Who solved the protein folding problem?

For the third time, techniques for the prediction of three-dimensional structures of proteins were critically assessed in a worldwide blind test. Steady progress is undeniable. How did this happen and what are the implications?     

Is the unfolded state the Rosetta Stone of the protein folding problem?

Solving the protein folding problem is one of the most challenging tasks in the post genomic era. Identification of folding-initiation sites is very important in order to understand the protein folding mechanism. Detection of residual structure in unfolded proteins can yield important clues to the initiation sites in protein folding. A substantial number of studied proteins possess residual structure in hydrophobic regions clustered together in the protein core. These stable structures can work as seeds in the folding process. In addition, local preferences for secondary structure in the form of turns for beta-sheet initiation and helical turns for alpha-helix formation can guide the folding reaction. In this respect the unfolded states, studied at increasing structural resolution, can be the Rosetta Stone of the protein folding problem.     

Are synthetic proteins relevant to the problem of protein folding?

The possibility to derive the analogs of native proteins by the chemical synthesis is considered to be a serious argument for the concept of posttranslational protein folding. The present paper analyzes for the first time chemically synthesized proteins to reveal whether they are relevant to the problem of protein folding. The results enable the following conclusions to be drawn. The acquisition of the peculiar conformations by the chemically synthesized proteins to exhibit the specific functions is conditioned by the highly marked features of the secondary and tertiary structures of the corresponding native proteins. These features will make themselves evident only if favorable conditions are carefully chosen during the experiments for each individual protein. Thus, in our opinion, the possibility to derive a synthetic protein is hardly evidence for the posttranslational folding of proteins.     

The protein folding problem: when will it be solved?

The protein folding problem can be viewed as three different problems: defining the thermodynamic folding code; devising a good computational structure prediction algorithm; and answering Levinthal's question regarding the kinetic mechanism of how proteins can fold so quickly. Once regarded as a grand challenge, protein folding has seen much progress in recent years. Folding codes are now being used to successfully design proteins and non-biological foldable polymers; aided by the Critical Assessment of Techniques for Structure Prediction (CASP) competition, protein structure prediction has now become quite good. Even the once-challenging Levinthal puzzle now seems to have an answer--a protein can avoid searching irrelevant conformations and fold quickly by making local independent decisions first, followed by non-local global decisions later.     

Is protein folding rate dependent on number of folding stages? Modeling of protein folding with ferredoxin-like fold

Statistical analysis of protein folding rates has been done for 84 proteins with available experimental data. A surprising result is that the proteins with multi-state kinetics from the size range of 50–100 amino acid residues (a.a.) fold as fast as proteins with two-state kinetics from the same size range. At the same time, the proteins with two-state kinetics from the size range 101–151 a.a. fold faster than those from the size range 50–100 a.a. Moreover, it turns out unexpectedly that usually in the group of structural homologs from the size range 50–100 a.a., proteins with multi-state kinetics fold faster than those with two-state kinetics. The protein folding for six proteins with a ferredoxin-like fold and with a similar size has been modeled using Monte Carlo simulations and dynamic programming. Good correlation between experimental folding rates, some structural parameters, and the number of Monte Carlo steps has been obtained. It is shown that a protein with multi-state kinetics actually folds three times faster than its structural homologs.     

Is protein unfolding the reverse of protein folding? A lattice simulation analysis.

Simulations and experiments that monitor protein unfolding under denaturing conditions are commonly employed to study the mechanism by which a protein folds to its native state in a physiological environment. Due to the differences in conditions and the complexity of the reaction, unfolding is not necessarily the reverse of folding. To assess the relevance of temperature initiated unfolding studies to the folding problem, we compare the folding and unfolding of a 125-residue protein model by Monte Carlo dynamics at two temperatures; the lower one corresponds to the range used in T -jump experiments and the higher one to the range used in unfolding simulations of all-atom models. The trajectories that lead from the native state to the denatured state at these elevated temperatures are less diverse than those observed in the folding simulations. At the lower temperature, the system unfolds through a mandatory intermediate that corresponds to a local free energy minimum. At the higher temperature, no such intermediate is observed, but a similar pathway is followed. The structures contributing to the unfolding pathways resemble most closely those that make up the "fast track" of folding. The transition state for unfolding at the lower temperature (above Tm) is determined and is found to be more structured than the transition state for folding below the melting temperature. This shift towards the native state is consistent with the Hammond postulate. The implications for unfolding simulations of higher resolution models and for unfolding experiments of proteins are discussed.     

How do cofactors modulate protein folding?

Cofactors are essential components of many proteins for biological activity. Characterization of several cofactor-binding proteins has shown that cofactors often have the ability to interact specifically with the unfolded polypeptides. This suggests that cofactor-coordination prior to polypeptide folding may be a relevant path in vivo. By binding before folding, the cofactor may affect both the mechanism and speed of folding. Here, we discuss three different cofactors that modulate protein-folding processes in vitro.     

Is protein folding hierarchic? I. Local structure and peptide folding

The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Nevertheless, we argue that both classes fold hierarchically and that folding begins locally. If this is the case, then the secondary structure of a protein is determined largely by local sequence information. Experimental data and theoretical considerations support this argument. Part I of this article reviews the relationship between secondary structures in proteins and their counterparts in peptides.     

Is there a unifying mechanism for protein folding?

Proteins appear to fold by diverse pathways, but variations of a simple mechanism - nucleation-condensation - describe the overall features of folding of most domains. In general, secondary structure is inherently unstable and its stability is enhanced by tertiary interactions. Consequently, an extensive interplay of secondary and tertiary interactions determines the transition-state for folding, which is structurally similar to the native state, being formed in a general collapse (condensation) around a diffuse nucleus. As the propensity for stable secondary structure increases, folding becomes more hierarchical and eventually follows a framework mechanism where the transition state is assembled from pre-formed secondary structural elements.     

How cooperative are protein folding and unfolding transitions?

A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two‐state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non‐cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier‐less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non‐cooperative processes may be ubiquitous features on the free energy landscape of protein folding.     

Membrane protein folding: how important are hydrogen bonds?

Water is an inhospitable environment for protein hydrogen bonds because it is polarizable and capable of forming competitive hydrogen bonds. In contrast, the apolar core of a biological membrane seems like an ideal environment for hydrogen bonds, and it has long been assumed that hydrogen bonding should be a powerful force driving membrane protein folding. Nevertheless, while backbone hydrogen bonds may be much stronger in membrane proteins, experimental measurements indicate that side chain hydrogen bond strengths are not strikingly different in membrane and water soluble proteins. How is this possible? I argue that model compounds in apolar solvents do not adequately describe the system because the protein itself is ignored. The protein chain provides a rich source of competitive hydrogen bonds and a polarizable environment that can weaken hydrogen bonds. Thus, just like water soluble proteins, evolution can drive the creation of potent hydrogen bonds in membrane proteins where necessary, but mitigating forces in their environment must still be overcome.