Mallela M.G. Krishna

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.

Cooperative Omega Loops in Cytochrome c: Role in Folding and Function

Hydrogen exchange experiments under slow exchange conditions show that an omega loop in cytochrome c (residues 40-57) acts as a cooperative unfolding/refolding unit under native conditions. This unit behavior accounts for an initial step on the unfolding pathway, a final step in refolding, and a number of other structural, functional and evolutionary properties.

Cytochrome c folding pathway: Kinetic native-state hydrogen exchange

Native-state hydrogen exchange experiments under EX1 conditions can distinguish partially unfolded intermediates by their formation rates and identify the amide hydrogens exposed and protected in each. Results obtained define a cytochrome c intermediate seen only poorly before and place it early on the major unfolding pathway. Four distinct unfolding steps are found to be kinetically ordered in the same pathway sequence inferred before.

Folding units govern the cytochrome c alkaline transition.

The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.

A unified mechanism for protein folding: Predetermined pathways with optional errors

There is a fundamental conflict between two different views of how proteins fold. Kinetic experiments and theoretical calculations are often interpreted in terms of different population fractions folding through different intermediates in independent unrelated pathways (IUP model). However, detailed structural information indicates that all of the protein population folds through a sequence of intermediates predetermined by the foldon substructure of the target protein and a sequential stabilization principle. These contrary views can be resolved by a predetermined pathway—optional error (PPOE) hypothesis. The hypothesis is that any pathway intermediate can incorporate a chance misfolding error that blocks folding and must be reversed for productive folding to continue. Different fractions of the protein population will then block at different steps, populate different intermediates, and fold at different rates, giving the appearance of multiple unrelated pathways. A test of the hypothesis matches the two models against extensive kinetic folding results for hen lysozyme which have been widely cited in support of independent parallel pathways. The PPOE model succeeds with fewer fitting constants. The fitted PPOE reaction scheme leads to known folding behavior, whereas the IUP properties are contradicted by experiment. The appearance of a conflict with multipath theoretical models seems to be due to their different focus, namely on multitrack microscopic behavior versus cooperative macroscopic behavior. The integration of three well‐documented principles in the PPOE model (cooperative foldons, sequential stabilization, optional errors) provides a unifying explanation for how proteins fold and why they fold in that way.

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway–optional error (PPOE) model but contrary to the properties implied in IUP models.

Branching in the sequential folding pathway of cytochrome c

Previous results indicate that the folding pathways of cytochrome c and other proteins progressively build the target native protein in a predetermined stepwise manner by the sequential formation and association of native‐like foldon units. The present work used native state hydrogen exchange methods to investigate a structural anomaly in cytochrome c results that suggested the concerted folding of two segments that have little structural relationship in the native protein. The results show that the two segments, an 18‐residue omega loop and a 10‐residue helix, are able to unfold and refold independently, which allows a branch point in the folding pathway. The pathway that emerges assembles native‐like foldon units in a linear sequential manner when prior native‐like structure can template a single subsequent foldon, and optional pathway branching is seen when prior structure is able to support the folding of two different foldons.

Translational diffusion of fluorescent probes on a sphere: Monte Carlo simulations, theory, and fluorescence anisotropy experiment

Translational diffusion of fluorescent molecules on curved surfaces (micelles, vesicles, and proteins) depolarizes the fluorescence. A Monte Carlo simulation method was developed to obtain the fluorescence anisotropy decays for the general case of molecular dipoles tilted at an angle α to the surface normal. The method is used to obtain fluorescence anisotropy decay due to diffusion of tilted dipoles on a spherical surface, which matched well with the exact solution for the sphere. The anisotropy decay is a single exponential for α=0°, a double exponential for α=90°, and three exponentials for intermediate angles. The slower decay component(s) for α≠0 arise due to the geometric phase factor. Although the anisotropy decay equation contains three exponentials, there are only two parameters, namely α and the rate constant, Dtr/R2, where Dtr is the translational diffusion coefficient and R is the radius of the sphere. It is therefore possible to determine the orientation angle and translational diffusion coef...

Rotational dynamics of surface probes in lipid vesicles

Translational and rotational diffusion of fluorescent molecules on the surface of small biological systems such as vesicles, proteins and micelles depolarize the fluorescence. A recent study has treated the case of the translational dynamics of surface probes (M.M.G. Krishna, R. Das, N. Periasamy and R. Nityananda, J. Chem. Phys., 112 (2000) 8502-8514) using Monte Carlo and theoretical methods. Here we extend the application of the methodologies to apply the case of rotational dynamics of surface probes. The corresponding fluorescence anisotropy decays were obtained using the Monte Carlo simulation methods for the two cases: surface probes undergoing rotational dynamics on a plane and on a sphere. The results were consistent with the theoretical equations which show that Monte Carlo methods can be used to simulate the surface diffusion problems. The anisotropy decay for the rotational diffusion of a molecule on a planar surface is single exponential and the residual anisotropy is zero. However, residual anisotropy is finite for the case of rotational diffusion on a sphere because of the spatial averaging of the anisotropy function. The rotational correlation time in both the cases is (4Drot)(-1) with Drot being the rotational diffusion coefficient. Rotational dynamics of a surface bound dye in a single giant liposome and in sonicated vesicles were studied and the results were explained according to the theoretical equations. A fast component of fluorescence depolarization was also observed for sonicated vesicles which was interpreted as wobbling-in-cylinder dynamics of the surface-bound dye.

Location and orientation of DODCI in lipid bilayer membranes: effects of lipid chain length and unsaturation

The location and orientation of a linear dye molecule, DODCI, in lipid bilayer membrane were determined by the effect of viscosity and refractive index of the aqueous medium on the fluorescence properties of the dye bound to the membrane. The membrane-bound dye is solubilized in two sites, one near the surface (short fluorescence lifetime) and another in the interior of the membrane (long lifetime). The ratio of the dye in the two locations and the orientation of the dye (parallel or perpendicular to the membrane) are sensitive to the lipid chain length and unsaturation in the alkyl chain. The fraction of the dye in the interior region is higher for short alkyl chains (C12>C14>C16>>C18C20) and in unsaturated lipids (C14:1>C14:0, C16:1>C16:0). These experimental results are consistent with the general principle that the penetration of an amphiphilic organic molecule in the interior region of the membrane is more when the structure of th bilayer is more fluid-like.