Protein folding and misfolding: mechanism and principles

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.     

Folding funnels, binding funnels, and protein function.

Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly, with a range of complexed conformations. Hence, knowledge of the shape of the folding funnels is biologically very useful. The converse also holds: If kinetic and thermodynamic data are available, hints regarding the role of the protein and its binding selectivity may be obtained. Thus, the utility of the concept of the funnel carries over to the origin of the protein and to its function.     

Protein folding simulation with solvent-induced force field: folding pathway ensemble of three-helix-bundle proteins

We propose a coarse-grained model of proteins that take into account solvent effects and apply it for simulating folding of a three-helix-bundle protein. The energy functional form, refined from our previous work (Takada et al., J Chem Phys 1999;110:11616-11629), tries to closely imitate real physico-chemical interactions. In particular, the hydrogen bond that depends on local dielectric constant, the helix capping effect, and side-chain entropic effects are included. With use of the model, we simulate folding of the GA module of an albumin binding domain, 1prb(7-53), finding most trajectories reach at the native topology within 1 micros. In the simulation, helices 1 and 3 are mostly formed earlier accompanied by non-specific collapse, while second helix is intrinsically less stable and is formed with the help of tertiary contacts at later stage. We compute an analog of the transition state ensemble and compare it with those of other three-helix-bundle proteins. The transition state of 1prb(7-53) includes a few specific tertiary contacts of C terminus of helix 3 with the loop region between helices 1 and 2. This resembles, but is not equivalent to, an early formed region of fragment B of staphylococcal protein A, but is quite different from the folding transient structures of a de novo designed three-helix-bundle peptide.     

Folding funnels and binding mechanisms.

The long-held views on lock-and-key versus induced fit in binding arose from the notion that a protein exists in a single, most stable conformation, dictated by its sequence. However, in solution proteins exist in a range of conformations, which may be described by statistical mechanical laws and their populations follow statistical distributions. Upon binding, the equilibrium will shift in favor of the bound conformation from the ensemble of conformations around the bottom of the folding funnel. Hence here we extend the implications and the usefulness of the folding funnel concept to explain fundamental binding mechanisms.     

Entropic barriers, transition states, funnels, and exponential protein folding kinetics: a simple model

This paper presents an analytically tractable model that captures the most elementary aspect of the protein folding problem, namely that both the energy and the entropy decrease as a protein folds. In this model, the system diffuses within a sphere in the presence of an attractive spherically symmetric potential. The native state is represented by a small sphere in the center, and the remaining space is identified with unfolded states. The folding temperature, the time-dependence of the populations, and the relaxation rate are calculated, and the folding dynamics is analyzed for both golf-course and funnel-like energy landscapes. This simple model allows us to illustrate a surprising number of concepts including entropic barriers, transition states, funnels, and the origin of single exponential relaxation kinetics.     

Protein modeling with reduced representation: statistical potentials and protein folding mechanism

A high resolution reduced model of proteins is used in Monte Carlo dynamics studies of the folding mechanism of a small globular protein, the B1 immunoglobulin-binding domain of streptococcal protein G. It is shown that in order to reproduce the physics of the folding transition, the united atom based model requires a set of knowledge-based potentials mimicking the short-range conformational propensities and protein-like chain stiffness, a model of directional and cooperative hydrogen bonds, and properly designed knowledge-based potentials of the long-range interactions between the side groups. The folding of the model protein is cooperative and very fast. In a single trajectory, a number of folding/unfolding cycles were observed. Typically, the folding process is initiated by assembly of a native-like structure of the C-terminal hairpin. In the next stage the rest of the four-ribbon beta-sheet folds. The slowest step of this pathway is the assembly of the central helix on the scaffold of the beta-sheet.     

A common folding mechanism in the cytochrome c family

Of the globular proteins, cytochrome c (cyt c) has been used extensively as a model system for folding studies. Here we analyse the folding pathway of different cyt c proteins from prokaryotes and eukaryotes, and attempt to single out general correlations between structural determinants and folding mechanisms. Recent studies provide evidence that the folding pathway of several cyt c proteins involves the formation of a partially structured intermediate. Using state-of-the-art kinetic analysis on published data, we show that such a folding intermediate is an obligatory on-pathway species that might represent either a defined local minimum in the reaction coordinate or an unstable high-energy state. Available data also indicate that some essential structural features of the folding intermediate and transition states are highly conserved across this protein family. Thus, cyt c proteins share a consensus folding mechanism in spite of large differences in physico-chemical properties and thermodynamic stability. This novel outlook on the folding of cyt c can shed light on much published data and might offer a general scheme by which to plan new experiments.     

Protein folding and binding in confined spaces and in crowded solutions

Simple theoretical models are presented to illustrate the effects of spatial confinement and macromolecular crowding on the equilibria and rates of protein folding and binding. Confinement is expected to significantly stabilize the folded state, but for crowding only a marginal effect on protein stability is expected. In confinement the unfolded chain is restricted to a cage but in crowding the unfolded chain may explore different interstitial voids. Because confinement and crowding eliminate the more expanded conformations of the unfolded state, folding from the compact unfolded state is expected to speed up. Crowding will shift the binding equilibrium of proteins toward the bound state. The significant slowing down in protein diffusion by crowding, perhaps beneficial for chaperonin action, could result in a decrease in protein binding rates.     

Dynamics of protein folding and cofactor binding monitored by single-molecule force spectroscopy

Many proteins in living cells require cofactors to carry out their biological functions. To reach their functional states, these proteins need to fold into their unique three-dimensional structures in the presence of their cofactors. Two processes, folding of the protein and binding of cofactors, intermingle with each other, making the direct elucidation of the folding mechanism of proteins in the presence of cofactors challenging. Here we use single-molecule atomic force microscopy to directly monitor the folding and cofactor binding dynamics of an engineered metal-binding protein G6-53 at the single-molecule level. Using the mechanical stability of different conformers of G6-53 as sensitive probes, we directly identified different G6-53 conformers (unfolded, apo- and Ni²⁺-bound) populated along the folding pathway of G6-53 in the presence of its cofactor Ni²⁺. By carrying out single-molecule atomic force microscopy refolding experiments, we monitored kinetic evolution processes of these different conformers. Our results suggested that the majority of G6-53 folds through a binding-after-folding mechanism, whereas a small fraction follows a binding-before-folding pathway. Our study opens an avenue to utilizing force spectroscopy techniques to probe the folding dynamics of proteins in the presence of cofactors at the single-molecule level, and we anticipated that this method can be used to study a wide variety of proteins requiring cofactors for their function.     

Exploring structures in protein folding funnels with free energy functionals: the denatured ensemble

We discuss the formulation of free energy functionals that describe the formation of structure in partially folded proteins. These free energy functionals take into account the inhomogeneous nature of contact energies, chain entropy and cooperative contributions reflecting the many body character of some folding forces like hydrophobicity, but do not directly account for non-native contacts because they assume the validity of the minimal frustration principle. We show how the free energy functionals can be used to interpret experiments on partially folded proteins that probe the fractional occupancy of specific local structures. In particular, we study the hydrogen protection factors in lysozyme studied in transient experiments by Gladwin and Evans and by Nash and Jonas using equilibrium pressure denaturation and the NMR order parameters measured by Dobson and Kim for the homologous protein alpha-lactalbumin.     

Membrane traffic: a driving force in cytokinesis

Dividing animal and plant cells maintain a constant chromosome content through temporally separated rounds of replication and segregation. Until recently, the mechanisms by which animal and plant cells maintain a constant surface area have been considered to be distinct. The prevailing view was that surface area was maintained in dividing animal cells through temporally separated rounds of membrane expansion and membrane invagination. The latter event, known as cytokinesis, produces two physically distinct daughter cells and has been thought to be primarily driven by actomyosin-based constriction. By contrast, membrane addition seems to be the primary mechanism that drives cytokinesis in plants and, thus, the two events are linked mechanistically and temporally. In this article (which is part of the Cytokinesis series), we discuss recent studies of a variety of organisms that have made a convincing case for membrane trafficking at the cleavage furrow being a key component of both animal and plant cytokinesis.     

Transduction of enzyme-ligand binding energy into catalytic driving force.

We propose a testable general mechanism by which ligand binding energy can be used to drive a catalytic step in an enzyme catalyzed reaction or to do other forms of work involving protein molecules. This energy transduction theory is based on our finding of the widespread occurrence of ligand binding-induced protein macrostate interconversions each having a large invariant delta H0 accompanied by a small but highly variable delta G0. This phenomenon, which can be recognized by the large delta Cp0's it generates, can provide the necessary energy input step but is not in itself sufficient to constitute a workable transduction mechanism. A viable mechanism requires the additional presence of an 'energy transmission step' which is terminated to trigger the 'power' stroke at a precise location on the reaction coordinate, followed by an energetically inexpensive 'return' step to restore the machine to its initial conditions. In the model we propose here, these additional steps are provided by the existence of ligand inducible 2-state transitions in the free enzyme and in each of the enzyme complexes that occur along the reaction coordinate, and by the selective blocking of certain of these interconversions by high energetic barriers. We provide direct experimental evidence supporting the facts that these additional mechanistic components do exist and that the liver glutamate dehydrogenase reaction is indeed driven by just such machinery. We describe some aspects of the chemical nature of these transitions, and evidence for their occurrence in other systems.