Detection of cis and trans X-Pro peptide bonds in proteins by 13C NMR: application to collagen.

Abstract

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...

Solution structure of the inhibitory phosphorylation domain of myosin phosphatase targeting subunit 1

“…Hence, we concluded that Pro 672 is in the trans conformation and not in a cis-trans equilibrium state 17,18…”

Hypothesis about the function of membrane-buried proline residues in transport proteins.

“…Cis peptide bonds involving proline arise in proteins (e.g., see ref. 27) and in synthetic linear and cyclic peptides (28).…”

The contribution of solid-state NMR spectroscopy to understanding biomineralization: Atomic and molecular structure of bone

“…Seminal work on the structure and dynamics of the extracellular collagen matrix in bone and other tissues was published as far back as 1980 by Denis Torchia and his group [31][32][33][34][35][36][37] and earlier than that on the characterization of proline structure and conformation in peptides [38,39] (albeit in solution), work that has significant relevance for understanding the structural properties of collagen in bone as will become apparent through this discussion. That many of these papers are not highly cited in the biological literature is most likely a consequence of his work being far ahead of its time.…”