NMR structural refinement of an extrahelical adenosine tridecamer d(CGCAGAATTCGCG)2 via a hybrid relaxation matrix procedure

Until very recently interproton distances from NOESY experiments have been derived solely from the two-spin approximation method. Unfortunately, even at short mixing times, there is a significant error in many of these distances. A complete relaxation matrix approach employing a matrix eigenvalue/eigenvector solution to the Bloch equations avoids the approximation of the two-spin method. We have calculated the structure of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex, d(CGCAGAATTCGCG)2, by an iterative refinement approach using a hybrid relaxation matrix method combined with restrained molecular dynamics calculations. Distances from the 2D NOESY spectra have been calculated from the relaxation rate matrix which has been evaluated from a hybrid NOESY volume matrix comprising elements from the experiment and those calculated from an initial structure. The hybrid matrix derived distances have then been used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure is then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. Although the crystal structure of the tridecamer clearly shows the extrahelical adenosine looped out way from the duplex, the NOESY distance restrained hybrid matrix/molecular dynamics structural refinement establishes that the extrahelical adenosine stacks into the duplex.     

Strategy for supplementing structure calculations using limited data with hydrophobic distance restraints

Introductory biochemistry texts often note that the fold of a protein is completely defined when the dihedral angles phi and psi are known for each amino acid. This assertion was examined with torsion angle dynamics and simulated annealing (TAD/SA) calculations of protein G using only dihedral angle restraints. When all dihedral angles were restrained to within 1 degrees of the values of the X-ray structure, the TAD/SA structures gave a backbone root mean square deviation to the target of 4 A. Factors that contributed to divergence from the correct solution include deviations of peptide bonds from planarity, internal conflicts resulting from the nonuniform energies of different phi, psi combinations, and relaxation to extended conformations in the absence of long-range constraints. Simulations including hydrogen-bond restraints showed that even a few long-range contacts constrain the fold better than a complete set of accurate dihedral restraints. A procedure is described for TAD/SA calculations using hydrogen-bond restraints, idealized dihedral restraints for residues in regular secondary structures, and "hydrophobic distance restraints" derived from the positions of hydrophobic residues in the amino acid sequence. The hydrogen-bond restraints are treated as inviolable, whereas violated hydrophobic restraints are removed following reduction of restraint upper bounds from 2 to 1 times the predicted radius of gyration. The strategy was tested with simulated restraints from X-ray structures of proteins from different fold classes and NMR data for cold shock protein A that included only backbone chemical shifts and hydrogen bonds obtained from a long-range HNCO experiment.     

Nuclear Overhauser effect and cross-relaxation rate determinations of dihedral and transannular interproton distances in the decapeptide tyrocidine A.

The following interproton distances are reported for the decapeptide tyrocidine A in solution: (a) r(phi) distances between NH(i) and H alpha (i), (b) r(psi) distances between NH (i + 1) and H alpha (i), (c) r(phi psi) distances between NH(i + 1) and NH(i), (d) NH in equilibrium NH transannular distances, (e) H alpha in equilibrium H alpha transannular distances, (f) r x 1 distances between H alpha and H beta protons, (g) NH(i) in equilibrium H beta (i) distances, (h) NH (i + 1) in equilibrium H beta (i) distances, (i) carboxamide-backbone protons and carboxamide-side chain proton distances, (j) side chain proton-side chain proton distances. The procedures for distance calculations were: NOE ratios and calibration distances, sigma ratios and calibration distances, and correlation times and sigma parameters. The cross-relaxation parameters were obtained from the product, say, of NOE 1 leads to 2 and the monoselective relaxation rate of proton 2; the NOEs were measured by NOE difference spectroscopy. The data are consistent with a type I beta-turn/ type II' beta-turn/ approximately antiparallel beta-pleated sheet conformation of tyrocidine A in solution and the NOEs, cross-relaxation parameters, and interproton distances serve as distinguishing criteria for beta-turn and beta-pleated sheet conformations. It should be borne in mind that measurement of only r phi and r psi distances for a decapeptide only defines the ( phi, psi)-space in terms of 4(10) possible conformations; the distances b-j served to reduce the degeneracy in possible (phi, psi)-space to one tyrocidine A conformation. The latter conformation is consistent with that derived from scalar coupling constants, hydrogen bonding studies, and proton-chromophore distance measurement, and closely resembles the conformation of gramicidin S.     

Smoluchowski dynamics of the vnd/NK‐2 homeodomain from Drosophila melanogaster: First‐order mode‐coupling approximation

This work is the first in a series devoted to applying mode coupling diffusion theory to the derivation of local dynamics properties of proteins in solution. The first‐order mode‐coupling approximation, or optimized Rouse–Zimm local dynamics (ORZLD), is applied here to derive the rotational dynamics of the bonds and compare the calculated with the experimental nmr ¹⁵N spin–lattice relaxation time behavior of the vnd/NK‐2 homeodomain from Drosophila melanogaster. The starting point for the calculations is the experimental three‐dimensional structure of the homeodomain determined by multidimensional nmr spectroscopy. The results of the computations are compared with experimentally measured ¹⁵N spin–lattice relaxation times T₁, at 34.5 and 60.8 MHz, to check the first‐order approximation. To estimate the relative importance of internal and overall rotation, both rigid and fluctuating dynamic models are examined, with fluctuations evaluated using molecular dynamics (MD) simulations. The correlation times for the fundamental bond vector time correlation function and for the second‐order bond orientational TCF are obtained as a function of the residue number for vnd/NK‐2. The stability of the corresponding local dynamics pattern for the fluctuating structure as a function of the length of the MD trajectory is presented. Diffusive dynamics, which is essentially free of model parameters even at first order in the mode‐coupling diffusion approach, confirm that local dynamics of proteins can be described in terms of rotational diffusion of a fluctuating quasi‐rigid structure. The comparison with the nmr data shows that the first‐order mode coupling diffusion approximation accounts for the correct order of magnitude of the results and of important qualitative aspects of the data sensitive to conformational changes. Indications are obtained from this study to efficiently extend the theory to higher order in the mode‐coupling expansion. These results demonstrate the promise of the mode‐coupling approach, where the local dynamics of proteins is described in terms of rotational diffusion of a fluctuating quasi‐rigid structure, to analyze nmr spin–lattice relaxation behavior. © 1999 John Wiley & Sons, Inc. Biopoly 49: 235–254, 1999     

A fiber matrix model for interstitial fluid flow and permeability in ligaments and tendons

Collagen fibrils in ligaments and tendons are highly organized into parallel arrays which influence interstitial fluid transport. Finite element (FE) models were developed analogous to the fibrillar arrays in ligaments and tendons to investigate interstitial fluid flow and tissue permeability as a function of interfibrillar spacing and fluid properties. Collagen fibrils were assumed to be a periodic square array of impermeable cylinders. A two-dimensional FE model was used to study transverse fluid flow and a three-dimensional model was used to study flow parallel to the collagen fibrils. Parametric FE analysis provided data to formulate empirical expressions for permeability (kappa) as a function of porosity (phi). Results show that longitudinal permeability (kappa = 1.1.10(-15)phi 2.5[1 - phi]-0.333) can be up to 50 times higher than transverse permeability (kappa = 1.2.10(-15)phi 0.5[phi - phi min]2.5) in a compact array. Maximum fluid shear stresses occur at the narrowest zones of adjacent fibrils (1.21 Pa or 12.1 dyn/cm2 at 10 microns/s of average transverse influx). If interstitial fluid is highly non-Newtonian, the permeability should be considered as flow (shear)-dependent. The computational results suggest that tissue permeability in ligaments and tendons is highly anisotropic, porosity-dependent, and can be estimated by analytic expressions.     

How accurately can oligonucleotide structures be determined from the hybrid relaxation rate matrix/NOESY distance restrained molecular dynamics approach?

The accuracy and precision of structures derived from a combined hybrid relaxation rate matrix/NOESY distance restrained molecular dynamics methodology were examined with simulations that included typical experimental errors. NOESY data were simulated for a DNA dodecamer duplex, d-(CGCGAATTCGCG)2, with added volume error of approximately 20% and low-level thermal noise. Distances derived from a hybrid relaxation matrix analysis of the NOE data were used as constraints in molecular dynamics driven structural refinements of several initial model geometries. The final structures were compared against results obtained from the traditional isolated two-spin approximation treatment of these NOESY volumes and also against refined structures that employed error-free data. Results show that the structures derived from the relaxation rate matrix analysis of the NOESY data are more accurate than those derived from a simple two-spin approximation analysis and it is possible to achieve refinement to the level of simulated experimental error. Results may be significantly improved with the use of either more accurately measured NOESY volumes or additional matrix-derived constraints. Many of the helical parameters and backbone torsional angles may be accurately reproduced by the hybrid matrix methodology.     

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.     

Dynamics of erabutoxin b as studied by nuclear magnetic resonance. Relaxation studies of methyl proton resonances

Longitudinal and transverse relaxation times were measured for well-resolved and assigned methyl proton resonances of erabutoxin b at 270 MHz, 300 MHz and 500 MHz. Both longitudinal and transverse magnetization decay curves are non-exponential due to cross-relaxation and cross-correlation effects. The longitudinal and transverse relaxation rates were obtained from the initial slope of both magnetization decay curves. The correlation times for the isotropic tumbling motion of the protein were determined to be 2.82 ns at 300 K and 1.62 ns at 330 K from the analysis of the relaxation data of some alpha protons. Using these values, the relaxation data of methyl protons were fitted to various theoretical models. Most of the methyl resonances could be fitted well to a model which allowed methyl rotation (in the range 0.01-0.05 ns) and an external contribution from protons assumed to be in positions derived from X-ray coordinates. The data for a few methyl groups, however, could not be fitted in this way. For these a smaller number of external protons than predicted by the X-ray coordinates was assumed. Additionally, a larger amplitude motion had to be introduced into the model for particular residues. This additional motion requires concerted protein motion close to these residues, since the X-ray structure suggests that steric hindrance would prevent local motion. These results are consistent with the idea of a flexible and dynamic structure for proteins.     

Separation of the variables of time and temperature in the mechanical properties of high sugar/polysaccharide mixtures

Experimental results from previous studies were analyzed in order to separate the dynamic mechanical properties of high sugar/polysaccharide mixtures into a basic function of temperature alone and a basic function of time alone. In doing so, the energy of vitrification as derived from the Williams, Landel, and Ferry equation, and the distribution function of relaxation times were used. It was found that the temperature course of vitrification depends on the nature of the polymer and the composition of the mixture. Thus, at the same level of cosolute, the glass transition temperature of the mixture is determined by the structural behavior of the macromolecule and, it appears, that cation-mediated associations--for example, of kappa-carrageenan--are more efficient "vitrifiers" than the neutral associations of agarose. Regardless of the glass transition temperature, vitrification requires five times the activation energy of elementary flow in the melt or of the viscoelastic relaxation in the rubbery state. In the region of long time scales of measurement, the time function is determined by the molecular weight distribution and the ability of the polysaccharide to form a three-dimensional network. In the area of short times, free volume effects leading to vitrification are similar for all materials.     

Relaxation data in NMR structure determination: model calculations for the lysozyme-Gd3+ complex

The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 A resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid "distance geometry-dynamic simulated annealing" procedure. A limited set of 769 NOEs was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 phi angles were also restrained on the basis of amide-alpha coupling constants calculated from the X-ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3+ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.     

Sampling of protein dynamics in nanosecond time scale by 15N NMR relaxation and self-diffusion measurements.

This paper presents a procedure for detection of intermediate nanosecond internal dynamics in globular proteins. The procedure uses 1H-15N relaxation measurements at several spectrometer frequencies and hydrodynamic calculations based on experimental self-diffusion coefficients. New heteronuclear experiments, using pulse field gradients, are introduced for the measurement of translation diffusion coefficients of 15N labeled proteins. An advanced interpretation of recently published (Luginbühl et al., Biochemistry, 36, 7305-7312 (1997)) backbone amide 15N relaxation data, measured at two spectrometers (400 and 750 MHz for 1H) for N-terminal DNA-binding domain (1-63) of 434 repressor, is presented. Non-applicability of commonly used fast (picosecond) dynamics model (FD) was justified by (i) poor fit of relaxation data by the FD model-free spectral density function both for isotropic and anisotropic models of the overall molecular tumbling; (ii) specific dependence of the overall rotation correlation times calculated from T1/T2 ratio on the spectrometer frequency; (iii) mismatch of the ratio of longitudinal 15N relaxation times T1, measured at different spectrometer frequencies, in comparison with that anticipated for the FD model; (iv) significantly underestimated overall rotation correlation time provided by the FD model (5.50+/-0.15 and 5.80+/-0.15 ns for 750 and 400 MHz spectrometer frequency respectively) in comparison with correlation time obtained from hydrodynamics. On the other hand, all relaxation and hydrodynamics data are in good correspondence with the model of intermediate (nanoseconds) dynamics. Overall rotation correlation time of 7.5+/-0.7 ns was calculated from experimental translation self-diffusion rate using hydrodynamics formalism (Garcia de la Torre, J. and Bloomfield, V.A. Quart. Rev. Biophys., 14, 81-139 (1981)). The statistical analysis of 15N relaxation data along with the hydrodynamic consideration clearly revealed that most of the residues in 434(1-63) repressor are involved in the nanosecond internal dynamics characterized by the the mean order parameters of 0.59+/-0.06 and the correlation times of ca. 5 ns.     

Molecular dynamics through 2H spin-lattice relaxation time measurements within an enzyme-inhibitor complex: Lysozyme and methyl N-acetyl glucosamin

Deuteron spin-lattice relaxation times of specifically labelled methyl N-acetyl-D-glucosaminides associated to lysozyme were measured from ¹H and ²H NMR spectra through bandshape analysis and FT inversion-recovery technique, respectively. Model calculations were carried out in order to assess the limits of the extreme narrowing approximation for the systems studied. Rotational correlation times of the acelamido methyl groups were analyzed in terms of anisolropic overall reorientation combined with internal rotation. The acetamido methyl group undergoes fast internal rotation in the α-glycoside complex about an axis nearly parallel with the major ellipsoidal axis of lysozyme. More rotational freedom is likely to occur in the β-glycoside complex.     

Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function

The effects of granulocyte-macrophage (GM)-CSF on the synthesis of MHC class II molecules and on the Ag presentation capacity by bone marrow derived macrophages (BMM phi) was investigated. BMM phi obtained by in vitro culture in the presence of macrophage-CSF were negative for synthesis of I-A molecules and induced the Ag-mediated proliferation of insulin-specific T clone cells with lower efficiency than splenic accessory cells. After pulse treatment with GM-CSF for 24 to 48 h, day 12 BMM phi exhibited highly efficient Ag presentation function which was superior to that induced by IFN-gamma. Expression of membrane-bound IL-1 was augmented significantly by GM-CSF, but not by IFN-gamma. However, the T cell clone used to probe for accessory cell function of BMM phi was not dependent on IL-1 for optimal proliferation. Concomitantly, GM-CSF induced the de novo synthesis of I-A molecules, although to a lesser extent than optimal doses of IFN-gamma. Thus GM-CSF appears to elicit properties in addition to Ia molecule synthesis and membrane IL-1 expression in BMM phi being essential for efficient accessory cell function to the T clone cells. The activation of BMM phi by GM-CSF was reversible and could be repeated. These data show that GM-CSF exerts a modulatory influence on preformed BMM phi, reversibly activating cells to Ia biosynthetic potential and pronounced accessory cell capacity, thus rendering the explanation unlikely that differentiation of precursor cells into a constitutively functional state had occurred.     

Phase fluorometric studies of spectral relaxation at the lipid-water interface of phospholipid vesicles

We examined the dynamic properties of the lipid-water interface region of model membranes using the probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). For comparison we also examined the temperature-dependent spectral properties of TNS in the viscous solvent glycerol. Fluorescence phase shift and demodulation measurements were used to prove that the membranes relax around the excited state of TNS on the ns time scale. The rate of spectral relaxation is thought to reflect the mobility of the polar interface region of the membranes on this same time scale. The spectral relaxation times were estimated by the use of phase-sensitive detection of fluorescence. Using this method one may directly record, in an approximate fashion, the emission spectra of the relaxed and the initially excited states of TNS. The relative intensities of these phase-sensitive spectra, in combination with the measured phase and modulation values on the short and long wavelength sides of the emission, yield the spectral relaxation times. For saturated and unsaturated phosphatidylcholines, at temperatures ranging from 5 to 50°C, the relaxation times ranged from 5 to 1 ns. The activation energies for spectral relaxation were near 4 kcal/mol. Surprisingly, the relaxation times decreased smoothly with increasing temperature, and did not change abruptly at the phase transition temperatures. These results indicate that the small molecular motions of the interface region of membranes, which are responsible for spectral relaxation, are not dramatically influenced by the phase state of the acyl side chain region of the membranes.     

Electrodiffusion of ions approaching the mouth of a conducting membrane channel.

The movement of ions in the aqueous medium as they approach the mouth (radius a) of a conducting membrane channel is analyzed. Starting with the Nernst-Planck and Poisson equations, we derive a nonlinear integrodifferential equation for the electric potential, phi(r), a less than or equal to r less than infinity. The formulation allows deviations from charge neutrality and dependence of phi(r) on ion flux. A numerical solution is obtained by converting the equation to an integral equation that is solved by an iterative method for an assumed mouth potential, combined with a shooting method to adjust the mouth potential until the numerical solution agrees with an asymptotic expansion of the potential at r-a much greater than lambda (lambda = Debye length). Approximate analytic solutions are obtained by assuming charge neutrality (Läuger, 1976) and by linearizing. The linear approximation agrees with the exact solution under most physiological conditions, but the charge-neutrality solution is only valid for r much greater than lambda and thus cannot be used unless a much greater than lambda. Families of curves of ion flux vs. potential drop across the electrolyte, phi(infinity)-phi (a), and of permeant ion density at the channel mouth, n1(a), vs. flux are obtained for different values of a/lambda and S = a d phi/dr(a). If a much greater than lambda and S = O, the maximum flux (which is approached when n1(a)----0) is reduced by 50% compared to the value predicted by the charge-neutrality solution. Access resistance is shown to be a factor a/[2 (a + lambda)] times the published formula (Hille, 1968), which was derived without including deviations from charge neutrality and ion density gradients and hence does not apply when there is no counter-ion current. The results are applied to an idealized diffusion-limited channel with symmetric electrolytes. For S = O, the current/voltage curves saturate at a value dependent on a/lambda; for S greater than O, they increase linearly for large voltage.     

A model of physiological and allometric factors in the self-thinning curve

A generalized self-thinning curve for plants is derived from the modified Von Bertallanfy equation. When an asymptotic relation between photosynthesis per unit of leaf area and stocking density is assumed, the self-thinning curve thus derived is also asymptotic on a log-log scale but is fitted quite well by a log-linear approximation. The model predicts that the slope of the log-linear approximation is a function of (a) photosynthetic response to density and (b) the relation between leaf area and total aboveground biomass. Intercept of the log-linear approximation is a function of these plus maximum attainable biomass, site productivity, density at which maximum photosynthesis is attained, and the nature of carbon loss within the plant community. Linkages between various parameters within the model act to reduce differences in slope and intercept for species with different life history's and physiological requirements.     

Rate of excitation propagation in a reduced Hodgkins-Huxley model. II. Slow relaxation of the sodium current]

The influence of sodium current activation on the value of nerve excitation conduction velocity is investigated on the basis of Hodgkin-Huxley model. The potassium activation and sodium inactivation are considered as slow processes which do not develop to an appreciable extent in the region of conduction velocity formation. The system of equations was derived and solved analytically after neglecting the dependency of sodium relaxation time on potential; the approximation of steady-state sodium activation was also used with the help of Hevyside function. The algebraic equation for conduction velocity was obtained; its solution has a simple analytical form in two limits of rapid and slow sodium current relaxation. The comparison with the experimental data has shown that at not very high temperatures the slow (compared to the potential dynamics) sodium current relaxation approximation is more appropriate. The dependency of impulse velocity on capacitance and conductance of the fiber was analyzed.     

Cell wall yield properties of growing tissue : evaluation by in vivo stress relaxation

Growing pea stem tissue, when isolated from an external supply of water, undegoes stress relaxation because of continued loosening of the cell wall. A theoretical analysis is presented to show that such stress relaxation should result in an exponential decrease in turgor pressure down to the yield threshold (Y), with a rate constant given by ε where is the metabolically maintained irreversible extensibility of the cell wall and ε is the volumetric elastic modulus of the cell. This theory represents a new method to determine in growing tissues.

Stress relaxation was measured in pea (Pisum sativus L.) stem segments using the pressure microprobe technique. From the rate of stress relaxation, of segments pretreated with water was calculated to be 0.08 per megapascal per hour while that of auxin-pretreated tissue was 0.24 per megapascal per hour. These values agreed closely with estimates of made by a steady-state technique. The yield threshold (0.29 megapascal) was not affected by auxin. Technical difficulties with measuring by stress relaxation may arise due to an internal water reserve or due to changes in subsequent to excision. These difficulties are discussed and evaluated.

A theoretical analysis is also presented to show that the tissue hydraulic conductance may be estimated from the T_½ of tissue swelling. Experimentally, pea stems had a swelling T_½ of 2.0 minutes, corresponding to a relative hydraulic conductance of about 2.0 per megapascal per hour. This value is at least 8 times larger than . From these data and from computer modeling, it appears that the radial gradient in water potential which sustains water uptake in growing pea segments is small (0.04 megapascal). This means that hydraulic conductance does not substantially restrict growth. The results also demonstrate that the stimulation of growth by auxin can be entirely accounted for by the change in .

     

Principles of quantitative analysis of two-dimensional spectra of nuclear Overhauser effect in evaluation of protein and peptide conformation

To elucidate potentialities of two-dimensional homonuclear Overhauser effect (NOESY) spectra of peptides and proteins for their spatial structure determination, impact of experimental parameters and intrinsic properties of the investigated molecule on proton cross-peak volumes in NOESY spectra was analysed. Recommendations which could increase accuracy of cross-peak volume measurements were suggested. Influence of intrinsic properties of a molecule (spin-lattice relaxation times T1, correlation time tau C and surrounding protons) on the volume of cross-peak for particular protons was analyzed using a complete relaxation matrix of the (formula; see text) helix of gramicidin A. Nonselective relaxation time T1 of the protons was found to affect only slightly the results of cross-peak volumes computer simulation, whereas correlation time tau C and surrounding protons seriously influenced cross-peak volumes. Nevertheless, cross-peak volumes between NH, C alpha H and C beta H protons of a dipeptide fragment of the entire molecule could be accurately simulated using the relaxation matrix of the individual dipeptide. Thus local conformations (torsion angles phi, psi and chi 1) of amino acid residues could be deduced independently of one another and prior to the complete analysis of a molecular structure. The result can be obtained even in the presence of spin-diffusion at mixing times providing maximal volumes of cross-peaks in NOESY spectra.