Boat conformations. Analysis and simulation of the complex 1H NMR spectrum of methyl 2,6:3,4-dianhydro-alpha-D-altropyranoside

The complex 1H NMR spectrum of methyl 2,6:3,4-dianhydro-alpha-D-altropyranoside (1) has been analyzed and simulated in detail by using input parameters derived from experimental 1H chemical shifts, long- and short-range coupling constants, spin-lattice relaxation times, and effective, spin-spin relaxation times obtained by trial and error matching of the experimental and simulated spectra. The 13C spin-lattice relaxation times of 1 have also been measured, and along with the 1H-1H long- and short-range coupling constants, have been interpreted in terms of the geometry of 1 defined by molecular dynamics with simulated annealing.     

Cell relaxation after electrodeformation: effect of latrunculin A on cytoskeletal actin

Precise measurement of the mechanical properties of a cell provides useful information about its structural organization and physiological state. It is interesting to understand the effect of individual components on the mechanical properties of the entire cell. In this study, we investigate the influence of the cytoskeletal actin on the viscoelastic properties of a cell. Actin-specific agents, including latrunculin A and jasplakinolide, are used to alter the organization of the cytoskeletal actin. Brassica oleracea protoplasts are treated with the drugs and deformed under an external electric potential. The relaxation processes of single protoplasts after electrodeformation are measured. The data are analyzed by a model-independent spectrum recovery algorithm. Two distinct characteristic time constants are obtained from the relaxation spectra. Treatment with latrunculin A increases both of the relaxation time constants. The longest relaxation times for control, latrunculin A treated, and jasplakinolide treated cells are determined to be 0.28, 1.0, and 0.21 s, respectively.     

Nucleosome structure relaxation during DNA unwrapping: Molecular dynamics simulation study

Effects of local relaxation of the nucleosome structure after DNA unwrapping from the histone octamer are considered in this paper. The influence of charge distribution in histones on the kinetics of DNA rewrapping was studied. It was shown that ionic environment rapidly stabilizes during relaxation simulation of the system by molecular dynamics. In the case of short relaxation, a rapid irreversible restoration of the structure, which is similar to a crystal one, occurs. In the case of longer relaxation, DNA rewrapping does not occur despite the absence of apparent differences in the ionic environment of DNA. The change in the quadrupole moment of the system during relaxation was shown.     

The Photocycle of Bacteriorhodopsin in the Two-Dimensional Orthorombic Crystal Form of Purple Membrane

Laser flash photolysis and low-temperature absorption studies of the photocycle of orthorhombic purple membrane (o-PM) reveal the existence of the same K, L, and M intermediates as found in the native hexagonal purple membrane (h-PM). However, the 0 intermediate is missing in the o-PM. The absorption spectrum of the K intermediate of o-PM is blueshifted by ~15 nm relative to the K intermediate found in the hexagonal purple membrane. The decay relaxation time constants of M in the o-PM are higher by more than an order of magnitude than the corresponding relaxation time constants in the h-PM. Similarly to the h-PM, the decay of M depends on the pulse width of excitation. The time-independent anisotropy factor obtained in photoselection studies of the M intermediate demonstrates the complete immobility of bacteriorhodopsin (bR) within the o-PM matrix. The same anisotropy factor of 0.3 obtained for o-PM and for h-PM suggests that in both crystalline lattices the transition moment of the retinal chromophore has similar angles with the plane of the membrane. The dependence of the decay kinetics of M on its occupancy may suggest the existence of kinetic coupling between neighboring bR molecules.     

Fluorescence correlation spectroscopy. I. Conceptual basis and theory

We describe a method for determining chemical kinetic constants and diffusion coefficients by measuring the rates of decay of spontaneous concentration fluctuations. The equilibrium of the system is not disturbed during the measurement. We measure the number of molecules of a specified type in a defined open volume as a function of time and compute the time course of the deviations from the thermodynamic mean concentration. The method is based on the principle that the rates of decay of spontaneous microscopic fluctuations are determined by the same phenomenological rate coefficients as those of macroscopic departures from equilibrium which result from external perturbations. Hence, an analysis of fluctuations yields the same chemical rate constants and diffusion coefficients as are measured by conventional procedures. In practice the number of the specified molecules is measured by a property such as absorbance or fluorescence which is specific and sensitive to chemical change. The sample volume is defined by a light beam which traverses the cell. As the molecules appear in or disappear from the light beam, either due to diffusion or chemical reaction, their concentration fluctuations give rise to corresponding fluctuations of the intensity of absorbed or emitted light. This paper presents the theory needed to derive chemical rate constants and diffusion coefficients from these fluctuations in light intensity. The theory is applied to three examples of general interest: pure diffusion in the absence of chemical reaction; the binding of a small rapidly diffusing ligand to a larger slowly diffusing macromolecule; and a unimolecular isomerization. The method should be especially useful in studying highly cooperative systems, relatively noncooperative systems with intermediate states closely spaced in free energy, small systems, and systems not readily subject to perturbations of state.     

Kinetics of enzyme-coenzyme interactions by NMR spectroscopy.

The kinetics for the binding of coenzymes to H4 and M4 lactate dehydrogenase from chicken were investigated by nuclear magnetic resonance spectroscopy. With detailed computer analysis, some kinetic parameters were extracted from the chemical shifts and the linewidth of the observed coenzyme resonances at various enzyme/coenzyme ratios and temperatures. The results of the analysis indicated that the dissociation rates of coenzymes from the enzyme/coenzyme complexes are slower with the H4 isozyme than those involving the M4 isozyme. The lifetimes for the NAD+-enzyme complexes are on the order of 1 msec while those for the NADH-enzyme complexes are on the order of 10 ms (at room temperature). Much shorter transverse relaxation times of the coenzyme resonances were observed in NADH-enzyme complexes than those in the NAD+-enzyme complexes. The calculated kinetic constants are in good agreement with the previous studies by stopped-flow and temperature jump methods. A generalized NMR kinetic treatment for the binding of small molecules to a macromolecule is presented.     

Electrostatic Potential of B-DNA: Effect of Interionic Correlations

Modified Poisson-Boltzmann (MPB) equations have been numerically solved to study ionic distributions and mean electrostatic potentials around a macromolecule of arbitrarily complex shape and charge distribution. Results for DNA are compared with those obtained by classical Poisson-Boltzmann (PB) calculations. The comparisons were made for 1:1 and 2:1 electrolytes at ionic strengths up to 1 M. It is found that ion-image charge interactions and interionic correlations, which are neglected by the PB equation, have relatively weak effects on the electrostatic potential at charged groups of the DNA. The PB equation predicts errors in the long-range electrostatic part of the free energy that are only ∼1.5 kJ/mol per nucleotide even in the case of an asymmetrical electrolyte. In contrast, the spatial correlations between ions drastically affect the electrostatic potential at significant separations from the macromolecule leading to a clearly predicted effect of charge overneutralization.     

Dielectric increment and dielectric dispersion of solutions containing simple charged linear macromolecules. II. Experimental results with synthetic polyelectrolytes

The electric permittivity of aqueous solutions of different synthetic polyelectrolytes have been measured as a function of frequency in the range 5 kHz up to 100 MHz in the absence of added salt. Solutions of polymethacrylic acid and polyacrylic acid of different degrees of polymerization, both partially neutralized with NaOH, were investigated as well as solutions of Na-polystyrenesulphonate at different concentrations.For all systems a dispersion profile with two separated dispersion regions was obtained with a molecular weight dependent value of the static electric permittivity. The low frequency dispersion region was found to be characterized by a molecular weight dependent mean relaxation time while for the high frequency dispersion region both the mean relaxation time and the dielectric increment are molecular weight independent. It is shown that the reciprocal values of the specific increments and of the relaxation times depend linearly on the macromolecular concentration. Extrapolation of the corresponding quantities to infinite dilution was found to be possible. A comparison of these extrapolated values with calculated ones according to the previously derived theory also applicable to flexible macromolecules establishes that this theory describes satisfactorily the dielectric behaviour of the systems investigated.The conclusion is reached that the high frequency dispersion and relaxation can be attributed to fluctuations in the distribution of bound counterions along limited parts of the macromolecule. The relaxation time of the low frequency dispersion region seems to be essentially determined by the rotation of the complete molecule and the static electric permittivity can he explained in terms of fluctuations in the counterion density extending over the whole macromolecule.     

On the reality of transition state dissociation constants

Transition state dissociation constants are currently considered, utilizing stopped flow equipment. The underlying theory is briefly reviewed, relating the ideas to steady state kinetics of enzyme systems. The ideas are further analyzed under the consideration of chemical relaxation. Test conditions are described which would allow an investigation of the concepts of transition state dissociation constants by chemical relaxation techniques. A discussion concerning the way in which the concepts of transition state dissociation constants relate to other theories which assume short-lived, but real, dissociation constants is included. The theory is rigorously analyzed (in a second part), revealing the nature of the assumption of a transition state dissociation constant: While they may be written in a formal manner, they are not based on reality—on kinetic grounds direct interconversions between transition states are practically impossible. This applies also to transition state dissociation constants involving protons.     

Comparison of time constants of single channel patches,quantum bumps,and noise analysis inLimulus ventral photoreceptors

Summary The characteristic time constants derived from three different experimental procedures for measuring light-evoked currents in photoreceptors are compared; these procedures include single-channel patch-clamp measurements, noise analysis, and current relaxation studies. Recent patch-clamp measurements of the mean open times of single light-activated channels in the ventral photoreceptor ofLimulus (Bacigalupo, J., Lisman, J.E. (1983),Nature (London) 304:268–270) yield a disagreement of the measured mean open time with the relaxation time of the falling phase of quantum bumps and with the inverse characteristic frequency of the noise power spectrum, measured by Wong (Wong, F. (1978),Nature (London) 276:76–79). We present new experimental results which show that the relaxation time of the falling phase of bumps is markedly shortened by light-adaptation. Hence the state of light-adaptation has to be taken into account when comparing different experiments. Secondly, we investigate three simple models for the mechanism of channel opening and closing, and conclude that an agreement of the mean open time of single channels, the relaxation time of the falling phase of bumps, and the inverse characteristic frequency of the noise power spectrum cannot be expected.     

Kinetics of allosteric conformational transition of a macromolecule prior to ligand binding

Two fundamentally different mechanisms of ligand binding are commonly encountered in biological kinetics. One mechanism is a sequential multistep reaction in which the bimolecular binding step is followed by first-order steps. The other mechanism includes the conformational transition of the macromolecule, before the ligand binding, followed by the ligand binding process to one of the conformational states. In stopped-flow kinetic studies, the reaction mechanism is established by examining the behavior of relaxation times and amplitudes as a function of the reactant concentrations. A major diagnostic tool for detecting the presence of a conformational equilibrium of the macromolecule, before the ligand binding, is the decreasing value of one of the reciprocal relaxation times with the increasing [ligand]. The sequential mechanism cannot generate this behavior for any of the relaxation times. Such dependence is intuitively understood on the basis of approximate expressions for the relaxation times that can be comprehensively derived, using the characteristic equation of the coefficient matrix and polynomial theory. Generally, however, the used approximations may not be fulfilled. On the other hand, the two kinetic mechanisms can always be distinguished, using the approach based on the combined application of pseudo-first-order conditions, with respect to the ligand and the macromolecule. The two experimental conditions differ profoundly in the extent of the effect of the ligand on the protein conformational equilibrium. In a large excess of the ligand, the conformational equilibrium of the macromolecule, before the ligand binding, is strongly affected by the binding process. However, in a large excess of the macromolecule, ligand binding does not perturb the internal equilibrium of the macromolecule. As a result, the normal mode, affected by the conformational transition, is absent in the observed relaxation process. In the case of a sequential mechanism, the number of relaxation times is not altered by different pseudo-first-order conditions. Thus, the approach provides a strong diagnostic criterion for detecting the presence of the conformational transition of the macromolecule and establishing the correct mechanism. Application of this approach is illustrated for the binding of 3'-O-(N-methylantraniloyl)-5'-diphosphate to the E. coli DnaC protein.     

In vivo brain macromolecule signals in healthy and glioblastoma mouse models: 1H magnetic resonance spectroscopy,post‐processing and metabolite quantification at 14.1 T

In ¹H magnetic resonance spectroscopy, macromolecule signals underlay metabolite signals, and knowing their contribution is necessary for reliable metabolite quantification. When macromolecule signals are measured using an inversion‐recovery pulse sequence, special care needs to be taken to correctly remove residual metabolite signals to obtain a pure macromolecule spectrum. Furthermore, since a single spectrum is commonly used for quantification in multiple experiments, the impact of potential macromolecule signal variability, because of regional differences or pathologies, on metabolite quantification has to be assessed. In this study, we introduced a novel method to post‐process measured macromolecule signals that offers a flexible and robust way of removing residual metabolite signals. This method was applied to investigate regional differences in the mouse brain macromolecule signals that may affect metabolite quantification when not taken into account. However, since no significant differences in metabolite quantification were detected, it was concluded that a single macromolecule spectrum can be generally used for the quantification of healthy mouse brain spectra. Alternatively, the study of a mouse model of human glioma showed several alterations of the macromolecule spectrum, including, but not limited to, increased mobile lipid signals, which had to be taken into account to avoid significant metabolite quantification errors.     

Assignment of methylene proton resonances in NMR spectra of embryonic and transformed cells to plasma membrane triglyceride

Some biological characteristics of cancer cells and solid tumors are identifiable by the high resolution NMR relaxation behavior of their nonaqueous components. Chemical analysis and two-dimensional scalar correlated (COSY) NMR spectroscopy show these resonances arise from neutral lipid in the plasma membrane. Triglyceride is shown to be the main plasma membrane component giving rise to the NMR spectrum, while soluble nonmembrane components account for 90% of the remaining resonances in the spectrum of intact cells. The presence of triglyceride has been detected by chemical analysis in highly purified plasma membranes from two different cell lines. The COSY spectra of cancer cells are comparable with that obtained for the triglyceride-rich very low density human lipoprotein.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Detection of thymosin beta 4 in situ in a guinea pig cerebral cortex preparation using 1H NMR spectroscopy.

In the present work we have investigated the macromolecules that contribute to the brain 1H NMR spectrum. The cerebral cortex showed distinct resonances at the uncrowded methyl- and methylene chemical shift scale of the spin-echo 1H NMR spectrum. The peaks at 1.22 and 1.40 ppm (relative to the methyl protons of N-acetyl aspartate at 2.02 ppm) arise from cerebral macromolecules without evidence for co-resonances from low molecular weight metabolites as shown by the spin-spin relaxation decays of these resonances. In addition to these NMR signals, peaks at 0.9 and 1.7 ppm from macromolecules were detected. These resonances are from proteins, and we have identified the polypeptides that contributed to the 1H NMR peaks. Two proteins that were present at concentrations of 250 and 350 micrograms/g of dryed tissue showed 1H NMR spectra that resembled the macromolecular pattern in the cerebral 1H NMR spectrum. They were identified as thymosin beta 4 and histone H1, respectively. Thymosin beta 4 was present in soluble high speed cytoplasmic fraction and in P2 pellet, whereas histone H1 was detected in nuclear enriched fraction. A chemical shift-correlated two-dimensional 1H NMR spectrum of thymosin beta 4 in vitro revealed a coupling pattern that matched the macromolecule in the cerebral cortex which we have previously noted (Kauppinen R. A., Kokko, H., and Williams, S. R. (1992) J. Neurochem. 58, 967-974). On the basis of both one- and two-dimensional NMR evidence, subcellular distribution and high concentration, we assign the 1H NMR signals at 0.9, 1.22, 1.40, and 1.7 ppm in the cerebral cortex to thymosin beta 4.     

Nuclear magnetic resonance conformational studies on the chemotactic tripeptide formyl-L-methionyl-L-leucyl-L-phenylalanine. A small beta sheet.

Previous work by several groups has shown that the combination of spin--spin coupling constants and spectral density components (derived from spin--lattice relaxation and/or nuclear Overhauser measurements) may aid in the task of conformational determination of peptides in solution. Using the peptide formyl-L-methionyl-L-leucyl-L-phenylalanine, which is a potent specific chemotactic agent for leucocytes, we show the following: (a) that 3JNHCH coupling constants are consistent with a high degree of rigidity in the peptide backbone in solution, (b) that 3H isotopic substitution in combination with relaxation data taken at different Larmor frequencies enables spectral density, and thence conformational, information to be obtained, (c) that side-chain conformations for this molecule mirror, in some aspects, those found in the solid state for other peptides containing the same residues, and (d) that temperature dependence of amide chemical shifts does not have direct implication concerning the existence of intramolecular hydrogen bonds in peptides. We are able to propose a family of conformations which appear to interchange rapidly on the NMR time scale and are characterized by a distribution of side-chain rotamers. The basic backbone conformation is, or closely approximates, a small beta antiparallel pleated sheet and as such suggests a possible mode of receptor--chemotactic peptide interaction.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.