The Folding Pathway of T4 Lysozyme: The High-resolution Structure and Folding of a Hidden Intermediate

Abstract

Folding intermediates have been detected and characterized for many proteins. However, their structures at atomic resolution have only been determined for two small single domain proteins: Rdapocytochrome b 562 and engrailed homeo domain. T4 lysozyme has two easily distinguishable but energetically coupled domains: the N-and C-terminal domains. An early native-state hydrogen exchange experiment identified an intermediate with the C-terminal domain folded and the Nterminal domain unfolded. We have used a native-state hydrogen exchange-directed protein engineering approach to populate this intermediate and demonstrated that it is on the folding pathway and exists after the rate-limiting step. Here, we determined its high-resolution structure and the backbone dynamics by multi-dimensional NMR methods. We also characterized the folding behavior of the intermediate using stopped-flow fluorescence, protein engineering, and native-state hydrogen exchange. Unlike the folding intermediates of the two single-domain proteins, which have many nonnative side chain interactions, the structure of the hidden folding intermediate of T4 lysozyme is largely native-like. It folds like many small single domain proteins. These results have implications for understanding the folding mechanism and evolution of multi-domain proteins.

Biophysics of protein evolution and evolutionary protein biophysics

“…In fact, in some experiments, mutations were intentionally introduced to stabilize various folding intermediates to facilitate their characterization [84,85]. In one case, swapping certain hydrophobic core residues between two related proteins could also swap the associated folding intermediates [86].…”

High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins

“…T4L is exploited as a model system in development of SDSL technology because of the extensive data base of WTand mutant crystal structures, including many bearing the R1 side chain (19,20,23,24), as well as solution NMR (30)(31)(32) and hydrogen exchange data (33)(34)(35). The enzyme consists of two independently folding subdomains (N and C).…”

“…The much smaller ΔV o compared to that of 118R1 suggests that the equilibrium observed is not that for the N ⇌ U equilibrium, but rather an N ⇌ I equilibrium, where I is an intermediate with incomplete unfolding. It is known that the stability of the N subdomain is lower than that for the C subdomain (38) and that intermediate folding states (I) exist for T4L in which the N and C terminal subdomains are unfolded and folded, respectively (30,33,38,39). The broad transition region of the CD-detected urea denaturation curve of 46R1 also suggests a partially unfolded intermediate state (SI Text).…”

Protein folding: Then and now

“…In recent years, it has been found that even for the conventionally observed small two-state proteins (~100 amino acids or less) there exist partially unfolded intermediates on the folding pathways. These intermediates are generally undetectable in kinetic folding experiments [8,116,[164][165][166][167][168] and, therefore, are called "hidden intermediates". In addition, mounting evidence has indicated that the intermediate states formed during protein folding and unfolding may have significant roles in protein functions (Fig.…”