Folding Circular Permutants of IL-1β: Route Selection Driven by Functional Frustration

Dominique T. Capraro,Patricia A. Jennings,Shachi Gosavi,Melinda Roy,José N. Onuchic,Dafydd Jones

doi:10.1371/journal.pone.0038512

Abstract

Interleukin-1β (IL-1β) is the cytokine crucial to inflammatory and immune response. Two dominant routes are populated in the folding to native structure. These distinct routes are a result of the competition between early packing of the functional loops versus closure of the β-barrel to achieve efficient folding and have been observed both experimentally and computationally. Kinetic experiments on the WT protein established that the dominant route is characterized by early packing of geometrically frustrated functional loops. However, deletion of one of the functional loops, the β-bulge, switches the dominant route to an alternative, yet, as accessible, route, where the termini necessary for barrel closure form first. Here, we explore the effect of circular permutation of the WT sequence on the observed folding landscape with a combination of kinetic and thermodynamic experiments. Our experiments show that while the rate of formation of permutant protein is always slower than that observed for the WT sequence, the region of initial nucleation for all permutants is similar to that observed for the WT protein and occurs within a similar timescale. That is, even permutants with significant sequence rearrangement in which the functional-nucleus is placed at opposing ends of the polypeptide chain, fold by the dominant WT “functional loop-packing route”, despite the entropic cost of having to fold the N- and C- termini early. Taken together, our results indicate that the early packing of the functional loops dominates the folding landscape in active proteins, and, despite the entropic penalty of coalescing the termini early, these proteins will populate an entropically unfavorable route in order to conserve function. More generally, circular permutation can elucidate the influence of local energetic stabilization of functional regions within a protein, where topological complexity creates a mismatch between energetics and topology in active proteins.

Highlights

Experimental studies using circularly permuted protein variants have given insight into the folding landscape and potential routes and mechanisms during folding for a variety of protein families [1,2,3]
Recent experiments on S6 have shown that some proteins can switch routes if an alternate folding nucleus becomes available when the native N- and C-termini are linked [6]
If a protein has one dominant folding route, it is possible that a change in the connectivity of the secondary structural elements can influence a change in folding rate [1,7,8]

Summary

Introduction

Experimental studies using circularly permuted protein variants have given insight into the folding landscape and potential routes and mechanisms during folding for a variety of protein families [1,2,3]. If a protein has one dominant folding route, it is possible that a change in the connectivity of the secondary structural elements can influence a change in folding rate [1,7,8]. Despite conservation of protein topology, we’ve shown that switching of the folding route occurs as a result of conversion of agonist to antagonist activity in IL-1b, via deletion of a functional loop [9]. This result suggests that a better understanding of the folding landscape may give novel insights into the production of designer proteins with partial agonist/ antagonist activities

Methods

Results

Conclusion