Molecular dynamics study of water channels in natural and synthetic amyloid-ß fibrils.

We compared all-atom explicit solvent molecular dynamics simulations of three types of Aß(1-40) fibrils: brain-seeded fibrils (2M4J, with a threefold axial symmetry) and the other two, all-synthetic fibril polymorphs (2LMN and 2LMP, made under different fibrillization conditions). Fibril models were constructed using either a finite or an infinite number of layers made using periodic images. These studies yielded four conclusions. First, finite fibrils tend to unravel in a manner reminiscent of fibril dissolution, while infinite fibrils were more stable during simulations. Second, salt bridges in these fibrils remained stable in those fibrils that contained them initially, and those without salt bridges did not develop them over the time course of the simulations. Third, all fibrils tended to develop a "stagger" or register shift of ß-strands along the fibril axis. Fourth and most importantly, the brain-seeded, 2M4J, infinite fibrils allowed bidirectional transport of water in and out of the central longitudinal core of the fibril by rapidly developing gaps at the fibril vertices. 2LMP fibrils also showed this behavior, although to a lesser extent. The diffusion of water molecules in the fibril core region involved two dynamical states: a localized state and directed diffusion in the presence of obstacles. These observations provided support for the hypothesis that Aß fibrils could act as nanotubes. At least some Aß oligomers resembled fibrils structurally in having parallel, in-register ß-sheets and a sheet-turn-sheet motif. Thus, our findings could have implications for Aß cytotoxicity, which may occur through the ability of oligomers to form abnormal water and ion channels in cell membranes.

Ionic strength independence of charge distributions in solvation of biomolecules.

Electrostatic forces enormously impact the structure, interactions, and function of biomolecules. We perform all-atom molecular dynamics simulations for 5 proteins and 5 RNAs to determine the dependence on ionic strength of the ion and water charge distributions surrounding the biomolecules, as well as the contributions of ions to the electrostatic free energy of interaction between the biomolecule and the surrounding salt solution (for a total of 40 different biomolecule/solvent combinations). Although water provides the dominant contribution to the charge density distribution and to the electrostatic potential even in 1M NaCl solutions, the contributions of water molecules and of ions to the total electrostatic interaction free energy with the solvated biomolecule are comparable. The electrostatic biomolecule/solvent interaction energies and the total charge distribution exhibit a remarkable insensitivity to salt concentrations over a huge range of salt concentrations (20 mM to 1M NaCl). The electrostatic potentials near the biomolecule's surface obtained from the MD simulations differ markedly, as expected, from the potentials predicted by continuum dielectric models, even though the total electrostatic interaction free energies are within 11% of each other.

Protein folding intermediates: native-state hydrogen exchange.

The hydrogen exchange behavior of native cytochrome c in low concentrations of denaturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.

Mechanisms and uses of hydrogen exchange.

Recent work has largely completed our understanding of the hydrogen-exchange chemistry of unstructured proteins and nucleic acids. Some of the high-energy structural fluctuations that determine the hydrogen-exchange behavior of native macromolecules have been explained; others remain elusive. A growing number of applications are exploiting hydrogen-exchange behavior to study difficult molecular systems and elicit otherwise inaccessible information on protein structure, dynamics and energetics.

Reduced contact order and RNA folding rates.

We investigated the relationship between RNA structure and folding rates accounting for hierarchical structural formation. Folding rates of two-state folding proteins correlate well with relative contact order, a quantitative measure of the number and sequence distance between tertiary contacts. These proteins do not form stable structures prior to the rate-limiting step. In contrast, most secondary structures are stably formed prior to the rate-limiting step in RNA folding. Accordingly, we introduce "reduced contact order", a metric that reflects only the number of residues available to participate in the conformational search after the formation of secondary structure. Plotting the folding rates and the reduced contact order from ten different RNAs suggests that RNA folding can be divided into two classes. To examine this division, folding rates of circularly permutated isomers are compared for two RNAs, one from each class. Folding rates vary by tenfold for circularly permuted Bacillus subtilis RNase P RNA isomers, whereas folding rates vary by only 1.2-fold for circularly permuted catalytic domains. This difference is likely related to the dissimilar natures of their rate-limiting steps.

Stepwise conversion of a mesophilic to a thermophilic ribozyme.

A fundamental question in RNA folding is the mechanism of thermodynamic stability. We investigated the equilibrium folding of a series of sequence variants in which one to three motifs of a 255-nucleotide mesophilic ribozyme were substituted with the corresponding motifs from its thermophilic homologue. Substitution of three crucial motifs individually or in groups results in a continual increase in the stability and folding cooperativity in a stepwise fashion. We find an unexpected relationship between stability and folding cooperativity. Without changing the folding cooperativity, RNAs having a similar native structure can only achieve moderate change in stability and likewise, without changing stability, RNAs having a similar native structure can only achieve moderate change in folding cooperativity. This intricate relationship must be included in the predictions of tertiary RNA stability.

Structures of fd gene 5 protein.nucleic acid complexes: a combined solution scattering and electron microscopy study.

Small-angle scattering and electron microscopy studies of fd gene 5 protein (g5p) and reconstituted g5p.nucleic acid complexes have been used to test models for the complexes and evaluate their uniqueness. In addition, we have obtained new information on the dependence of nucleotide type and protein/nucleotide (P/N) ratio on the structure of the complexes. Reconstituted complexes were made with single-stranded fd viral DNA (fd ssDNA), poly[d(A)] and poly[r(A)]. All complexes form similar left-handed, flexible superhelices having approximately the same diameter, but the pitch differs among these complexes. The g5p protein is a dimer in solution and the dimers associate to form a superhelical framework to which the polynucleotide is attached. The combined X-ray and neutron scattering data confirm the nucleic acid is inside the protein superhelix. A Monte Carlo integration modeling procedure applied to the scattering data was used to systematically test large numbers of possible models for each complex, and previously proposed models based on parameters obtained from electron microscopy were found to be essentially correct and unique. The data on the complexes with different P/N ratios showed that mass per unit length values decreased while the rise per dimer and pitch of the superhelix increased for g5p.fd-ssDNA complexes with decreasing P/N ratios.

Hydrogen exchange: the modern legacy of Linderstrøm-Lang.

This discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstrøm-Lang, shows how hydrogen exchange approaches initially conceived and implemented by Lang and his colleagues some 50 years ago are contributing to current progress in structural biology. Examples are chosen from the active protein folding field. Hydrogen exchange methods now make it possible to define the structure of protein folding intermediates in various contexts: as tenuous molten globule forms at equilibrium under destabilizing conditions, in kinetic intermediates that exist for less than one second, and as infinitesimally populated excited state forms under native conditions. More generally, similar methods now find broad application in studies of protein structure, energetics, and interactions. This article considers the rise of these capabilities from their inception at the Carlsberg Labs to their contemporary role as a significant tool of modern structural biology.

Characterization of tertiary folding of RNA by circular dichroism and urea.

CD spectroscopy can be used to monitor RNA tertiary folding transitions that may not be observable by absorbance spectroscopy. With the use of computer-controlled titrators, data can be acquired rapidly, and accurate thermodynamic properties can be obtained over a wide variety of conditions. Thus, CD spectroscopy provides a useful complement to site-resolved or chemical modification methods.

Distinguishing between two-state and three-state models for ubiquitin folding.

Conflicting results exist regarding whether the folding of mammalian ubiquitin at 25 degrees C is a simple, two-state kinetic process or a more complex, three-state process with a defined kinetic intermediate. We have measured folding rate constants up to about 1000 s(-1) using conventional rapid mixing methods in single-jump, double-jump, and continuous-flow modes. The linear dependence of folding rates on denaturant concentration and the lack of an unaccounted "burst-phase" change for the fluorescence signal indicate that a two-state folding model is adequate to describe the folding pathway. This behavior also is seen for folding in the presence of the stabilizing additives 0.23 M sodium sulfate and 1 M sodium chloride. These results stress the need for caution in interpreting deviations from ideal two-state "chevron" behavior when folding is heterogeneous or folding rate constants are near the detection limit.

Denatured states of ribonuclease A have compact dimensions and residual secondary structure.

Using small-angle X-ray scattering and Fourier transform infrared spectroscopy, we have determined that the thermally denatured state of native ribonuclease A is on average a compact structure having residual secondary structure. Under strongly reducing conditions, the protein further unfolds into a looser structure with larger dimensions but still retains a comparable amount of secondary structure. The dimensions of the thermally and chemically denatured states of the reduced protein are different but both are more compact than is predicted for a random coil of the same length. These results demonstrate that thermal denaturation in ribonuclease A is not a simple two-state transition from a native to a completely disordered random coil state.

Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity.

The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 degrees C, the addition of Mg2+ (Kd approximately 50 microM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] approximately 0.8 mM, Hill coefficient approximately 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; approximately 30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.

Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil.

We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, and stability, earlier attempts assumed that the cosolvent and denaturant interact to the same degree in the transition state. Applying this analysis to GCN4-p2' yielded a nearly 1/eta dependence between folding rates and viscosity for both the dimeric and the cross-linked, monomeric versions of the coiled coil, but it revealed no such coherent relationship for cytochrome c. We also developed a method to determine the relative viscosity dependence of the dimeric and monomeric forms of the coiled coil independent of the assumption concerning the transition state's relative interaction with cosolvents and denaturants. Application of this method indicated that the effect of viscosity on both the folding and the unfolding rates was the same for the dimeric and monomeric versions, further supporting the view that the folding of the dimeric version is folding-limited rather than encounter-limited. The finding that GCN4-p2' folding appears to exhibit a 1/eta viscosity dependence implies that the rate-limiting step in folding is opposed predominantly by solvent-derived rather than internal frictional forces. These results are interpreted in relation to various models for protein folding.

Trifluoroethanol promotes helix formation by destabilizing backbone exposure: desolvation rather than native hydrogen bonding defines the kinetic pathway of dimeric coiled coil folding.

We measure the effects of low concentrations of helix-stabilizing cosolvents, including 2,2,2-trifluoroethanol (TFE), on the thermodynamics and kinetics of folding of the dimeric alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. The change in kinetic behavior upon addition of 5% (v/v) TFE indicates that it stabilizes the transition state to the same degree as the fully helical native state. However, folding rates are largely insensitive to alanine to glycine mutagenesis, indicating that the majority of helical structure is formed after the transition state. Equilibrium hydrogen isotope partitioning measurements indicate that intramolecular hydrogen bonds are not strengthened by TFE and that amide hydrogen bonds in the transition state are nearly the same strength as those in the unfolded state. Thus, the mechanism by which TFE exerts its helix-stabilizing effects can be divorced from helix formation and does not depend on the strengthening of intrahelical hydrogen bonds. Rather, TFE increases the structure of the binary alcohol/water solvent, thereby increasing the energetic cost associated with solvation of the polypeptide backbone. At low concentrations, TFE destabilizes the unfolded species and thereby indirectly enhances the kinetics and thermodynamics of folding of the coiled coil. A high degree of polypeptide backbone desolvation, and not the formation of regular helical structure and native strength hydrogen bonds, is the critical feature of the transition state for folding of this small dimeric protein.

Engineered metal binding sites map the heterogeneous folding landscape of a coiled coil.

To address whether proteins fold along multiple pathways, i,i+4 bi-histidine metal binding sites are introduced into dimeric and crosslinked versions of the leucine zipper region of the growth control transcription factor, GCN4. Divalent metal ion binding enhances both the equilibrium and folding activation free energies for GCN4. The enhancement of folding rates quantifies the fraction of molecules that have the binding site in a helical geometry in the transition state. Hence, this new method, termed Psi-analysis, identifies the degree of pathway heterogeneity for a protein that folds in a two-state manner, a capability that is generally unavailable even with single molecule methods. Adjusting metal ion concentration continuously varies the stability of the bi-histidine region without additional structural perturbation to the protein. For dimeric and crosslinked versions, the accompanying changes in kinetic barrier heights at each metal ion concentration maps the folding landscape as well as establishes the importance of connectivity in pathway selection. Furthermore, this method can be generalized to other biophysical studies, where the ability to continuously tune the stability of a particular region with no extraneous structural perturbation is advantageous.

The burst phase in ribonuclease A folding and solvent dependence of the unfolded state.

Submillisecond burst phase signals measured in kinetic protein folding experiments have been widely interpreted in terms of the fast formation of productive folding intermediates. Experimental comparisons with non-folding polypeptide chains show that, for ribonuclease A and cytochrome c, these signals in fact reflect a shift from one biased ensemble of the unfolded state to another as a function of change in denaturant concentration.

Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding.

The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.