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D A Torchia

Publications and source records attributed to D A Torchia.

At least 73 records · Page 4Linked to original sources
PubMed1983

Solid state 13C NMR study of collagen molecular dynamics in hard and soft tissues.

The molecular dynamics of the collagen backbone in intact connective tissues has been elucidated using 13C line shape analysis. Since one-third of the amino acid residues in collagen are glycines, we have labeled: (a) reconstituted lathrytic (uncross-linked) chick calvaria collagen fibrils; (b) rat tail tendon (cross-linked); and (c) rat calvaria (cross-linked and mineralized) collagen with [1-13C]glycine. The proton-enhanced and normal 90 degrees - t proton-decoupled spectrum of each collagen sample shows an asymmetric chemical shift powder pattern for the glycine carbonyl carbon. The powder line width, delta, (delta = sigma zz - sigma xx) at 22 degrees C for the uncross-linked reconstituted collagen fibril is 108 ppm, whereas the maximum value of delta (140 ppm) is observed for the cross-linked and mineralized collagen fibrils in rat calvaria. The powder line widths for the cross-linked fibrils in tail tendons and demineralized calvaria are 124 and 120 ppm, respectively. However, since the same line shape and line width (145 ppm) are observed for all samples at -35 degrees C, the difference in delta values observed at room temperature is attributed to differences in molecular mobility of collagen in various samples. The line shapes are analyzed using a dynamic model in which azimuthal orientation of the collagen backbone is assumed to fluctuate as a consequence of reorientation about the helix axis. The observed line shapes are sensitive to motions having correlation times less than approximately 10(-4) s and the analysis provides the values of the root mean square fluctuation in azimuthal angle, gamma rms, due to such motions. It is found that gamma rms equals 41 degrees, 33 degrees, and 14 degrees for the uncross-linked, cross-linked, and mineralized collagens, respectively. These results provide the first information about the extent that cross-linking and mineralization restrict molecular motion in collagen.

Animals↗
PubMed1983

Mobility and function in elastin and collagen.

13C- and 2H-labelled amino acids have been incorporated into elastin and collagen and rotational correlation times of the labelled sites have been derived from an analysis of nuclear magnetic resonance relaxation parameters and line-shapes. The elastin experiments were designed to discriminate between the various models that have been proposed to account for the rubber-like elasticity of elastin. The correlation times of carbonyl carbons of the elastin backbone show that elastin chains are very flexible at the molecular level. In addition, the molecular dynamics and viscoelastic behaviour of elastin are well correlated over a wide range of temperatures and solvents. These results all support the rubber-like network model of elastin structure. The collagen experiments were designed to investigate the intermolecular interactions between molecules in collagen fibres. Correlation times of labelled sites in the collagen backbone and side-chains show that substantial flexibility, especially of the side-chains, takes place in reconstituted (non-cross-linked) collagen fibrils. Therefore, the interactions between side-chains that presumably direct and stabilize the fibrillar assembly take place in fluid domains. The molecular flexibility is not affected by the presence of cross-links but is absent when the collagen is mineralized.

Animals↗
PubMed1983

Intracellular polymerization of sickle hemoglobin. Effects of cell heterogeneity.

To determine the extent to which the broad distribution in intracellular hemoglobin concentrations found in sickle erythrocytes affects the extent of intracellular polymerization of hemoglobin S, we have fractionated these cells by density using discontinuous Stractan gradients. The amount of polymer formed in the subpopulations was experimentally measured as a function of oxygen saturation using 13C nuclear magnetic resonance spectroscopy. The results for each subpopulation are in very good agreement with the theoretical predictions based on the current thermodynamic description for hemoglobin S gelation. We further demonstrate that the erythrocyte density profile for a single individual with sickle cell anemia can be used with the theory to predict the amount of polymer in unfractionated cells. We find that heterogeneity in intracellular hemoglobin concentration causes the critical oxygen saturation for formation of polymer to shift from 84 to greater than 90%; polymer is formed predominantly in the dense cells at the very high oxygen saturation values. The existence of polymer at arterial oxygen saturation values has significance for understanding the pathophysiology of sickle cell anemia. The utility of these techniques for assessing various therapeutic strategies is discussed.

Anemia, Sickle Cell↗
PubMed1982

Characterization of leucine side-chain reorientation in collagen-fibrils by solid-state 2H NMR.

We have used 2H quadrupole-echo NMR spectroscopy to study the molecular dynamics of the leucine side chain in collagen fibrils labeled with [2H10]leucine. X-ray crystallographic studies of leucine and small leucyl-containing peptides and proteins [Benedetti, C. (1977) in Proceedings of the Fifth American Peptides Symposium, eds, Goodman, M. & Meienhofer, J. (Wiley, New York), pp. 257--274; Janin, J., Wodak, S., Levitt, M. & Maigret, B. (1978) J. Mol. Biol. 125, 357--386] show that the amino acid side chain exists predominantly in only two of the nine possible conformations. 2H NMR spectra of polycrystalline D,L [2H10]leucine obtained from -45 degrees C to +100 degrees C showed that interconversion of the two conformations did not take place on the 2H NMR timescale in this temperature range. In contrast, experimental lineshapes observed for [2H10]leucine-labeled collagen fibrils from -85 degrees C to +30 degrees C were simulated by using a model in which the side chain hops at various rates between the two predominant conformations found by the x-ray studies. A small difference between calculated and observed linewidths above the freezing point of water can be accounted for by backbone reorientation or by the presence of a small percentage of other side-chain conformations. Thus, these results provide strong evidence that the two predominant x-ray conformations not only exist in the fibrils as the preferred orientations but interconvert at rates that are proportional to temperature over the range - 85 degrees C to +30 degrees C. These observations concur with previous NNR studies of collagen fibrils that demonstrated a mobile contact region between collagen molecules.

Animals↗
PubMed1982

Determination of sickle hemoglobin polymer in SS and AS erythrocytes.

Using 13C nuclear magnetic resonance techniques, we have studied the polymerization of hemoglobin S and intracellular polymerization in SS and AS erythrocytes. For SS erythrocytes, polymer could be detected at high oxygen saturations (greater than 90%) and increased with decreasing oxygen saturation to 70% of the total hemoglobin at complete deoxygenation. These results are consistent with the non-ideal behavior of hemoglobin S at the high intracellular concentration of 34 g/dl. The amount of polymer in AS erythrocytes was also measured as a function of oxygen saturation and is in agreement with the amount of polymer determined for homolysates using ultracentrifuge sedimentation. In general, these results on intracellular gelation are in good agreement with results predicted by cell-free studies extrapolated to the high intracellular hemoglobin concentration (34 g/dl). The significance of the detection of polymer at high oxygen saturation of SS erythrocytes is discussed in terms of morphologic deformation and erythrocyte transit through the circulation. We propose that intracellular polymerization is important in abnormal cell rheology and manifestation of disease.

Anemia, Sickle Cell↗
PubMed1981

13C nuclear magnetic resonance suggests a flexible proteoglycan core protein.

13C NMR was used to study the molecular dynamics of the chick limb bud proteoglycan core protein. Because only about 10% of the proteoglycan is protein, [2-13C]glycine and [3-13C]serine were incorporated into the core protein of the chick limb bud proteoglycan monomer using a chondrocyte culture system. The purified labeled monomer was studied in solution (50 mg/ml, 0.05 M sodium acetate/0.05 M sodium phosphate, pH 7.4) at 37 degrees C. Spin-lattice relaxation times, line widths, and nuclear Overhauser enhancements were measured for the labeled carbons in the protein and for the natural abundance carbons in the glycosaminoglycan chains. Analyses of these data show that correlation times for backbone reorientation of protein and polysaccharide chains in the intact monomer are predominantly in the range of 0.5-5.0 ns. These correlation times are 10(2)-10(3) times smaller than the minimum correlation time calculated for a rigid monomer, indicating that the core protein and polysaccharide backbones are segmentally flexible. Signal intensity data show that at least 80% of the protein backbone is flexible but do not exclude the possibility that 20% of the protein backbone has ordered structure. We observe both broad and narrow signal components in the spectrum of the intact monomer, showing that backbone motion is heterogeneous. The broad signal component is not observed after the monomer is digested with chondroitinase. This result and the strong concentration dependence of the 13C line width observed in solutions of chondroitin 4-sulfate suggest an assignment of the broad signal component to residues near the protein-polysaccharide linkage region. The difference in NMR parameters observed for free and substituted serine carbons confirms that motion of the substituted serine side chain is restricted.

Animals↗
PubMed1981

Polymerization of hemoglobin in sickle trait erythrocytes and lysates.

We report 13C/1H magnetic double resonance measurements of polymer in sickle trait erythrocytes and direct measurements of solubility on lysates of these cells as a function of oxygen saturation. Polymerized hemoglobin is detected by these two methods only at oxygen saturation values below 60% and increases to 0.4 of the total hemoglobin at complete deoxygenation. These results confirm the validity of the NMR approach in determining values of polymer fraction. We have also found that the thermodynamic theory, that successfully describes gelation in sickle erythrocytes, does not predict the experimental results for sickle trait erythrocytes and must be refined.

Anemia, Sickle Cell↗
PubMed1980

2H NMR study of molecular motion in collagen fibrils.

Collagen was labelled through tissue culture with [3,3,3-d3]alanine. 2HNMR spectra were obtained of the labelled collagen as fibrils and in solution using the quadrupolar echo technique for solids. The 2H NMR data were analysed in terms of a model for reorientation in which the molecule is considered to jump between two orientations in a time which is short compared to the residence time in each site, and short compared to (delta vq)-1. The best fit of the data indicates that the collagen molecule in the fibrils experiences reorientation about its long axis over an angular range of approximately 30--40 degrees. The T2 for [3,3,3-d3]alanine-labelled collagen fibrils is estimated to be approximately 110 mus.

Alanine↗
PubMed1980

Deuterium nuclear magnetic resonance of specifically labeled native collagen. Investigation of protein molecular dynamics using the quadrupolar echo technique.

Collagen was labeled with [3,3,3-d3]alanine and with [d10]leucine via tissue culture. 2H nuclear magnetic resonance (NMR) spectra were obtained of collagen in solution and as fibrils using the quadrupolar echo technique. The 2H NMR data for [3,3,3-d3]alanine-labeled collagen fibrils were analyzed in terms of a model for motion in which the molecule is considered to jump between two sites, separated azimuthally by an angle 2 delta, in a time which is rapid compared with the residence time in both sites. The data suggest that the molecule undergoes reorientation over an angle, 2 delta, of approximately 30 degrees in the fibrils, and that the average angle between the alanine C alpha--C beta bond axis and the long axis of the helix is approximately 75 degrees. Reorientation is possibly segmental. The T2 for [3,3,3-d3]alanine-labeled collagen fibrils was estimated to be 105 mus. The 2H NMR data for the methyl groups of [d10]leucine-labeled collagen were analyzed qualitatively. These data established that for collagen in solution and as fibrils, rotation occurs about the leucine side-chain bonds, in addition to threefold methyl rotation and reorientation of the peptide backbone. The T2 for the methyl groups of leucine-labeled collagen is estimated to be approximately 130 mus. Taken together, these data provide strong evidence that both polypeptide backbone reorientation and amino acid side-chain motion occur in collagen molecules in the fibrils. Stabilizing interactions that determine fibril structure must therefore depend upon at least two sets of contacts in any given local region.

Alanine↗
PubMed1980

Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation.

We have used 13C/1H magnetic double-resonance spectroscopy to measure the amount of sickle hemoglobin polymer within sickle erythrocytes as a function of oxygen saturation. We previously showed that the methods of cross-polarization and scalar decoupling could be used to measure accurately the polymer fraction in deoxygenated sickle hemoglobin solutions [Noguchi, C.T., Torchia, D.A. & Schechter, A.N. (1979) Proc. Natl. Acad. Sci. USA 76, 4936-4940]. Our measurements show that the amount of intracellular deoxyhemoglobin S polymer increases monotonically with decreasing oxygen saturation. Polymer can be detected at oxygen saturation values above 90%. This result can be theoretically explained by the excluded volume effect of the oxyhemoglobin S in the cell. The very high total intracellular hemoglobin concentration (34 g/dl) reduces the amount of soluble deoxyhemoglobin S to about 3 g/dl at 90% oxygen saturation. The agreement between theory and experiment indicates that the equilibrium properties of intracellular polymerization can be described by the analyses resulting from studies of concentrated sickle hemoglobin solutions. The curve for polymer formation as a function of oxygen saturation is roughly hyperbolic whereas that for cell sickling is sigmoidal; the difference is most apparent for measurements at pH 7.65. Intracellular polymer formation may in general have a different relationship to oxygen saturation than cell sickling and may be a more meaningful parameter of the pathophysiological process in sickle cell anemia than cell morphology. In addition, measurements of intracellular polymer should be useful in evaluating potential therapeutic agents.

Anemia, Sickle Cell↗
PubMed1979

13C NMR quantitation of polymer in deoxyhemoglobin S gels.

13C/1H magnetic double-resonance spectroscopy has been used to quantitate the amount of polymerized hemoglobin S in deoxygenated gels at 30 degrees C, for samples whose hemoglobin concentration range from 21 to 32 g/dl. Scalar- and dipolar-decoupled spectra and a 13C proton-enhanced dipolar-decoupled spectrum were recorded for each sample as was a scalar-decoupled spectrum for a matching oxyhemoglobin S control. The difference between the oxyhemoglobin S and deoxyhemoglobin S scalar-decoupled spectra was used to determine the polymer fraction, and this value was compared with the polymer fraction determined by using ultracentrifugation sedimentation on the same sample (assuming a two-phase model). The polymer fraction value determined by uncorrected sedimentation averaged 0.15 more than the value obtained from NMR. The discrepancy between the two techniques was largely removed when the analysis of the sedimentation data included a correction for depletion of hemoglobin in the supernatant or sol phase due to sedimentation of free molecules. The best fit to both the sedimentation and NMR data was obtained by using a solubility of deoxyhemoglobin S at 30 degrees C of 17.3 +/- 1 g/dl. These results indicate that the NMR techniques, which do not require separation of the sample into a sol phase and a pellet phase, provide quantitative information about the deoxyhemoglobin S polymer and will be useful for studies of sickle erythrocytes.

Erythrocytes↗
PubMed1979

Enamel matrix: structural proteins.

Cell-free, fetal bovine enamel tissue was examined intact by high resolution. 13C Fourier transform, nuclear magnetic resonance spectroscopy. Two types of protein chains were observed under these conditions, one exhibiting rapid mobility and accounting for approximately two-thirds of the enamel matrix, while the other exhibited restricted or anisotropic segmental motion and accounted for the remaining third of the matrix. Sequential extraction of this fetal enamel under non-degradative conditions with dissociative solvents yielded two biochemically distinct populations of matrix protein. As expected, the bulk of the matrix consisted of proline-rich amelogenins, although the SDS-gel electrophoresis molecular weights for these proteins were somewhat higher than those reported using other extraction methods. Approximately fifteen percent of the total matrix consisted of much higher molecular weight phosphoproteins (46,000-72,000 daltons) whose amino acid composition closely resembled that reported for mature enamel protein. These high molecular weight proteins were tightly bound to the fetal enamel apatite crystallites.

Animals↗