The Denatured State Dictates the Topology of Two Proteins with Almost Identical Sequence but Different Native Structure and Function

Abstract

The protein folding problem is often studied by comparing the mechanisms of proteins sharing the same structure but different sequence. The recent design of the two proteins G A 88 and G B 88, displaying different structures and functions while sharing 88% sequence identity (49 out of 56 amino acids), allows the unique opportunity for a complementary approach. At which stage of its folding pathway does a protein commit to a given topology? Which residues are crucial in directing folding mechanisms to a given structure? By using a combination of biophysical and computational techniques, we have characterized the folding of both G A 88 and G B 88. We show that, contrary to expectation, G B 88, characterized by a native ␣؉␤ fold, displays in the denatured state a content of native-like helical structure greater than G A 88, which is all-␣ in its native state. Both experiments and simulations indicate that such residual structure may be tuned by changing pH. Thus, despite the high sequence identity, the folding pathways for these two proteins appear to diverge as early as in the denatured state. Our results suggest a mechanism whereby protein topology is committed very early along the folding pathway, being imprinted in the residual structure of the denatured state.

Folding pathways of proteins with increasing degree of sequence identities but different structure and function

“…Two-state folding was further confirmed by the excellent agreement between the thermodynamic parameters obtained by equilibrium and kinetic data. These data parallel earlier studies on G A 88 (14) and confirm that this protein system folds via a two-state folding pathway with an unstructured denatured state.…”

“…Furthermore, additional support for the presence of residual structure in the denatured state of G B 88 is represented by the presence of three nonstandard values of Φ (i.e., L20A, V21A, and Y33F; Table S2). Remarkably, these positions are located at the N-and C-terminal regions of the central helix of the G B fold, which was observed by molecular dynamics simulation to be partially formed in the denatured state of G B 88 (14). Overall, despite the very high level of sequence identities, when comparing the folding of each heteromorphic pair, no common intermediate was detected, suggesting that they fold via completely independent paths.…”

“…In a preliminary study, we compared the folding and unfolding kinetics of G A 88 and G B 88 at a variety of different experimental conditions (14). We observed that a detectable residual structure is present in the denatured state of G B 88, while the denatured state of G A 88 is essentially unstructured.…”

“…Recent molecular dynamics simulations on G A 88 and G B 88 suggested the long and stable helix in the central region of the sequence of G B 88 preventing the polypeptide chain to form the loop connecting helix 1 and helix 2 in G A 88 and thus to fold into a fully helical structure (14). This finding is further corroborated by the high helical propensity of the only α-helix of all the G B proteins as predicted by AGADIR (28), when compared to the G A counterparts.…”

“…We have recently analyzed the folding mechanisms of G A 88 and G B 88 at a variety of different conditions by experiments and simulations (14). We observed that despite the high level of identity of the primary structures of these two proteins (49 out of 56 residues), their folding pathways appear to diverge as early as in the denatured state, which in G B 88 but not in G A 88 displays a detectable residual structure.…”