Following co-operative formation of secondary and tertiary structure in a single protein module

Self Cite

112

Experimental data from protein engineering studies and NMR spectroscopy have been used by theoreticians to develop algorithms for helix propensity and to benchmark computer simulations of folding pathways and energy landscapes. Molecular dynamic simulations of the unfolding of chymotrypsin inhibitor 2 (CI2) have provided detailed structural models of the transition state ensemble for unfolding͞folding of the protein. We now have used the simulated transition state structures to design faster folding mutants of CI2. The models pinpoint a number of unfavorable local interactions at the carboxyl terminus of the single ␣-helix and in the protease-binding loop region of CI2. By removing these interactions or replacing them with stabilizing ones, we have increased the rate of folding of the protein up to 40-fold ( ‫؍‬ 0.4 ms). This correspondence, and other examples of agreement between experiment and theory in general, ⌽-values and molecular dynamics simulations, in particular, suggest that significant progress has been made toward describing complete folding pathways at atomic resolution by combining experiment and simulation.

Section: Resultsmentioning

confidence: 96%

Synergy between simulation and experiment in describing the energy landscape of protein folding

Ladurner

Itzhaki

Daggett

et al. 1998

Self Cite

112

“…These patches of structure do not exist in the denatured state (Figs. 1 and 5), and fragment studies show that there is little drive for such structure (10,32). Instead, the nucleation site remains embryonic until sufficient long-range contacts are made (the nucleation-condensation mechanism).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, the denatured state of CI2 appears to be largely unstructured as probed by NMR studies of various fragments (9)(10)(11). However, there appears to be some tendency for very weak native helical structure and some weak clustering of hydrophobic residues, particularly near the center of the protein.…”

mentioning

confidence: 99%

Protein folding from a highly disordered denatured state: The folding pathway of chymotrypsin inhibitor 2 at atomic resolution

Kazmirski

Wong

Freund

et al. 2001

Self Cite

159

153

Previous experimental and theoretical studies have produced highresolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation-condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding͞ unfolding pathway of CI2 at atomic resolution.CI2 ͉ nuclear magnetic resonance ͉ molecular dynamics simulations ͉ conformational transitions ͉ nucleation-condensation P rotein folding is a rapid and complex process that is difficult to characterize. To add to this difficulty, the denatured state consists of a large ensemble of conformations interconverting at a rapid rate. The denatured state is often assumed to be devoid of intramolecular interactions, such that the stability of a protein can be explained purely in terms of interactions in the native state. In recent years, it has become apparent that many proteins contain residual structure in the denatured state (ref. 1 and refs. therein). However, detailed characterization of this structure is very challenging, if not impossible in many cases. As such, during folding one follows the transition of a diverse system from an unknown starting point to a well-ordered native state. Further information about the diversity, dynamics, and structure of the denatured state is necessary to characterize and understand better this process.The simplest folding pathway to define is two state, i.e., involving only the denatured and native states, which are separated by the energetically unfavorable transition state. Chymotrypsin inhibitor 2 (CI2) was the first protein shown to fold by a two-state mechanism, and it has since been the focus of a number of experimental and theoretical studies. It is a 64-residue protein that consists of an ␣-helix and a three-stranded ␤-sheet (Fig. 1). The main hydrophobic core is formed by the packing of the ␣-helix against the ␤-sheet.Experimentally, the structure of the transition state has been studied by the protein engineering (⌽-value) method (2). In combination with molecular dynamics (MD) simulations, an atomic-resolution model of the transition state has been proposed (3-7) and verified (8). The rate-limiting step for the folding of CI2 involves the final expulsion of water molecules from the exposed nonpolar side chains and the tight packing of the hydrophobic core. The transition state is similar to an expanded native state with some disruption of the secondary structure.In contrast, the denatured state of CI2 appears to be largely unstructured as probed by NMR studies of variou...

“…Denatured state. Experimental NMR studies of protein fragments demonstrate that in denatured conditions CI2 is highly unstructured (43,44), with a very slight tendency for the native helical structure and a minor hydrophobic clustering near the center of the chain. Likewise, a compilation of NMR experiments and MD simulations on a full-length CI2 indicate a highly unfolded denatured state with the residual structure mentioned above (7).…”

Section: Ci2mentioning

confidence: 99%

Characterization of protein-folding pathways by reduced-space modeling

Kmiecik

Koliński²

2007

102

Ab initio simulations of the folding pathways are currently limited to very small proteins. For larger proteins, some approximations or simplifications in protein models need to be introduced. Protein folding and unfolding are among the basic processes in the cell and are very difficult to characterize in detail by experiment or simulation. Chymotrypsin inhibitor 2 (CI2) and barnase are probably the best characterized experimentally in this respect. For these model systems, initial folding stages were simulated by using CA-CB-side chain (CABS), a reduced-space protein-modeling tool. CABS employs knowledge-based potentials that proved to be very successful in protein structure prediction. With the use of isothermal Monte Carlo (MC) dynamics, initiation sites with a residual structure and weak tertiary interactions were identified. Such structures are essential for the initiation of the folding process through a sequential reduction of the protein conformational space, overcoming the Levinthal paradox in this manner. Furthermore, nucleation sites that initiate a tertiary interactions network were located. The MC simulations correspond perfectly to the results of experimental and theoretical research and bring insights into CI2 folding mechanism: unambiguous sequence of folding events was reported as well as cooperative substructures compatible with those obtained in recent molecular dynamics unfolding studies. The correspondence between the simulation and experiment shows that knowledge-based potentials are not only useful in protein structure predictions but are also capable of reproducing the folding pathways. Thus, the results of this work significantly extend the applicability range of reduced models in the theoretical study of proteins.protein structure prediction ͉ Monte Carlo simulations ͉ protein denatured state ͉ folding nucleus ͉ residual structure A large number of folded protein structures was determined by x-ray crystallography or NMR. For a few proteins, the folding intermediates were characterized by using protein engineering (1) and NMR techniques (2, 3). The major transition states (TS) of chymotrypsin inhibitor 2 (CI2) and barnase were mapped at the level of individual residues by protein engineering (4, 5). Much less is known, however, about early folding events. It is very important to understand how protein folding is initiated and how the native structure emerges subsequently. The denatured state, an ensemble of partially folded, highly mobile conformations, is very difficult to study, although there have been recent reports of NMR studies of residual structure in denatured proteins. Such structures, along with hydrophobic clusters, were discovered even under highly denaturing conditions (6, 7). Moreover, denatured proteins can exhibit a longrange ordering of native-like topology (8). Therefore, the folding process can be directed from the very beginning when starting from a specific structure (9, 10). It becomes evident that the denatured state plays a crucial role in all aspects of protein ...