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

Abstract

Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by Φ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called G A (a 3-α helix fold) and G B (an α∕β fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the G A domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the G B s, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed.kinetics | protein engineering | protein folding | protein G T he ultimate goal of a biophysical study is to extract general rules from the analysis of simple systems, a task that is particularly challenging in the case of protein folding. In fact, when considering different globular proteins, complexity stems by and large from the difference in sequence but also the multiplicity of structures of the native and denatured states. A suitable strategy to tackle the problem is to study proteins that differ in sequence but share the same overall fold, i.e., members of the same protein family (1-8). Over the past two decades, this approach allowed drawing some general conclusions about the correlation between 3D structure and sequence composition. In particular, it was reported that the mechanism of folding is, generally, conserved for members of the same protein family (9, 10) supporting the idea that native topology is often a main factor in controlling folding pathway and speed (11).A sophisticated protein engineering approach allowed Bryan and co-workers to obtain pairs of proteins with an increasing degree of sequence identity (up to the extraordinary value of 95%), but different 3D structure and function (12, 13). The primary structure of two domains from the streptococcal protein G, sharing 16% sequence identity, was subjected to extensive site-directed mutagenesis cycles, leading to the synthesis of pairs of variants with an increasing level of sequence identity (30%, 77%, 88%, and 95%, respectively) (12, 13). The two wild-type protein domains are called G A , displaying a three-helix bundle fold, and G B , displaying a α þ β ubiquitin-like fold. Therefore, the different variants (Fig. 1) were identified as G ...

Take home lessons from studies of related proteins

“…In a more recent extensive study of the designed system Gianni and co-workers have shown that G A 88 folds using a robust transition state to a three helical bundle, while G B 88 folds over a very malleable energy landscape to a ubiquitin-like (mostly β-sheet) topology [79]. This malleability is assigned to the presence of multiple, competing foldons.…”

Molecular dynamics simulations on the conformational transitions from the GA98 (GA88) to GB98 (GB88) proteins

“…It has been confirmed that the N and C termini in the conversion of G A to G B play a critical role in fold switching with hydrophobic core converting . The studies on the presence of an intermediate with reduced native contacts during the unfolding process from G B state to its unfolded state, and further investigations of the similar folding directions for G A 88 or G B 88 state have been performed by Gianni, Samuel, and co‐workers using equilibrium and kinetic folding/unfolding experiments, and molecular dynamics (MD) simulations . The folding mechanisms and stability for the G A 98, G B 98 states and their mutants were investigated using the free energy surface calculations and the all‐atom/coarse‐grained models .…”

Structural Role of Hydrophobic Core in Proteins-Selected Examples

“…The presented analysis concerns the well-known problem of correlating the protein's amino acid sequence with its 3D structure [1][2][3][4]. The search for algorithms which can be used to translate the former into the latter is a fundamental problem in proteomics [5,6] and often yields useful insight into the specific properties of individual proteins [7].…”