Characterization of the Folding Energy Landscapes of Computer Generated Proteins Suggests High Folding Free Energy Barriers and Cooperativity may be Consequences of Natural Selection

Protein folding has become one of the best understood biochemical reactions from a kinetic viewpoint. The funneled energy landscape, a consequence of the minimal frustration achieved by evolution in sequences, explains how most proteins fold efficiently and robustly to their functional structure and allows robust prediction of folding kinetics. The folding of Rop (repressor of primer) dimer is exceptional because some of its mutants with a redesigned hydrophobic core both fold and unfold much faster than the WT protein, which seems to conflict with a simple funneled energy landscape for which topology mainly determines the kinetics. We propose that the mystery of Rop folding can be unraveled by assuming a double-funneled energy landscape on which there are two basins that correspond to distinct but related topological structures. Because of the near symmetry of the molecule, mutations can cause a conformational switch to a nearly degenerate yet distinct topology or lead to a mixture of both topologies. The topology predicted to have the lower free-energy barrier height for folding was further found by all-atom modeling to give a better structural fit for those mutants with the extreme folding and unfolding rates. Thus, the non-Hammond effects can be understood within energy-landscape theory if there are in fact two different but nearly degenerate structures for Rop. Mutations in symmetric and regular structures may give rise to frustration and thus result in degeneracy.

Section: Discussionmentioning

confidence: 76%

Symmetry and frustration in protein energy landscapes: A near degeneracy resolves the Rop dimer-folding mystery

Levy

Cho

Shen

et al. 2005

“…Top7 is a de novo designed protein that consists of 92 residues and shows significant thermodynamic stability yet complex kinetic behavior (21,22). Top7 is a ␣/␤ protein and shows a novel protein topology (21) that has not been sampled by nature through evolution.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule force spectroscopy reveals a mechanically stable protein fold and the rational tuning of its mechanical stability

Sharma

Perišić

Peng

et al. 2007

122

135

It is recognized that shear topology of two directly connected force-bearing terminal ␤-strands is a common feature among the vast majority of mechanically stable proteins known so far. However, these proteins belong to only two distinct protein folds, Ig-like ␤ sandwich fold and ␤-grasp fold, significantly hindering delineating molecular determinants of mechanical stability and rational tuning of mechanical properties. Here we combine singlemolecule atomic force microscopy and steered molecular dynamics simulation to reveal that the de novo designed Top7 fold [Kuhlman B, Dantas G, Ireton GC, Varani G, Stoddard BL, Baker D (2003) Science 302:1364 -1368] represents a mechanically stable protein fold that is distinct from Ig-like ␤ sandwich and ␤-grasp folds. Although the two force-bearing ␤ strands of Top7 are not directly connected, Top7 displays significant mechanical stability, demonstrating that the direct connectivity of force-bearing ␤ strands in shear topology is not mandatory for mechanical stability. This finding broadens our understanding of the design of mechanically stable proteins and expands the protein fold space where mechanically stable proteins can be screened. Moreover, our results revealed a substructure-sliding mechanism for the mechanical unfolding of Top7 and the existence of two possible unfolding pathways with different height of energy barrier. Such insights enabled us to rationally tune the mechanical stability of Top7 by redesigning its mechanical unfolding pathway. Our study demonstrates that computational biology methods (including de novo design) offer great potential for designing proteins of defined topology to achieve significant and tunable mechanical properties in a rational and systematic fashion.atomic force microscopy ͉ computational design ͉ mechanical unfolding ͉ unfolding pathway P rotein mechanics is an important aspect of biology. Many proteins have evolved to sense, generate and bear mechanical forces (1) in a variety of biological processes, such as cellular adhesion (2), muscle contraction (3), and ligand-receptor interactions (4). Elastomeric proteins constitute a unique class of mechanical proteins with diverse functions ranging from molecular springs to structural materials of superb mechanical properties. Recent development in single-molecule force spectroscopy has made it possible to study the mechanical properties of proteins at singlemolecule level (5, 6). These studies not only revealed rich information about the architectural design of elastomeric proteins and shed light on the biophysical principles underpinning various biological processes, but also revealed promising prospect of using engineered elastomeric proteins for nanomechanical applications (7-9). However, in comparison to chemical and thermodynamic properties of proteins, experimental data on mechanical properties of proteins remains rather limited and molecular determinants of mechanical properties of proteins are less well understood. These factors have made it difficult or impossible to predict...

“…Generally, proteins fold cooperatively, either at the global, domain, or subglobal level (24)(25)(26). Therefore, downhill folding is likely to be limited to a few atypical systems, such as ''molecular rheostats'' (4) or, possibly, designed proteins with an unusually high hydrophobic content (27).…”

Section: Resultsmentioning

confidence: 99%

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

Meisner

Sosnick

2004

Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to ''zipper up,'' while having an extrapolated folding rate of kf ‫؍‬ 2 ؋ 10 5 s ؊1 . These properties make it likely to attain the elusive ''downhill folding'' limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. protein folding ͉ EX1 ͉ stretched exponential ͉ coiled-coil T he two-state approximation of protein folding, in which the unfolded and native states are separated by a single freeenergy barrier and no other species accumulate to a significant degree, appears to be adequate for most small proteins (1). Nevertheless, it is unsatisfying because it generally precludes identifying the individual steps involved in the folding process. To overcome this problem, experiments (2-6) have pursued the elusive theoretical prediction of ''downhill folding'' (7-9) (Fig. 1). If attainable, a downhill energy surface may allow the experimental identification of a series of intermediate states along the folding pathways that likely exists but largely has been observed only in computer simulations.Downhill-folding behavior is predicted to occur when folding rates approach the value of the attempt frequency of the barrier crossing process,Here, the barrier height does not contribute to the rate, and ⌬G ‡ Ϸ0. This limit may be likened to extreme Hammond behavior (10) in which the folding transition state moves so far to the starting condition in response to heightened stability that the transition state coincides with the unfolded state.A barrier-free reaction must be extremely rapid, but how fast is fast enough? Estimates from measured rates of helix, loop, and hairpin formation, reaction-rate and polymer-collapse theories, and folding simulations suggest that the minimum possible folding time constant is on the order of 1 s (6). Because of the increased ruggedness of the energy landscape, downhill folders of high stability would have proportionally slower folding rates, as would those with longer sequences and greater ␤-sheet content. After correcting for length and stability, 12 proteins with predicted barrier-free time constants of Ͻ100 s have been identified as potential downhill folders (6).The folding speed and stability of the covalently cross-linked variant of the dimeric yeast transcription factor GCN4 coiledcoil places it with the top members of this group (11,12). For the version GCN4p2C, with the Gly-Gly-Cys tether located at the C terminus, the extrapolated folding time constant is f Ϸ 10 s and Ͻ1 s when normalized for the high stability of the molecule (6). This fast folding rate may be due to a simple topology that requires that the helices only ''zipper-up.'' These qualities identify it among the most likely candidates for downhill folding. Here, we invest...