Ordering Of Side Chains Research Articles

Side chain dynamics in unfolded protein states: an NMR based 2H spin relaxation study of delta131delta.

NMR relaxation data on disordered proteins can provide insight into both structural and dynamic properties of these molecules. Because of chemical shift degeneracy in correlation spectra, detailed site-specific analyses of side chain dynamics have not been possible. Here, we present new experiments for the measurement of side chain dynamics in methyl-containing residues in unfolded protein states. The pulse schemes are similar to recently proposed methods for measuring deuterium spin relaxation rates in (13)CH(2)D methyl groups in folded proteins.(1) However, because resolution in (1)H-(13)C correlation maps of unfolded proteins is limiting, relaxation data are recorded as a series of (1)H-(15)N spectra. The methodology is illustrated with an application to the study of side chain dynamics in delta131delta, a large disordered fragment of staphylococcal nuclease containing residues 1-3 and 13-140 of the wide-type protein. A good correlation between the order parameters of the symmetry axes of the methyl groups and the backbone (1)H-(15)N bond vectors of the same residue is observed. Simulations establish that such a correlation is only possible if the unfolded state is comprised of an ensemble of structures which are not equiprobable. A motional model, which combines wobbling-in-a-cone and Gaussian axial fluctuations, is proposed to estimate chi(1) torsion angle fluctuations, sigma(chi)()1, of Val and Thr residues on the basis of the backbone and side chain order parameters. Values of sigma(chi)()1 are approximately 10 degrees larger than what has previously been observed in folded proteins. Of interest, the value of sigma(chi)()1 for Val 104 is considerably smaller than for other Val or Thr residues, suggesting that it may be part of a hydrophobic cluster. Notably large (15)N transverse relaxation rates are observed in this region. To our knowledge, this is the first time that side chain dynamics in an unfolded state have been studied in detail by NMR.

Variable-temperature UV and Raman study of complicated thermochromic phase transition of order-disorder type in poly(di-n-decylsilane) [(n-C10H21)2Si

A complicated thermochromic phase transition (PT) of order—disorder type in poly(di-n-decylsilane) has been studied in detail by variable-temperature UV and Raman spectroscopy. Not less than five polymer modifications were shown to participate in this PT. Above the equilibrium PT temperature Tc (∼60 °C), the polymer exists as a hexagonal columnar mesophase (HCM) with a disordered silicon backbone and disordered side chains. PT proceeds not abruptly but over a temperature range 60—5 °C and does not reach completion, being stopped by glassification. At room temperature, a coexistence in the polymer of HCM and a few crystalline phases with ordered backbone is observed. The latter are separated in space, what was evidenced by Raman micromapping of a polymer film. The temperature intervals of existence of different ordered phases also do not coincide, this enables one to identify in the UV and Raman spectra the components, corresponding to the modifications possessing trans (anti) and, supposedly, AD+AD– main chain conformations. As the relaxation processes in this comb-like polymer occur slowly, the quantitative ratio of different phases is not a single-valued function of temperature but depends on the thermal prehistory of the sample. Ordering of long side chains on cooling was demonstrated by the Raman method.

Glassy dynamics of side-chain ordering in a simple model of protein folding.

We introduce a modified version of protein lattice models in which monomers have several spin states, representing side-chain rotamers. Completion of folding corresponds to reaching the native backbone configuration with complete ordering of side chains. We find that as temperature is lowered, side-chain ordering becomes much slower than backbone folding. The presence of side chains leads to nonexponential kinetics and a broad distribution of relaxation times.

Iron Cluster and Microstructure Formation in Metal-Centered Star Block Copolymers: Amphiphilic Iron Tris(bipyridine)-Centered Polyoxazolines

The microstructure of a metal-centered six-arm star block copolymer, amphiphilic iron tris(bipyridine)-centered poly(2-ethyl-2-oxazoline)-b-poly(2-undecyl-2-oxazoline), [Fe{bpy(PEOX−PUOX)2}3]2+, was investigated with small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Cylindrical PEOX microdomains were observed in a matrix of PUOX. A similar cylindrical morphology was noted for bpy(PEOX−PUOX)2, the metal-free macroligand subunit of which the Fe star is comprised. Thin films simple-cast from chloroform solution onto carbon substrates annealed at 160 °C for ≈2 days revealed in both TEM and AFM the formation of nanoscale iron clusters (≈20−40-nm diameter) distributed randomly across the surface of the film. No clusters were evident in “as-cast” films subjected to TEM analysis without the annealing step, suggesting the importance of thermal treatment for nanocrystal formation. For bulk films, larger micrometer-scale (1−2 μm) iron aggregates formed and segregated to the surface of the film after thermal treatment for ≈1 week. A liquid crystal-like mesophase due to undecyl alkyl side chain ordering was evidenced by SAXS and WAXS in the bulk sample.

Observation of Surface Ordering of Alkyl Side Chains in Polystyrene/Polyelectrolytes Diblock Copolymer Langmuir Films

Block polyelectrolytes P(Sx-b-VP/RXy) composed of polystyrene (PS) blocks (x = 200, 260) and alkylated (R = Cn) poly(vinylpyridine) (PVP) (X = I or Br; y = 119−270; n = 4, 10, and 18) have been studied using the Langmuir film balance technique and in situ X-ray and neutron reflectivity measurements. Previous studies showed that the self-assembled surface micelles (with PS core and RPV+PX- corona) at the air/water interface revealed a characteristic transition (when n > 6) without any significant hydration or the submersion of the corona chains. Combined X-ray and neutron reflectivity studies of P(Sx-b-VP/RXy) polyelectrolytes show that although the polyelectrolyte block is water soluble, it remains adsorbed to the water surface. The thickness of the adsorbed layer measured by X-ray reflectivity is in agreement with that measured using neutron reflectivity. This agreement indicates that the counterions are closely associated with the alkylated PVP chains, localized at the air/water interface, and are not d...

Thermal and structural properties of biologically derived monodisperse hairy-rod polymers.

Monodisperse derivatives of poly(gamma-4-(hexadecyloxy)benzyl alpha,L-glutamate) (PHBG-X, X = 3 or 4) with backbone sequence GluAsp(Glu17Asp)xGluGlu were prepared by reaction of 4-(hexadecyloxy)phenyldiazomethane with the corresponding monodisperse poly(alpha,L-glutamate) (PLGA) derivatives (PLGA-X, X = 3 or 4). PHBG-3 and -4 exhibited strong endotherms near 45 degrees C and weak endotherms near 86 degrees C when analyzed by differential scanning calorimetry. X-ray diffraction suggested that these polymers aggregate to form layerlike solid structures at room temperature, with extended alkyl side chains forming paraffinlike crystallites. Most of the side chain order disappears at the first melting transition; however, the layerlike structure remains. Both polymers are isotropic above the second melting transition; no ordered melts were observed at higher temperatures, possibly due to the small aspect ratios of PHBG-3 and -4. In contrast, polydisperse poly(gamma-4-(hexadecyloxy)benzyl alpha,L-glutamate) (PDI = 1.2, DP = 98) (PHBG-P1), prepared from commercial PLGA, formed liquid crystalline (LC) phases between 97 and 105 degrees C.

Molecular dynamics simulation of hen egg white lysozyme: A test of the GROMOS96 force field against nuclear magnetic resonance data

Biomolecular force fields for use in molecular dynamics (MD) simulations of proteins, DNA, or membranes are generally parametrized against ab initio quantum-chemical and experimental data for small molecules. The application of a force field in a simulation of a biomolecular system, such as a protein in solution, may then serve as a test of the quality and transferability of the force field. Here, we compare various properties obtained from two MD simulations of the protein hen egg white lysozyme (HEWL) in aqueous solution using the latest version, GROMOS96, of the GROMOS force field and an earlier version, GROMOS87+, with data derived from nuclear magnetic resonance (NMR) experiments: NOE atom–atom distance bounds, 3JHNα-coupling constants, and backbone and side-chain order parameters. The convergence of these quantities over a 2-ns period is considered, and converged values are compared to experimental ones. The GROMOS96 simulation shows better agreement with the NMR data and also with the X-ray crystal structure of HEWL than the GROMOS87+ simulation, which was based on an earlier version of the GROMOS force field. Proteins 2000;40:145–153. © 2000 Wiley-Liss, Inc.

Molecular dynamics simulation of hen egg white lysozyme: A test of the GROMOS96 force field against nuclear magnetic resonance data

Biomolecular force fields for use in molecular dynamics (MD) simulations of proteins, DNA, or membranes are generally parametrized against ab initio quantum-chemical and experimental data for small molecules. The application of a force field in a simulation of a biomolecular system, such as a protein in solution, may then serve as a test of the quality and transferability of the force field. Here, we compare various properties obtained from two MD simulations of the protein hen egg white lysozyme (HEWL) in aqueous solution using the latest version, GROMOS96, of the GROMOS force field and an earlier version, GROMOS87+, with data derived from nuclear magnetic resonance (NMR) experiments: NOE atom-atom distance bounds, (3)J(HNalpha)-coupling constants, and backbone and side-chain order parameters. The convergence of these quantities over a 2-ns period is considered, and converged values are compared to experimental ones. The GROMOS96 simulation shows better agreement with the NMR data and also with the X-ray crystal structure of HEWL than the GROMOS87+ simulation, which was based on an earlier version of the GROMOS force field.

Conformational analysis of long spacers in PROSITE patterns

To determine if variable sequences (spacers) between conserved positions in a sequence motif or pattern share a consensus structure, three-dimensional structures containing PROSITE patterns with spacers of fixed length greater than three residues were analyzed. Structural similarities of a given pattern were evaluated by computing the backbone φ, ψ and side-chain χ1 dihedral order parameters. The exact bias information in analyzing the conformational variability of the patterns was taken into account by introducing a new parameter, the bias coefficient, which describes the number and distribution of residue types found at each position of a pattern in the structures. The results of the analyses show that backbone conformational heterogeneity at a given position in a sequence motif does not necessarily correlate with the residue-type variability at that position, and the long spacer region can adopt a well-defined backbone conformation, in addition to the conserved residues. Furthermore, a PROSITE pattern may be redefined to yield two or more “refined” regular expressions, each corresponding to a distinct backbone conformation. A way in which the observed structural consensus in a pattern may be employed to improve the accuracy of function prediction from sequence is suggested.

Kinetics of crystallization of poly(octylthiophene) conducting gels. NMR approach

Abstract Conjugated poly(octylthiophene) (POT) gels are semi-crystalline. Their kinetics of crystallization are observed from 1 H-NMR versus temperature, T, and crosslink concentration. Crystalline ratios are quantitatively determined. They depend on the thermal gel history and on the crosslink ratio. Two mechanisms of crystallization are detected which are characterized by different crystallization rates. They are controlled either by a slow arrangement of main chains (T>100 °C) or by a fast ordering of side-chains (T

Dynamic Flexibility of Protein−Inhibitor Complexes: A Study of the HIV-1 Protease/KNI-272 Complex

The dynamics and flexibility of protein−ligand complexes is central to understanding and predicting binding geometries and energetics. We have calculated various measures of the dynamic flexibility of a pseudo-C2-symmetric protein, HIV-1 protease, complexed with the asymmetric inhibitor KNI-272 based on molecular dynamics simulations. This system is expected to be an excellent candidate for observing asymmetric dynamics between the two monomers due to the differences in the interactions between the two monomers of the protease and the inhibitor. Experimental methods have thus far been unable to observe the expected asymmetry in this system. Our calculated results are in excellent agreement with the available experimental data for the main-chain order parameters from a parallel 15N NMR study of the same inhibitor−protein complex, as well as the Debye−Waller temperature factors from X-ray crystallography. In our simulations, asymmetry between the monomers is found almost exclusively in the side-chain order parameters of the inhibitor and protease (especially residues 84A and 84B), for which experimental data are not yet available. We analyze the dynamic information obtained from the different methods and discuss protein−ligand interactions responsible for the dynamical behavior of the complex.

Liquid crystallinity and side chain order in partially substituted semi-flexible polymers

A series of hydroxypropyl cellulose (HPC) derivatives with, on the average, 3, 2, 3/2, 1, 1/2, 1/3 alkyl side chains (18 carbons) on each anhydroglucose unit were synthesized. X-ray studies indicate that layered structures prevail in most of these HPC derivatives, even in the sample containing only one alkyl side chain per two rings, i.e. C18(1/6)HPC. The polymers were able to pack in better ordered arrays in the solvent-cast films than in as-made samples obtained by precipitation from a nonsolvent. Side chain melting endotherms were observed for C18(3/3), C18(2/3), C18(1/6), and C18(1/9)HPC's. In C18(1/2) and C18(1/3)HPC's, side chain melting enthalpies were negligible but isotropization enthalpies were unexpectedly large. The isotropization temperatures and the associated enthalpies remained almost constant in differential scanning calorimetry (DSC) heating/cooling cycles. Distinct morphological features were observed under the cross polarized optical microscope (POM) after shearing the samples in the molten state. The degree of side chain substitution has a profound influence on the polymer morphology.

Liquid crystallinity and side chain order in partially substituted semi-flexible polymers

A series of hydroxypropyl cellulose (HPC) derivatives with, on the average, 3, 2, 3/2, 1, 1/2, 1/3 alkyl side chains (18 carbons) on each anhydroglucose unit were synthesized. X-ray studies indicate that layered structures prevail in most of these HPC derivatives, even in the sample containing only one alkyl side chain per two rings, i.e. C18(1/6)HPC. The polymers were able to pack in better ordered arrays in the solvent-cast films than in as-made samples obtained by precipitation from a nonsolvent. Side chain melting endotherms were observed for C18(3/3), C18(2/3), C18(1/6), and C18(1/9)HPC's. In C18(1/2) and C18(1/3)HPC's, side chain melting enthalpies were negligible but isotropization enthalpies were unexpectedly large. The isotropization temperatures and the associated enthalpies remained almost constant in differential scanning calorimetry (DSC) heating/cooling cycles. Distinct morphological features were observed under the cross polarized optical microscope (POM) after shearing the samples in the molten state. The degree of side chain substitution has a profound influence on the polymer morphology.

Protein folding: the endgame.

The last stage of protein folding, the "endgame," involves the ordering of amino acid side-chains into a well defined and closely packed configuration. We review a number of topics related to this process. We first describe how the observed packing in protein crystal structures is measured. Such measurements show that the protein interior is packed exceptionally tightly, more so than the protein surface or surrounding solvent and even more efficiently than crystals of simple organic molecules. In vitro protein folding experiments also show that the protein is close-packed in solution and that the tight packing and intercalation of side-chains is a final and essential step in the folding pathway. These experimental observations, in turn, suggest that a folded protein structure can be described as a kind of three-dimensional jigsaw puzzle and that predicting side-chain packing is possible in the sense of solving this puzzle. The major difficulty that must be overcome in predicting side-chain packing is a combinatorial "explosion" in the number of possible configurations. There has been much recent progress towards overcoming this problem, and we survey a variety of the approaches. These approaches differ principally in whether they use ab initio (physical) or more knowledge-based methods, how they divide up and search conformational space, and how they evaluate candidate configurations (using scoring functions). The accuracy of side-chain prediction depends crucially on the (assumed) positioning of the main-chain. Methods for predicting main-chain conformation are, in a sense, not as developed as that for side-chains. We conclude by surveying these methods. As with side-chain prediction, there are a great variety of approaches, which differ in how they divide up and search space and in how they score candidate conformations.

Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 °C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available.

Effect of temperature on the structure of poly (3-decylthiophene)

The results of the structure investigations of poly (3 -alkylthiophenes) (PATs ) are briefly reviewed. The importance of the side chain order on the experimental diffractograms of PATs is discussed and the model of the relation between the conformation of a polymer and its diffraction pattern is presented. Thermochromism in PATs is related to the conformational changes, as implied from the photoelectron spectroscopy measurements and from the quantum chemical calculations. Our investigations have shown that this phenomenon can be observed by X-ray diffraction. The temperature dependences of the diffractograms obtained for poly (3-decylthiophene) are consistent with the conformational model of thermochromism.

Conformational Study of Poly(p-phenylene) by Molecular Mechanics Minimization

Molecular mechanics minimizations were performed on alkyl-substituted biphenyls to explore the effect of the position and length of side chains on the planarity of the phenyl rings. This approach was extended to poly(p-2,5-di-n-alkylphenylene)s. The structural behavior was investigated by systematically modifying the length of the main chain and the side chains. Studies of substituted biphenyls showed that the methyl substituent at 2, 2‘, 6, and 6‘ positions much affected the torsion angle between the phenyls. The torsion angle decreased and converged to an asymptotic value of ∼70° from trans as the length of side chains was increased in 2,2‘,5,5‘-tetraalkylbiphenyls. The results of a single poly(p-2,5-di-n-alkylphenylene) chain clearly showed that the main chain and side chains cooperate on the molecular level to make a stable conformation. Poly(p-2,5-di-n-alkylphenylene)s with long side chains showed partial ordering of side chains with a plain plate structure, whereas those with short side chains did not show side-chain ordering and had cylindrical, or hairy-rod structure. The onset of side-chain ordering for this single-chain system was found to occur for a sexiphenylene unit with eight carbons in the side chains. The average torsion angles of poly(p-2,5-di-n-alkylphenylene)s decreased from 89° to 61° relative to trans as the length of side chains was increased from methyl to dodecyl.

Ordered Structure of Poly(1H,1H-fluoroalkyl α-fluoroacrylate)s

Structural studies for a homologous series of poly(1H,1H-fluoroalkyl α-fluoroacrylate)s [-{CH 2 CFC(O)OCH 2 (CF 2 ) n Y} p- , Y = H or F, Nc = 1 + n = 2-12] have been performed with X-ray diffraction, DSC, and polarized IR spectral measurements. At room temperature, all the homologues used have an ordered structure (crystallites) which consists of the ordering of main and fluoroalkyl side chains. The structure of crystallites depended on both length (N c ) of fluoroalkyl side chains and chemical structure. In the shorter fluoroalkyl homologues (N c < 7), the side chains are arranged in a double-layer packing at room temperature, while they are mostly in a single-layer packing in the longer fluoroalkyl homologues. As temperature increased, in the short fluoroalkyl homologues, the double-layer packing was more arranged around a temperature above T g and then gradually disturbed, while in the long fluoroalkyl homologues, the single layer packing transformed into a double-layer packing above T g . The layered structure and its change with temperature were discussed from the degree of orderings of the main backbone chains and the fluoroalkyl side chains.

Intermolecular Association and Supramolecular Organization in Dilute Solution. 1. Regioregular Poly(3-dodecylthiophene)

Static and dynamic polarized and depolarized light scattering characterization of dilute solutions of regioregular poly(3-dodecylthiophene) is described to study intermolecular association as a function of the thermal history of the solutions. It is shown that metastable aggregation obtains under all of the conditions studied, including temperatures as high as 65 °C. Under some conditions, the aggregated moiety appears to have a disklike shape at room temperature, with appreciable depolarized scattering, attributed to an extended chain structure, consistent with the formation of the lamellar suprastructure characteristic of the bulk, with the polythiophene chains adopting an extended conforma- tion and forming a nematic phase in a polythiophene lamella faced by lamellae rich in alkyl chains. Under other conditions, especially at low temperature, elongated supramolecular structures are formed, with the polythiophene chains in an extended conformation. The reversible thermochromic effect is associated with enhanced order of the alkyl side chains with decreasing temperature, facilitating coplanar conformers in the polythiophene backbones, with the attendant enhancement in the ﷿-﷿* transitions of the thiophene ring electronic absorption spectra. This behavior is analogous to the events in the thermochromic event in the bulk. Such supramolecular structures could intervene in normal film casting solution processing, with effects on the electronic or optical properties of the cast film. This interpretation of the light scattering data suggests strategies to enhance or suppress lamella formation in solvent-cast films.

Surface Structure Analysis of Polyacrylate Thin Films

The surface contact angles of poly(fluoroalkyl acrylate)s and poly(fluoroalkyl methacrylate)s vary with the cooling mode used after heat treatments. Grazing angle X-ray reflectivity measurement was applied to characterize the surface roughness of polyacrylate thin films on silicon wafers. Surface and interface roughnesses of polyacrylate thin films with large contact angles were measured as 0.7–1.2 nm, which are smaller than roughnesses (1.2–1.8 nm) with small contact angles. It was found that the ordering of side chains in polyacrylate thin films causes the surface to change the contact angles.