We use the PASADENA (parahydrogen and synthesis allow dramatically enhanced nuclear alignment) method to achieve 13C polarization of approximately 20% in seconds in 1-13C-succinic-d2 acid. The high-field 13C multiplets are observed as a function of pH, and the line broadening of C1 is pronounced in the region of the pK values. The 2JCH, 3JCH, and 3JHH couplings needed for spin order transfer vary with pH and are best resolved at low pH leading to our use of pH approximately 3 for both the molecular addition of parahydrogen to 1-13C-fumaric acid-d2 and the subsequent transfer of spin order from the nascent protons to C1 of the succinic acid product. The methods described here may generalize to hyperpolarization of other carboxylic acids. The C1 spin-lattice relaxation time at neutral pH and 4.7 T is measured as 27 s in H2O and 56 s in D2O. Together with known rates of succinate uptake in kidneys, this allows an estimate of the prospects for the molecular spectroscopy of metabolism.

Ala-OMe. In all these compounds the orientation dependence of 7j calculated (for 9 = 0°and = 90°) by using a three-site jump model is in good agreement with the orientation dependence of Tx measured over a large temperature range. Correlation times derived from the analysis of these data show that the activation energy is 8.5-11.4 kJ/mol for methyl reorientation in all cases except for L-[3,3,3-2H3]alanine, which has an activation energy of 22.6 kJ/mol. This abnormally large activation energy is correlated with the tight packing found in the crystal structure. This result suggests that measurement of methyl-group activation energies may be a useful way to locate tightly packed domains within proteins.We have used 2H NMR spectroscopy to investigate the effects of molecular packing on activation energy of methyl reorientation in polycrystalline amino acids and peptides. Activation energies can be obtained from rotational correlation times, rc, measured as a function of temperature. 2H NMR spectroscopy is a useful method for measuring rotational correlation times in solids.2-5 The values of correlation times in the range 10-4-10-7 can be determined from analysis of line shapes while correlation times in the range 10-7-10-12 s can be determined from analysis of spin-lattice relaxation times (7j).Since correlation times for methyl reorientation are normally less than 10-8 s at temperature above -150 °C,6 we have derived the correlation times for methyl reorientation from Tx measurements. In solids, the 2H 7j is orientation dependent and a measurement of the relaxation time at = 0°and = 90°u niquely defines the correlation time(s).7is the angle made by the methyl rotation axis and the external magnetic field. In addition, these Tx measurements permit one to discriminate among various models

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 ofleucine and small leucyl-containing peptides and proteins [Benedetti, C. (1977) at 313 nm to establish purity. The percentage incorporation of deuterium (43%) was determined by chemical ionization gas chromatography/mass spectrometry of N-acetylmethyl ester derivatives of the hydrolyzed protein. Radiotracer (3H and 14C) analysis showed that leucine was the only amino acid labeled during biosynthesis.2H NMR spectra were observed for polycrystalline D,L-[2H10]leucine over the temperature range -45°C to + 100°C (Fig. 2). Because of the size ofthe methylene and methine deuteron quadrupolar splitting, Avq(120 kHz), only a part of the spectrum arising from these deuterons is shown. Rapid threefold rotation of the methyl groups in leucine averages Avq to 38 kHz at -45°C, and these six deuterons display the classic axially symmetric quadrupolar powder pattern. Over the temperature range studied, very little additional side chain motion

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.