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

We have determined the amplitude of nanosecond fluctuations of the collagen azimuthal orientation in intact tissues and reconstituted fibers from an analysis of 13C NMR relaxation data. We have labeled intact rat calvaria and tibia collagen (mineralized and cross-linked), intact rat tail tendon and demineralized bone collagen (cross-linked), and reconstituted lathyritic (non-cross-linked) chick calvaria collagen with [2-13C]glycine. This label was chosen because one-third of the amino acid residues in collagen are glycine and because the 1H-13C dipolar coupling is the dominant relaxation mechanism. Spin-lattice relaxation times (T1) and nuclear Overhauser enhancements were measured at 15.09 and 62.98 MHz at 22 and -35 degrees C. The measured NMR parameters have been analyzed by using a dynamic model in which the azimuthal orientation of the molecule fluctuates as a consequence of reorientation about the axis of the triple helix. We have shown that if root mean square fluctuations in the azimuthal orientations are small, gamma rms much less than 1 rad, the correlation function decays with a single correlation time tau and T1 depends only upon tau and gamma rms and not the detailed model of motion. Our analysis shows that, at 22 degrees C, tau is in the 1-5-ns range for all samples and gamma rms is 10 degrees, 9 degrees, and 5.5 degrees for the non-cross-linked, cross-linked, and mineralized samples, respectively. At -35 degrees C, gamma rms is less than 3 degrees for all samples. These results show that mineral and low temperature significantly restrict the amplitude of nanosecond motions of the collagen backbone.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Heretofore the complexity of natural abundance spectra has precluded the use of 13C NMR to detect cis peptide bonds in proteins. We have incorporated [4-'3C~pro-line into chicken calvaria collagen and report here well-resolved Cy signals, arising from cis and trans X-Pro and X-Hyp peptide bonds (where X is any amino acid residue) in the 13C NMR spectrum of the thermally unfolded protein. Measurement of 13C signal areas shows that 16% of the X-Pro and 8% of X-Hyp bonds are cis in the unfolded collagen. These results strongly support the conclusion drawn from kinetic studies that cis-trans isomerization of peptide bonds is the rate-limiting step in helix propagation after nucleation. Our method can be applied to other proteins as well and should aid in testing the generality of the hypothesis of Brandts, Halvorson, and Brennan that cis-trans isomerization is the rate-limiting step in protein folding when proline is present. Fig. 1) was obtained on a Nicolet NT-500 spectrometer; 40,000 timedomain signals were collected in quadrature with 32,768 points per channel, a 30-kHz spectral window, and a delay time of 1 s between the 20-,us 900 pulses. Broad-band proton decoupling was used during data acquisition; otherwise lowlevel proton irradiation was used to obtain an Overhauser enhancement. The NMR sample contained 50% 2H20, which served as an internal deuterium lock. Sample temperature was maintained at 60 ± 20C with the spectrometer temperature controller.[4-13C]Proline, synthesized by the method of Young and Torchia (19), was incorporated at a level of 30% into lathyritic chicken calvaria collagen in culture. The labeled protein was prepared and characterized as described by Jelinski and Torchia (21), and the protein was hydrolyzed in 6 M HCl at 106'C for 24 hr; 62.98-MHz 13C spectra were obtained on a home-built spectrometer (22). Twenty thousand (see Fig. 2a) and twelve thousand (see Fig. 2b) time-domain signals were collected in quadrature with 2048 points per channel, a 10-kHz spectral window, and delay times of 0.7 s (see Fig. 2a) and 4.0 s (see Fig. 2b) between the 8-,us 900 pulses. The signal areas of Fig. 2a did not change when larger delay times were used. Broad-band proton decoupling was used during data acquisition; otherwise either low-level or no proton irradiation was used depending on whether or not an Overhauser enhancement was desired. NMR tubes (5-mm) containing 0.2 ml of solution were used in a solenoid coil. Samples were not spun, and a field-frequency lock was not used.Each solution contained ca. 0.2% 13CH3CN (90% enriched with 13C at the methyl carbon), and the methyl resonance (1 ppm, relative to external tetramethylsilane) served as an internal reference. Sample temperature was maintained constant within ± PC by using a Varian temperature controller. RESULTS AND DISCUSSIONCollagen was chosen for this initial study for the following reasons. First, almost one-quarter of the 1000 amino acid residues in each collagen chain are either prolyl or hydroxyprolyl (23). Second, after nucleati...