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

Abstract

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

Protein side-chain conformation: a systematic variation of χ1mean values with resolution – a consequence of multiple rotameric states?

“…, 1 2 = +60 ) and solid-state NMR results indicate that there is rapid interconversion between them (Batchelder et al, 1982;Colnago et al, 1987), though the motion may be more complex than described by this simple two-site jump model (Yang et al, 1998). Dunbrack & Karplus (1993) observed that of 19 leucine residues whose side-chain conformations they had been trying to predict in six proteins, nine of them could have had wrong dihedral angles in the published structures owing to errors in map interpretation arising from positional degeneracy.…”

Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides

“…2 H NMR has long been used to determine the degree to which amino acid side chain dynamics are sensitive to structural environment, for example, the perturbation of the motion of leucine side chains in collagen fibrils (30), the dynamic impact of polylysine adsorption onto silica and hydroxyapatite surfaces (31), and the dynamics of hydrophobic side chains in membrane-associated proteins (32)(33)(34). Additionally, 13 C-2 H NMR correlation techniques have been used by Reif and coworkers to identify the dynamics of deuterated valine and leucine side chains in uniformly 13 C-labeled micro-crystalline proteins (35,36).…”

“…Models for the motion of leucine side chains are usually based on the assumption that exchange occurs between a subset of these bond rotational isomers. For example, in a 2 H NMR study of helical collagen fibrils with L-leucine-2 H 10 uniformly incorporated, Batchelder et al (30) assumed exchange of the side chain between two conformers observed to be dominant in leucylpeptide crystal structures: tg þ and g − t, where g þ , g − , and t correspond to the gauche þ, gauche −, and trans rotational isomers of the C α -C β and C β -C γ bonds. The temperature variation of the 2 H line shape was modeled as variation in the rate of exchange between these two conformations of the leucine side chain, assumed to occur with equal a priori populations.…”

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

“…Moreover, reported 13 C NMR experiments did not display a sufficient spectral resolution to allow a clear distinction among the different amino acid carbon resonances (9,10). NMR studies using specific residue labeling have given valuable information on dynamics (11)(12)(13). To deduce the backbone conformation, Saitô et al (9) have compared 13 C chemical shifts of fibrillar collagen with those obtained on small triple helical peptides.…”