Co-populated Conformational Ensembles of β(2)-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry

Ordered assembly of monomeric human β(2)-microglobulin (β(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 °C), wild-type β(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, β(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of β(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of β(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid

Microscopic Theory of Protein Folding Rates.I: Fine Structure of the Free Energy Profile and Folding Routes from a Variational Approach

A microscopic theory of the free energy barriers and folding routes for minimally frustrated proteins is presented, greatly expanding on the presentation of the variational approach outlined previously [J. J. Portman, S. Takada, P. G. Wolynes, Phys. Rev. Lett. {\bf 81}, 5237 (1998)]. We choose the $\lambda$-repressor protein as an illustrative example and focus on how the polymer chain statistics influence free energy profiles and partially ordered ensembles of structures. In particular, we investigate the role of chain stiffness on the free energy profile and folding routes. We evaluate the applicability of simpler approximations in which the conformations of the protein molecule along the folding route are restricted to have residues that are either entirely folded or unfolded in contiguous stretches. We find that the folding routes obtained from only one contiguous folded region corresponds to a chain with a much greater persistence length than appropriate for natural protein chains, while the folding route obtained from two contiguous folded regions is able to capture the relatively folded regions calculated within the variational approach. The free energy profiles obtained from the contiguous sequence approximations have larger barriers than the more microscopic variational theory which is understood as a consequence of partial ordering.Comment: 16 pages, 11 figure

Mechanisms underlying sequence-independent beta-sheet formation

We investigate the formation of beta-sheet structures in proteins without taking into account specific sequence-dependent hydrophobic interactions. To accomplish this, we introduce a model which explicitly incorporates both solvation effects and the angular dependence (on the protein backbone) of hydrogen bond formation. The thermodynamics of this model is studied by comparing the restricted partition functions obtained by "unfreezing" successively larger segments of the native beta-sheet structure. Our results suggest that solvation dynamics together with the aforementioned angular dependence gives rise to a generic cooperativity in this class of systems; this result explains why pathological aggregates involving beta-sheet cores can form from many different proteins. Our work provides the foundation for the construction of phenomenological models to investigate the competition between native folding and non-specific aggregation.Comment: 20 pages, 5 figures, Revtex4, simulation mpeg movie available at http://www-physics.ucsd.edu/~guochin/Images/sheet1.mp

Protein folding tames chaos

Protein folding produces characteristic and functional three-dimensional structures from unfolded polypeptides or disordered coils. The emergence of extraordinary complexity in the protein folding process poses astonishing challenges to theoretical modeling and computer simulations. The present work introduces molecular nonlinear dynamics (MND), or molecular chaotic dynamics, as a theoretical framework for describing and analyzing protein folding. We unveil the existence of intrinsically low dimensional manifolds (ILDMs) in the chaotic dynamics of folded proteins. Additionally, we reveal that the transition from disordered to ordered conformations in protein folding increases the transverse stability of the ILDM. Stated differently, protein folding reduces the chaoticity of the nonlinear dynamical system, and a folded protein has the best ability to tame chaos. Additionally, we bring to light the connection between the ILDM stability and the thermodynamic stability, which enables us to quantify the disorderliness and relative energies of folded, misfolded and unfolded protein states. Finally, we exploit chaos for protein flexibility analysis and develop a robust chaotic algorithm for the prediction of Debye-Waller factors, or temperature factors, of protein structures

Molecular Dynamics Studies on HIV-1 Protease: Drug Resistance and Folding Pathways

Drug resistance to HIV-1 Protease involves accumulation of multiple mutations in the protein. Here we investigate the role of these mutations by using molecular dynamics simulations which exploit the influence of the native-state topology in the folding process. Our calculations show that sites contributing to phenotypic resistance of FDA-approved drugs are among the most sensitive positions for the stability of partially folded states and should play a relevant role in the folding process. Furthermore, associations between amino acid sites mutating under drug treatment are shown to be statistically correlated. The striking correlation between clinical data and our calculations suggest a novel approach to the design of drugs tailored to bind regions crucial not only for protein function but also for folding.Comment: Revtex, 14 pages, 7 eps figures. Proteins, Structure Function and Genetics, in press (2001

Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain. A study using electrospray ionization mass spectrometry and fluorescence binding studies

Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer

The denatured state of N-PGK is compact and predominantly disordered

The Organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I-b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I-k) and denatured (D)]. The transition to I-b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I-b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I-b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics Of I-b indicate that it forms from the fully denatured state within 100 mu s, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I-b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology. (C) 2008 Elsevier Ltd. All rights reserved

Hsp70 in mitochondrial biogenesis

The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology, Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, protein translocation across mitochondrial membranes, protein folding and finally the delivery of misfolded proteins to proteolytic enzymes in the mitochondrial matrix