How large is an alpha-helix? Studies of the radii of gyration of helical peptides by small-angle X-ray scattering and molecular dynamics.

Using synchrotron radiation and the small-angle X-ray scattering technique we have measured the radii of gyration of a series of alanine-based alpha-helix-forming peptides of the composition Ace-(AAKAA)(n)-GY-NH(2), n=2-7, in aqueous solvent at 10(+/-1) degrees C. In contrast to other techniques typically used to study alpha-helices in isolation (such as nuclear magnetic resonance and circular dichroism), small-angle X-ray scattering reports on the global structure of a molecule and, as such, provides complementary information to these other, more sequence-local measuring techniques. The radii of gyration that we measure are, except for the 12-mer, lower than the radii of gyration of ideal alpha-helices or helices with frayed ends of the equivalent sequence-length. For example, the measured radius of gyration of the 37-mer is 14.2(+/-0.6)A, which is to be compared with the radius of gyration of an ideal 37-mer alpha-helix of 17.6A. Attempts are made to analyze the origin of this discrepancy in terms of the analytical Zimm-Bragg-Nagai (ZBN) theory, as well as distributed computing explicit solvent molecular dynamics simulations using two variants of the AMBER force-field. The ZBN theory, which treats helices as cylinders connected by random walk segments, predicts markedly larger radii of gyration than those measured. This is true even when the persistence length of the random walk parts is taken to be extremely short (about one residue). Similarly, the molecular dynamics simulations, at the level of sampling available to us, give inaccurate values of the radii of gyration of the molecules (by overestimating them by around 25% for longer peptides) and/or their helical content. We conclude that even at the short sequences examined here (< or =37 amino acid residues), these alpha-helical peptides behave as fluctuating semi-broken rods rather than straight cylinders with frayed ends.

Unusual compactness of a polyproline type II structure.

Polyproline type II (PPII) helix has emerged recently as the dominant paradigm for describing the conformation of unfolded polypeptides. However, most experimental observables used to characterize unfolded proteins typically provide only short-range, sequence-local structural information that is both time- and ensemble-averaged, giving limited detail about the long-range structure of the chain. Here, we report a study of a long-range property: the radius of gyration of an alanine-based peptide, Ace-(diaminobutyric acid)2-(Ala)7-(ornithine)2-NH2. This molecule has previously been studied as a model for the unfolded state of proteins under folding conditions and is believed to adopt a PPII fold based on short-range techniques such as NMR and CD. By using synchrotron radiation and small-angle x-ray scattering, we have determined the radius of gyration of this peptide to be 7.4 +/- 0.5 angstroms, which is significantly less than the value expected from an ideal PPII helix in solution (13.1 angstroms). To further study this contradiction, we have used molecular dynamics simulations using six variants of the AMBER force field and the GROMOS 53A6 force field. However, in all cases, the simulated ensembles underestimate the PPII content while overestimating the experimental radius of gyration. The conformational model that we propose, based on our small angle x-ray scattering results and what is known about this molecule from before, is that of a very flexible, fluctuating structure that on the level of individual residues explores a wide basin around the ideal PPII geometry but is never, or only rarely, in the ideal extended PPII helical conformation.

Effects of nitration on the structure and aggregation of alpha-synuclein.

Substantial evidence suggests that the aggregation of the presynaptic protein alpha-synuclein is a key step in the etiology of Parkinson's disease (PD). Although the molecular mechanisms underlying alpha-synuclein aggregation remain unknown, oxidative stress has been implicated in the pathogenesis of PD. Here, we report the effects of tyrosine nitration on the propensity of human recombinant alpha-synuclein to fibrillate in vitro. The properties of nitrated alpha-synuclein were investigated using a variety of biophysical and biochemical techniques, which revealed that nitration led to formation of a partially folded conformation with increased secondary structure relative to the intrinsically disordered structure of the monomer, and to oligomerization at neutral pH. The degree of self-association was concentration-dependent, but at 1 mg/mL, nitrated alpha-synuclein was predominantly an octamer. At low pH, small-angle X-ray scattering data indicated that the nitrated protein was monomeric. alpha-Synuclein fibrillation at neutral pH was completely inhibited by nitrotyrosination and is attributed to the formation of stable soluble oligomers. The presence of heparin or metals did not overcome the inhibition; however, the inhibitory effect was eliminated at low pH. The addition of nitrated alpha-synuclein inhibited fibrillation of non-modified alpha-synuclein at neutral pH. Potential implications of these findings to the etiology of Parkinson's disease are discussed.

Probing counterion modulated repulsion and attraction between nucleic acid duplexes in solution.

Understanding biological and physical processes involving nucleic acids, such as the binding of proteins to DNA and RNA, DNA condensation, and RNA folding, requires an understanding of the ion atmosphere that surrounds nucleic acids. We have used a simple model DNA system to determine how the ion atmosphere modulates interactions between duplexes in the absence of specific metal ion-binding sites and other complicated interactions. In particular, we have tested whether the Coulomb repulsion between nucleic acids can be reversed by counterions to give a net attraction, as has been proposed recently for the rapid collapse observed early in RNA folding. The conformation of two DNA duplexes tethered by a flexible neutral linker was determined in the presence of a series of cations by small angle x-ray scattering. The small angle x-ray scattering profiles of two control molecules with distinct shapes (a continuous duplex and a mimic of the compact DNA) were in good agreement with predictions, establishing the applicability of this approach. Under low-salt conditions (20 mM Na+), the tethered duplexes are extended because of a Coulombic repulsion estimated to be 2-5 kT/bp. Addition of high concentrations of Na+ (1.2 M), Mg2+ (0.6 M), and spermidine3+ (75 mM) resulted in electrostatic relaxation to a random state. These results indicate that a counterion-induced attractive force between nucleic acid duplexes is not significant under physiological conditions. An upper limit on the magnitude of the attractive potential under all tested ionic conditions is estimated.

Random-coil behavior and the dimensions of chemically unfolded proteins.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R(G)) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R(G) of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r(2) = 0.988) by a power-law relationship with a best-fit exponent, 0.598 +/- 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

Stimulation of insulin fibrillation by urea-induced intermediates.

Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 m urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased alpha-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.

Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin.

The structure of C-terminal domain (CaD136, C-terminal residues 636-771) of chicken gizzard caldesmon has been analyzed by a variety of physico-chemical methods. We are showing here that CaD136 does not have globular structure, has low secondary structure content, is essentially noncompact, as it follows from high R(g) and R(S) values, and is characterized by the absence of distinct heat absorption peaks, i.e. it belongs to the family of natively unfolded (or intrinsically unstructured) proteins. Surprisingly, effective binding of single calmodulin molecule (K(d) = 1.4 +/- 0.2 microM) leads only to a very moderate folding of this protein and CaD136 remains substantially unfolded within its tight complex with calmodulin. The biological significance of these observations is discussed.

Partially folded intermediates in insulin fibrillation.

Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.

The fastest global events in RNA folding: electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme.

Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.

Nuclear localization of alpha-synuclein and its interaction with histones.

The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.

Prediction of the association state of insulin using spectral parameters.

Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state.

Rapid compaction during RNA folding.

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques.

The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radius of gyration and hydrodynamic radius derived from the SAXS and PFG-NMR data, respectively, and (ii) a histogram of the size distribution of structures obtained from preliminary analyses of structural parameters recorded on the unfolded state. Results show that this unfolded state, U(exch), which exists in equilibrium with the folded state, F(exch), under non-denaturing conditions, is relatively compact, with the average size of conformers within the unfolded state ensemble only 30-40% larger than the folded state structure. In addition, the model predicts a significant overlap in the size range of structures comprising the U(exch) state with those in a denatured state obtained by addition of 2 M guanidinium chloride.

Elucidation of the molecular mechanism during the early events in immunoglobulin light chain amyloid fibrillation. Evidence for an off-pathway oligomer at acidic pH.

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.

Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH.

Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivo fibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability, and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 m urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.

Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins.

The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous alpha-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, beta- and gamma-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. beta-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas alpha- and gamma-synucleins were slightly more compact and structured. gamma-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to alpha-synuclein, beta- And gamma-synucleins inhibit alpha-synuclein fibril formation. Complete inhibition of alpha-synuclein fibrillation was observed at 4:1 molar excess of beta- and gamma-synucleins. No significant incorporation of beta-synuclein into the fibrils was detected. The lack of fibrils formed by beta-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed.

Exploring the folding landscape of a structured RNA.

Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

Structural and functional properties of Yersinia pestis Caf1 capsular antigen and their possible role in fulminant development of primary pneumonic plague.

Yersinia pestis capsular antigen Caf1 is shown to be a beta-structural protein that in polymeric form possesses very high conformational stability. Different approaches show that a dimer is the minimal cooperative block of Caf1 adhesin. Caf1 dimer interacts effectively with IL-1 receptors of human macrophage and epithelial cells. The specificity of such interaction is confirmed by the inhibition of IL-1alpha binding by Caf1. The Caf1 role in pneumonic plague pathogenesis is discussed.