Highly populated turn conformations in natively unfolded tau protein identified from residual dipolar couplings and molecular simulation

Tau, a natively unstructured protein that regulates the organization of neuronal microtubules, is also found in high concentrations in neurofibrillary tangles of Alzheimer's disease and other neurodegenerative disorders. The conformational transition between these vastly different healthy and pathological forms remains poorly understood. We have measured residual dipolar couplings (RDCs), J-couplings, and nuclear Overhauser enhancement (NOE) in construct K18 of tau, containing all four repeat domains R1-R4. NHN RDCs were compared with prediction on the basis of a statistical model describing the intrinsic conformational sampling of unfolded proteins in solution. While local variation and relative amplitude of RDCs agrees with propensity-based prediction for most of the protein, homologous sequences in each repeat domain (DLKN, DLSN, DLSK, and DKFD in repeats R1-R4) show strong disagreement characterized by inversion of the sign of the central couplings. Accelerated molecular dynamic simulations (AMD) in explicit solvent revealed strong tendencies to form turns, identified as type I beta-turns for repeats R1-R3. Incorporation of the backbone dihedral sampling resulting from AMD into the statistical coil model closely reproduces experimental RDC values. These localized sequence-dependent conformational tendencies interrupt the propensity to sample more extended conformations in adjacent strands and are remarkably resistant to local environmental factors, as demonstrated by the persistence of the RDC signature even under harsh denaturing conditions (8 M urea). The role that this specific conformational behavior may play in the transition to the pathological form is discussed.     

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.     

Structure and dynamics of an unfolded protein examined by molecular dynamics simulation

The accurate characterization of the structure and dynamics of proteins in disordered states is a difficult problem at the frontier of structural biology whose solution promises to further our understanding of protein folding and intrinsically disordered proteins. Molecular dynamics (MD) simulations have added considerably to our understanding of folded proteins, but the accuracy with which the force fields used in such simulations can describe disordered proteins is unclear. In this work, using a modern force field, we performed a 200 μs unrestrained MD simulation of the acid-unfolded state of an experimentally well-characterized protein, ACBP, to explore the extent to which state-of-the-art simulation can describe the structural and dynamical features of a disordered protein. By comparing the simulation results with the results of NMR experiments, we demonstrate that the simulation successfully captures important aspects of both the local and global structure. Our simulation was ~2 orders of magnitude longer than those in previous studies of unfolded proteins, a length sufficient to observe repeated formation and breaking of helical structure, which we found to occur on a multimicrosecond time scale. We observed one structural feature that formed but did not break during the simulation, highlighting the difficulty in sampling disordered states. Overall, however, our simulation results are in reasonable agreement with the experimental data, demonstrating that MD simulations can already be useful in describing disordered proteins. Finally, our direct calculation of certain NMR observables from the simulation provides new insight into the general relationship between structural features of disordered proteins and experimental NMR relaxation properties.     

Relationships between unfolded configurations of proteins and dynamics of folding to the native state

We compare folding trajectories of chymotrypsin inhibitor (CI2) using a dynamic Monte Carlo scheme with Go-type potentials. The model considers the four backbone atoms of each residue and a sphere centered around C^β the diameter of which is chosen according to the type of the side group. Bond lengths and bond angles are kept fixed. Folding trajectories are obtained by giving random increments to the φ and ψ torsion angles with some bias toward the native state. Excluded volume effects are considered. Two sets of 20 trajectories are obtained, with different initial configurations. The first set is generated from random initial configurations. The initial configurations of the second set are generated according to knowledge-based neighbor dependent torsion probabilities derived from triplets in the Protein Data Bank. Compared to chains with randomly generated initial configurations, those generated with neighbor-dependent probabilities (i) fold faster, (ii) have better defined secondary structure elements, and (iii) have less number of non-native contacts during folding. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 3667–3678, 2006     

Modulation of structure and dynamics by disulfide bond formation in unfolded states

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.     

The dynamics of unfolded versus folded tRNA: the role of electrostatic interactions

The dynamics of RNA contributes to its biological functions such as ligand recognition and catalysis. Using quasielastic neutron scattering spectroscopy, we show that Mg(2+) greatly increases the picosecond to nanosecond dynamics of hydrated tRNA while stabilizing its folded structure. Analyses of the atomic mean-squared displacement, relaxation time, persistence length, and fraction of mobile atoms showed that unfolded tRNA is more rigid than folded tRNA. This same result was found for a sulfonated polystyrene, indicating that the increased dynamics in Mg(2+) arises from improved charge screening of the polyelectrolyte rather than specific interactions with the folded tRNA. These results are opposite to the relationship between structural compactness and internal dynamics for proteins in which the folded state is more rigid than the denatured state. We conclude that RNA dynamics are strongly influenced by the electrostatic environment, in addition to the motions of local waters.     

Synthesis, crystal structure, and different local conformations of pyridine-imide oligomers

In this article, we report a new approach toward synthesis of pyridine-imide oligomers (PIOs). Using this approach, both dimer and trimer were one-pot synthesized from acylation of monomeric monoamide with monomeric dichloride. The yield of trimer was dependent on the alkoxyl terminals: it was 30% for methoyl group, whereas it was 95% for 3-chloro-1-propoxyl terminal. Acylation of dimeric monoamide with monomeric dichloride produced trimer, tetramer, and pentamer in a yield of 34%, 33%, and 28%, respectively. The synthesis was proposed to be mediated through an exchange between pyridine-2-carboxamide and pyridine-2-carbonyl chloride, both forming intramolecular or intermolecular hydrogen-bonds between pyridine-nitrogen and pyridine-2-amide hydrogen atoms. Crystal structure from three trimers with different terminal groups was reported. Analysis on the crystal structures revealed that these three trimers had different local conformations. The different local conformations were originated from the structural tunability of the imide unit in either the coplanarity or bond parameters.     

Gas-phase and solution conformations of the alpha-L-iduronic acid structural unit of heparin

The IdoA2S structural unit of heparin (subunit G) may oscillate among the three conformations (4C1, 1C4, and 2So). Only the twisted boat conformation allowed the biologically active pentasaccharide unit of heparin (DEFGH) to bind to antithrombin. Our work reports, in detail, the results of systematic large-scale theoretical investigations of the three basic conformations (4C1, 1C4, and 2So) of the IdoA2S structural unit of heparin, its anionic forms, and its sodium salt using the B3LYP/6-311++G(d, p) and B3LYP/6-31+G(d) model chemistries. According to our calculations, the most stable structure of these molecules corresponds to the 2So skew-boat conformation. This form is also the most stable in a water solution. The 2So conformation of neutral molecules is not maintained in the anionic species. With anions, both 1C4 and 4C1 conformations are present. The relative stability of individual species of the substituted iduronic acid affects extra stabilization by means of intramolecular hydrogen bonds. The calculated macroscopic pKa of 1,4-DiOMe IdoA2S are as follows: pKa = 0.25 for the terminal C(2)-OSO3H group, pKa = 3.67 for the terminal C(5)-CO2H group, and pKa = 14.00 for the C(3)-OH hydroxyl group. The computed Gibbs interaction energies, DeltaGdegrees , for the reaction 1,4-DiOMe IdoA2S(2-) + 2Na+ <==> 1,4-DiOMe IdoA2SNa2 (4C1, 1C4, and 2So conformations) are negative and span a rather small energy interval (from -1244 to -1290 kJ mol(-1)).     

Comparison between self-guided Langevin dynamics and molecular dynamics simulations for structure refinement of protein loop conformations

This article presents a comparative analysis of two replica‐exchange simulation methods for the structure refinement of protein loop conformations, starting from low‐resolution predictions. The methods are self‐guided Langevin dynamics (SGLD) and molecular dynamics (MD) with a Nosé–Hoover thermostat. We investigated a small dataset of 8‐ and 12‐residue loops, with the shorter loops placed initially from a coarse‐grained lattice model and the longer loops from an enumeration assembly method (the Loopy program). The CHARMM22 + CMAP force field with a generalized Born implicit solvent model (molecular‐surface parameterized GBSW2) was used to explore conformational space. We also assessed two empirical scoring methods to detect nativelike conformations from decoys: the all‐atom distance‐scaled ideal‐gas reference state (DFIRE‐AA) statistical potential and the Rosetta energy function. Among the eight‐residue loop targets, SGLD out performed MD in all cases, with a median of 0.48 Å reduction in global root‐mean‐square deviation (RMSD) of the loop backbone coordinates from the native structure. Among the more challenging 12‐residue loop targets, SGLD improved the prediction accuracy over MD by a median of 1.31 Å, representing a substantial improvement. The overall median RMSD for SGLD simulations of 12‐residue loops was 0.91 Å, yielding refinement of a median 2.70 Å from initial loop placement. Results from DFIRE‐AA and the Rosetta model applied to rescoring conformations failed to improve the overall detection calculated from the CHARMM force field. We illustrate the advantage of SGLD over the MD simulation model by presenting potential‐energy landscapes for several loop predictions. Our results demonstrate that SGLD significantly outperforms traditional MD in the generation and populating of nativelike loop conformations and that the CHARMM force field performs comparably to other empirical force fields in identifying these conformations from the resulting ensembles. Published 2011 Wiley Periodicals, Inc. J Comput Chem, 2011     

Slow dynamics in folded and unfolded states of an SH3 domain.

(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.     

Native and unfolded cytochrome c--comparison of dynamics using 2D-IR vibrational echo spectroscopy

Unfolded vs native CO-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus ( Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs GuHCl concentration produces a sigmoidal curve that overlays the concentration-dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low- and high-concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from subpicosecond to greater than 100 ps through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the approximately 100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy-like state involving hydrophobic collapse around the heme.     

Amino acid and peptide derivatives of the indole series

Summary 1. A method for obtaining amino acid and peptide derivatives of -methyltryptamine have been described.2. In respect of their biological activity, the closest to Indopan are glycyl--methyltryptamine and L--glutamyl--methyltryptamine, which, at the same time, possess a weaker action on the peripheral adrenoreactive system.3. When amino acid residues are introduced into the molecule of -methyltryptamine, not only does the degree of biological activity of the compound change but, in some cases, new biological properties actually appear.Khimiya Prirodnykh Soedinenii, Vol. 3, No. 2, pp. 108–116, 1967     

Amino acid and peptide derivatives of fullerene

A general method for the synthesis of amino acid and peptide derivatives of fullerene (ADF) was developed, and the physicochemical properties of the compounds obtained were studied. ADF were shown to penetrate into liposomes and to exhibit adjuvant properties and antiviral activity. Deceased Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1050–1054, May, 1998.     

Conformational analysis of bradykinin by annealed molecular dynamics and comparison to NMR-derived conformations

Conformational analysis of bradykinin (BK), a nonapeptide of the sequence RPPGFSPFR, was accomplished using annealed molecular dynamics (AMD) at 1000 K in BIOGRAF 2.2. One hundred anneal cycles produced 100 conformations over approximately 2000 ps. These conformations were compared to structures derived by nuclear magnetic resonance (NMR) methods for similar shape and energy. Energy minimization of relevant conformations using both BIOGRAF 2.2 and AMBER 3.0a revealed that the AMD-determined conformations are in the same energy range as the NMR-determined structures. Also, the shape of the relevant conformations appeared similar, suggesting that AMD is a good tool for the conformational analysis of small peptide ligands. © 1993 John Wiley & Sons, Inc.     

Molecular dynamics simulations of the rehydration of folded and unfolded cytochrome C ions in the vapor phase.

Molecular dynamics (MD) simulations have been performed to study the rehydration of compact and unfolded cytochrome c ions in the vapor phase. Experimental studies have shown that the compact conformations adsorb many more water molecules than unfolded ones when exposed to water vapor. MD simulations performed with up to 150 water molecules reproduce the key experimental observations, including a partial refolding caused by hydration. According to the calculations it is more energetically favorable to hydrate the compact conformation in the initial stages of hydration, because it is easier for a water molecule to interact simultaneously with several polar groups (due to their proximity). The protonated side chains are not favored hydration sites in the simulations because they have "self-solvation" shells which must be disrupted for the water to penetrate. For both conformations, the adsorbed water molecules are mainly located in surface crevices.     

Spin-label ESR with nanochannels to improve the study of backbone dynamics and structural conformations of polypeptides

Nanochannels of mesoporous silica materials were previously found useful for reducing the tumbling motion of encapsulated biomolecules while leaving the biomolecular structure undisturbed. Here we show that experiments of cw-ESR distance measurement in nano-confinement can benefit immediately from the above mentioned features of sufficiently slow molecular tumbling, enabling more accurate determination of interspin distances throughout the temperature range, from 200 to 300 K. A 26-residue prion protein peptide, which can fold into either a helical or hairpin structure, as well as its variants, are studied by using ESR. By comparing the spectra obtained in vitrified bulk solutions vs. mesopores, the spectra from the latter display typical slow-motional lineshapes, thereby enabling dipolar anisotropy to be unambiguously revealed throughout the temperature range, whereas the spectra from the former are dominated by the disordering of the side chain and the rotational tumbling of the peptide. The spectral changes regarding the two secondary structures in nano-confinement are found to show a strong correlation with the dynamic properties of the backbones. The effect of viscosity agent perturbation on the motion of an R1 nitroxide side chain, a commonly employed probe, could be substantial in a bulk solution condition, though it is absolutely absent in nanochannels. Under nano-confinement, the probe is proven sufficiently sensitive to the backbone motions. Overall, the distance distributions determined from the mesopore studies not only describe the conformational structures (by average distances), but also the backbone dynamics (by distribution widths) of the spin-labeled peptides.     

Miscible blend dynamics and the length scale of local compositions

We consider the influence of a local, or effective, composition on dynamics in the miscible polymer blend PEO/PMMA. Quasielastic neutron scattering in combination with deuterium labeling is employed to determine characteristic relaxation times of the PEO component over spatial scales from 3 to 10 Å. Information about the distribution of relaxation times is obtained indirectly from the stretching parameters in a stretched exponential fit. We examine the behavior of these parameters with spatial scale and temperature, finding that their variation supports a distribution of PEO mobility in the blend which is far wider than pure PEO and narrows with decreasing temperature for small spatial scales. This is linked to the concept of local compositions defined over varying spatial scales, and indicates that the concept of a local composition, linked to PEO dynamics, is important in this system. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 2914–2923, 2005     

Molecular dynamics simulation study on zwitterionic structure to maintain the normal conformations of Glutathione

Molecular dynamics simulations were applied to normal conformational Glutathione (GSH) and GSH over zwitterionic and hydrophobic surfaces respectively. Conformational analysis of GSH during the simulation time on RMSD, conformational flexibility and dihedral distribution were performed. The re- sults showed that zwitterionic structure maintains the normal conformations of GSH to a better extent, which should be a first good proof of the hypothesis of "maintain of normal structure".