Protein folding and misfolding: mechanism and principles

Abstract

Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding. The protein folding problemThe search for protein folding pathways and the principles that guide them has proven to be one of the most difficult problems in all of structural biology. Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates only live for <1 s and they cannot be isolated and studied by the usual structural methods.The protein folding problem has been attacked by the entire range of fast spectroscopic methods which are able to track folding in real time. Much valuable information has been obtained but mainly of a kinetic nature. Kinetic methods do not describe the structure of the

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

“…The folding model pictured in Fig. 5B is identical to the extensively worked out case of cytochrome c for which a series of HX NMR studies defined four native-like foldon-based intermediates in a well-ordered pathway (24)(25)(26). It also is consistent with the finding of individual nativelike intermediates in many other proteins.…”

“…In the initial on-pathway step, a limited structural unit is assembled by a relatively unguided amino acid search (59), which makes the first on-pathway step the intrinsically slow step. In subsequent steps in which amino acid searching can be informed by existing structure, a faster assisted folding mode emerges called "sequential stabilization" (25,60). Like the process of coupled folding upon binding (61)(62)(63)(64), the prior structure will selectively guide and stabilize the units with which it interacts in the native protein.…”

“…It seems likely that the concurrent interfoldon interactions produced by the two factors just considered could provide the required degree of selectivity, whereas individual residue-level interactions would not. When the bias toward native interactions is insufficient, probabilistic misfolding is likely to occur (25).…”

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

“…Reynolds and coworkers showed that (1) the folding rate correlates extremely closely with total contact distance evaluated only over the lock residues, and (2) the lock residues tend to have high ϕ-values, "as would be expected for residues that play an important role in the transition structure for folding" (Chintapalli et al 2010). Reynolds further suggests that the closed loop hypothesis is able to give an alternative description of the data obtained by Englander's group (Bai et al 1995;Hoang et al 2003;Englander et al 2007) for cytochromes c and b562 (as well as for triosephosphate-isomerase) initially interpreted in terms of independent folding units (foldons). The closed loop hypothesis-based mechanism was said to be "as elegant as the published explanations as it does not invoke discontinuous foldons."…”

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

“…The two-state model is an invaluable tool in protein folding studies, and has provided significant insight into how both local and global properties of proteins influence their folding rates. [1][2][3] However, as a result of its simplicity, the two-state model provides only a snapshot (often through inferences made by perturbing the transition state ensemble 4 ; of the folding pathways that connect D to N, 5,6 and does not address kinetic features such as transient intermediates and kinetic traps. 7 Moreover, description of heterogeneity among partly folded states and folding via parallel pathways are both outside the confines of the two-state approximation.…”