Experimental evaluation of topological parameters determining protein-folding rates

Abstract

Recent work suggests that structural topology plays a key role in determining protein-folding rates and pathways. The refolding rates of small proteins that fold without intermediates are found to correlate with simple structural parameters such as relative contact order, long-range order, or the fraction of short-range contacts. To test and evaluate the role of structural topology experimentally, a set of circular permutants of the ribosomal protein S6 from Thermus thermophilus was analyzed. Despite a wide range of relative contact order, the permuted proteins all fold with similar rates. These results suggest that alternative topological parameters may better describe the role of topology in proteinfolding rates.T he amino acid sequence of a protein and its chemical environment determine its native structure (1), but how this structure is determined from sequence remains one of the major unsolved problems in biology. The process of protein folding cannot be accomplished by random search through all possible conformations, because the number of structures available to an unfolded protein is too large; therefore there must be a biased or directional search in order for a protein to reach its native state. Small Two-State Proteins Show a Wide Range in Folding RatesThe simplest models for studying the protein-folding process are those that refold without intermediates (2). To date there are more than 20 examples of such simple systems. These proteins all fold cooperatively in a mono-exponential fashion from the denatured state, but the rates at which they fold span 6 orders of magnitude. What causes this remarkable variety of refolding rates? Folding Algorithms Based on the Topology of the Native State Have Predictive ValueRecently, several folding algorithms based on native structural information have been used to predict folding rates and nucleation sites (3-6). These efforts suggest that the topology of the final structure is an important determinant in the mechanism of protein folding.If native topology plays a major role in protein folding, is there a simple structural parameter that will capture this feature and explain the large range of observed folding rates? Recently, several simple parameters defining topological features of the native state have been shown to correlate well with proteinrefolding rates. The first and most commonly used parameter is relative contact order (RCO; ref. 7). Remarkably, RCO, which represents the normalized average sequence separation between contacting residues, was observed to correlate extremely well with the folding rates of a small set of two-state proteins. The lower the RCO, the faster the protein folds. Although it is possible to alter folding rates significantly through point mutations that do not change RCO, and there are proteins with similar RCOs and different folding rates (8), in general this correlation has improved as more two-state proteins have been characterized (9) and can explain the difference in folding rates among a family of structurally homologous pro...

Competition between native topology and nonnative interactions in simple and complex folding kinetics of natural and designed proteins

“…Because db enhances folding cooperativity (7,33,35), the overall barriers in the db þ hϕ S6 models are significantly higher than that in the Gō model. Consistent with experiment (14,15,28,29), these model results demonstrate a lack of two-state folding cooperativity for Top7 and two-state-like folding for S6. Native topology is apparently a dominant factor that leads to the drastically different folding thermodynamics of Top7 and S6: Among the db þ hϕ models examined in Fig.…”

“…This finding prompted us to further pursue a recent native-centric model augmented by sequence-dependent nonnative hydrophobic interactions (27) and to use an improved version of this model to investigate the interplaying roles of native topology and placement of hydrophobic residues in Top7's noncooperative folding kinetics. As a control, we applied the same model to ribosomal protein S6 (28,29), which folds much more cooperatively. Among several cooperatively folding proteins that have secondary structure elements similar to those in Top7 (e.g., acylphosphatase), we chose to study S6 because of the computational tractability engendered by its relatively fast folding rate.…”

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

“…The strategy of circular permutation has been applied to a number of small protein domains, aiming at the investigation of the stability and folding mechanisms upon change of sequence connectivity (5)(6)(7)(8)(9). The most important theoretical breakthrough in the protein folding field is generally considered the concept of funneled energy landscape (29), which suggests the denatured chain to fold to its native conformation via an energy landscape displaying an overall funneled topography.…”

“…The notion that protein folding pathways are governed by protein topology (1) has recently been challenged by ingenious experiments using topological mutants such as circularly permuted variants (5)(6)(7)(8)(9). Despite the dramatic change experienced by the primary structure, circular permutations seem well tolerated by several protein sequences.…”

Changes of Protein Folding Pathways by Circular Permutation

“…2 and 3, it is apparent that circular permutation of the S6 structure leads to significant changes of the folding process (27,31). The effect is particularly clear in ␣-helix 1 and ␤-strand 4 where the -values display consistent changes of more than 0.2 units and for the radical response at positions 75 and 88 in P [13][14] .…”