applied to this minimally populated state, and lower resolution methods have produced divergent views. Even the existence of a generalized TS remains actively debated. In PNAS, Guinn et al. (2) dissect the burial properties of the TS for 13 proteins by analyzing the denaturant and temperature dependence of folding rates to distinguish the burial of hydrophobic surface from that of amide groups. With this capability, they propose that the TSs generally are very advanced and often contain the native 2° structure, a level higher than most prior methods have indicated. The analysis by Guinn et al. (2) indicates that amide surface is preferentially buried in the TS. On average, 77% of the total amide surface is buried in the TS, whereas a smaller fraction, 60%, of the total hydrophobic surface is buried at this point in the reaction (although ∼twofold more hydrophobic surface is buried in the TS in absolute terms). Notably, the amount of amide burial often can be accounted for through the formation of the entire native 2° structure. Accordingly, Guinn et al. propose that TSs often contain the native 2° structure with most of the tertiary contacts forming post-TS. Structural modeling indicates that this level of 2° structure organizes the chain into the native fold before the TS and the rate-limiting step is the formation of a key set of tertiary contacts. Because the amide and hydrocarbon burial are preferentially buried preand post-TS, respectively, the authors suggest that these two quantities along with total burial are natural reaction coordinates. The results by Guinn et al. emphasizing 2° structure over hydrophobicity (2) evoke a comparison with the long-standing debate on the determinants of protein stability. Pauling and others stressed the importance of hydrogen bonds (3), but Kauzmann later argued that the hydrophobic effect is the dominant factor (4). However, more recent work has revisited this conclusion (5). Analogously, early kinetics models focused on the role of 2° structure (6), whereas later work emphasized hydrophobic collapse and the possibility that 2°-structure formation is driven by the collapse process (7).