Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy

NMR measurements of a large set of protein backbone one-bond dipolar couplings have been carried out to refine the structure of the third IgG-binding domain of Protein G (GB3), previously solved by X-ray crystallography at a resolution of 1.1 A. Besides the commonly used bicelle, poly(ethylene glycol), and filamentous phage liquid crystalline media, dipolar couplings were also measured when the protein was aligned inside either positively or negatively charged stretched acrylamide gels. Refinement of the GB3 crystal structure against the (13)C(alpha)-(13)C' and (13)C'-(15)N dipolar couplings improves the agreement between experimental and predicted (15)N-(1)H(N) as well as (13)C(alpha)-(1)H(alpha) dipolar couplings. Evaluation of the peptide bond N-H orientations shows a weak anticorrelation between the deviation of the peptide bond torsion angle omega from 180 degrees and the angle between the N-H vector and the C'-N-C(alpha) plane. The slope of this correlation is -1, indicating that, on average, pyramidalization of the peptide N contributes to small deviations from peptide bond planarity ( = 179.3 +/- 3.1 degrees ) to the same degree as true twisting around the C'-N bond. Although hydrogens are commonly built onto crystal structures assuming the N-H vector orientation falls on the line bisecting the C'-N-C(alpha) angle, a better approximation adjusts the C(alpha)-C'-N-H torsion angle to -2 degrees. The (15)N-(1)H(N) dipolar data do not contradict the commonly accepted motional model where angular fluctuations of the N-H bond orthogonal to the peptide plane are larger than in-plane motions, but the amplitude of angular fluctuations orthogonal the C(alpha)(i-1)-N(i)-C(alpha)(i) plane exceeds that of in-plane motions by at most 10-15 degrees. Dipolar coupling analysis indicates that for most of the GB3 backbone, the amide order parameters, S, are highly homogeneous and vary by less than +/-7%. Evaluation of the H(alpha) proton positions indicates that the average C(alpha)-H(alpha) vector orientation deviates by less than 1 degrees from the direction that makes ideal tetrahedral angles with the C(alpha)-C(beta) and C(alpha)-N vectors.     

NMR analysis of interaction between styrene-divinylbenzene gel beads and small molecules

The interaction of small molecules such as cyclohexane, tetrahydrofuran, and acetonitrile with styrene-divinylbenzene copolymer gel beads were investigated by ¹³C-NMR spectroscopy. When the gel content was 0.1–0.3 g/mL, the ¹³C-NMR spectrum of the solvent displayed two peaks. A sharp peak at lower magnetic field was assigned to the free solvent, and a broad one to the solvent affected by the gel. This signal splitting is attributed to the upfield shift caused by aromatic rings of styrene units in the polymer chain. The nitrile carbon of acetonitrile showed the largest upfield shift. The mobility of small molecules in gel beads was also investigated using the nuclear magnetic relaxation method. In the case of good solvents for gel beads, the mobility was affected by the cross-linking density, whereas poor solvents exhibited little dependence of the cross-linking density. © 1995 John Wiley & Sons, Inc.     

Powder crystallography by proton solid-state NMR spectroscopy

The investigation of 1H-1H spin-diffusion build-up curves using a rate matrix analysis approach shows that high-resolution magic angle spinning NMR of protons, applied to powdered organic compounds, provides a method to probe crystalline arrangements. The comparison between experimental 1H data and simulation is shown to depend strongly on the parameters of the crystal structure, for example on the unit cell parameters or the orientation of the molecule in the unit cell, and those parameters are experimentally determined for a model organic compound.     

Diffusion of small molecules inside a peptide hydrogel

A set of four phenylalanine analogues experiences diffusion retardation when transferred from phosphate-buffered saline into a peptide hydrogel of the same pH and ionic strength. The extent of retardation increases linearly with logP(oct), their lipophilicity.     

Geometry determination of tetrasubstituted stilbenes by proton NMR spectroscopy

A simple spectroscopic method was applied to determine the geometry of tetrasubstituted alkenes. The observation of the ⁵J-coupling constants in proton NMR spectra on the ¹³C satellite signals could confirm the previous misassignment of 2,3-diphenylbutene. Hence, the (E)-isomer showed a 1.5 Hz coupling constant, whereas the (Z)-isomer showed a 1.1 Hz coupling constant. Based on this new assignment and a stereospecific preparation, we also propose a revision concerning the NMR data of 2,3-diphenyl-2,3-butanediol.     

Diffusion of small molecules in glassy polymers

Small molecules in glassy polymers are considered to occupy sites with a distribution of free energies of dissolution. Then their diffusivity depends on concentration and temperature in the same way as it has been derived for hydrogen atoms in metallic glasses. For hydrogen it was shown that the tracer diffusion coefficient is proportional to the activity coefficient of the solute atoms. The latter can be evaluated from measured data of sorption of the small molecules in the polymer. Knowing this quantity, the thermodynamic factor can be calculated and the concentration dependence of the mutual diffusion coefficient is obtained in excellent agreement with published experimental results. New experimental results are presented for the diffusion coefficient of CO₂ in Kapton and four polycarbonates (BPA-PC, BPZ-PC, TMBPA-PC, and TMC-PC) in the low CO₂ pressure range of a few mbar up to 1 bar. The results are in agreement with the model developed for hydrogen. The reference diffusion coefficient, which is a fitting parameter of the model that is independent of the distribution of free energies is smallest for the polycarbonate BPZ-PC having a high γ-relaxation temperature. This correlation between the diffusion coefficient and the dynamics of the polymer can be found for other substituted polycarbonates as well. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35 : 2397–2408, 1997     

Spin-pattern recognition in high-resolution proton NMR spectroscopy

Techniques are introduced for the recognition of specific spin patterns in complex overlapping proton NMR spectra. They are based on the efficient and selective excitation of high-order multiple quantum coherence in systems of specified coupling network topology, and allow the suppression of undesired spin patterns.     

Influence of kappa-carrageenan gel structures on the diffusion of probe molecules determined by transmission electron microscopy and NMR diffusometry

The influence of the microstructures of different kappa-carrageenan gels on the self-diffusion behavior of poly(ethylene glycol) (PEG) has been determined by nuclear magnetic resonance (NMR) diffusometry and transmission electron microscopy (TEM). It was found that the diffusion behavior was determined mainly by the void size, which in turn was defined by the state of aggregation of the kappa-carrageenan. The kappa-carrageenan concentration was held constant at 1 w/w%, and the aggregation was controlled by the amount of potassium and/or sodium chloride and, for samples containing potassium, also by the cooling rate. Gels containing potassium formed microstructures where kappa-carrageenan strands are rather evenly distributed over the image size, while sodium gels formed dense biopolymer clusters interspersed with large openings. In a gel with small void sizes, relatively slow diffusion was found for all PEG sizes investigated. Extended studies of the self-diffusion behavior of the 634 g mol(-)(1) PEG showed that there is a strong time dependence in the measured PEG diffusion. An asymptotic lower time limit of the diffusion coefficient was found in all gels when the diffusion observation time was increased. According to the ratio, D/D(0), where D(0) is the diffusion coefficient in D(2)O and D is the diffusion coefficient in the gels, the gels could be divided into three classes: small, medium, and large voids. For quenched kappa-carrageenan solutions with salt concentrations of 20 mM K(+), 100 mM K(+), or 20 mM K(+)/200 mM Na(+) as well as slowly cooled solutions with only 20 mM K(+), D/D(0) ratios between 0.18 and 0.29 were obtained. By quenching a kappa-carrageenan solution with 100 mM K(+), the D/D(0) was 0.5, while D/D(0) ratios between 0.9 and 1 were obtained in a quenched solution with 250 mM Na(+) and slowly cooled samples with 20 mM K(+)/200 mM Na(+) or 250 mM Na(+).     

Study on transport of molecules in gel by surface-enhanced Raman spectroscopy

Cellulose - Surface-enhanced Raman spectroscopy (SERS)-based biosensors have recently been extensively developed because of their high sensitivity and nondestructive nature. Conventional SERS...     

The structure of the proton skeleton in 1,3-butadieneiron Tricarbonyl as determined from the NMR spectra of oriented molecules

¹H NMR spectra of 1,3-butadieneiron tricarbonyl oriented in a nematic liquid crystal solvent have been analysed and used to derive the structure of the proton skeleton, which is found to be non-planar. The distance of the anti protons from the plane defined by the remaining four protons is 0.77 ± 0.06 ±.     

Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy

The dynamics of weakly bending polymers is analyzed on the basis of a Gaussian semiflexible chain model and the fluorescence correlation spectroscopy (FCS) correlation function is determined. Particular attention is paid to the influence of the rotational motion on the decay of the FCS correlation function. An analytical expression for the correlation function is derived, from which the averaged segmental mean square displacement can be determined independent of any specific model for the polymer dynamics. The theoretical analysis exhibits a strong dependence of the correlation function on the rotational motion for semiflexible polymers with typical lengths and persistence lengths of actin filaments or fd viruses. Hence, FCS allows for a measurement of the rotational motion of such semiflexible polymers. The theoretical results agree well with experimental measurements on actin filaments and confirm the importance of large relaxation times.     

Improved resolution in proton NMR spectroscopy of powdered solids

We present a new solid-state nuclear magnetic resonance experiment that yields, under CRAMPS decoupling conditions, a significant reduction in proton line widths for powdered organic solids. This experiment which relies on a constant-time acquisition of the proton transverse magnetization, removes the contribution of nonrefocusable broadening from the proton line widths. Although this new technique suffers from relatively low sensitivity, we demonstrate in this paper its feasibility on two model samples, L-alanine and the dipeptide Ala-Asp. In both cases a factor of between 2 and 3 in line width reduction is obtained for most of the proton resonances.     

Application of gel chromatography to small molecules

Summary Liuid chromatographic separations of hydrocarbons, alcohols, triglycerides and surfactants with molecular weights ranging from 100–1,000 were accomplished, using columns packed with porous polymeric beads. The fractionation capability of the porous gels has been extended to smaller molecules through the development of small porosity gels. The gels separated according to molecular size and are therefore useful both for separation and identification purposes. Since they require no stationary liquid phase, they afford long column life without need of a saturated carrier.

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Zusammenfassung Flüssigkeits-chromatographische Trennungen von Kohlenwasserstoffen, Alkoholen, Triglyceriden und oberflächenaktiven Substanzen mit Molekulargewichten von 100–1000 wurden mit Hilfe von porösen Polymerkörnern erreicht. Das Trennvermögen der porösen Gele wurde durch Entwicklung kleinporiger Typen auf kleinere Moleküle ausgedehnt. Da die Moleküle entsprechend ihrer Größe getrennt werden, kann außer der Trennung auch eine Identifizierung erreicht werden. Da keine flüssige stationäre Phase erforderlich ist, ist eine lange Lebensdauer der Säulen gewährleistet.

     

Assignment of proton NMR spectra and conformational analysis by two-dimensional heteronuclear relayed correlation NMR spectroscopy

The method of two-dimensional heteronuclear relayed correlation spectroscopy was used to establish the assignment of the severely overcrowded part of the proton spectrum of menthol by relating it to the previously assigned carbon spectrum. Extrapolation of the signal-to-noise ratio obtained with overnight data accumulation on a 10 mM solution suggests that this experiment should be feasible on as little as 10 mg of a moderate-sized organic compound.