Katsuyuki Nishimura

Inter- and intra-molecular contributions of neighboring dipolar pairs to the precise determination of interatomic distances in a simple [13C, 15N]-peptide by 13C, 15N-REDOR NMR spectroscopy

Abstract Both 13 C- and 15 N-REDOR experiments were performed to determine the 13 C- 15 N interatomic distance in [ 1- 13 C]N-acetyl-Pro-[ 15 N]Gly-Phe crystal and to evaluate the inter- or intra-molecular dipolar contributions leading to its errors. The interatomic distance was determined to be 3.43 A after removing the intermolecular contribution to the echo dephasing arising from the intermolecular dipolar interaction with labeled nuclei of the neighboring molecule. This contribution was estimated by extrapolation to infinite dilution of the labeled sample in unlabeled peptide. The theoretical formalism was developed for the nucleus coupled with two heteronuclei. The conformation of this peptide turned out to be β-turn type II on the basis of the abovementioned interatomic distance.

Pressure induced isomerization of retinal on bacteriorhodopsin as disclosed by Fast Magic Angle Spinning NMR

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light‐driven proton pump. We have detected pressure‐induced isomerization of retinal in bR by analyzing 15N cross polarization‐magic angle spinning (CP‐MAS) NMR spectra of [ζ‐15N]Lys‐labeled bR. In the 15N‐NMR spectra, both all‐trans and 13‐cis retinal configurations have been observed in the Lys Nζ in protonated Schiff base at 148.0 and 155.0u2003ppm, respectively, at the MAS frequency of 4u2003kHz in the dark. When the MAS frequency was increased up to 12u2003kHz corresponding to the sample pressure of 63 bar, the 15N‐NMR signals of [ζ‐15N]Lys in Schiff base of retinal were broadened. On the other hand, other [ζ‐15N]Lys did not show broadening. Subsequently, the increased signal intensity of [ζ‐15N]Lys in Schiff base of 13‐cis retinal at 155.0u2003ppm was observed when the MAS frequency was decreased from 12 to 4u2003kHz. These results showed that the equilibrium constant of [all‐trans‐bR]/[13‐cis‐bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen‐bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all‐trans to 13‐cis state in the membrane protein bR.

Natural abundance 13C REDOR coupled to a singly 15N-labeled nucleus: simultaneous determination of interatomic distances in crystalline ammonium [15N] l-glutamate monohydrate

Abstract REDOR technique was applied to natural abundance 13 C nuclei coupled to a singly labeled 15 N nucleus to determine the 13 C, 15 N interatomic distances simultaneously in crystalline ammonium [ 15 N] l -glutamate monohydrate ( 1 ). Consequently, the interatomic C–N distances between 15 N and 13 C O, 13 C α , 13 C β , 13 C γ , and 13 C δ carbon nuclei for 1 were determined with a precision of ±0.15xa0A, after the experimental conditions such as the location of samples in the rotor, length of π pulse etc. were carefully optimized. 13 C-REDOR factors for three spin system, (Δ S / S 0 ) CN1N2 , and the sum of two isolated 2-spin system, ( Δ S/S 0 ) ∗ =( Δ S/S 0 ) CN 1 +( Δ S/S 0 ) CN 2 , were further evaluated by the REDOR measurements on isotopically diluted 1 in a controlled manner. Subsequently, the intra- and intermolecular C–N distances were separated by searching the minima in the contour map of root mean square deviation (RMSD) between the theoretically and experimentally obtained ( Δ S/S 0 ) ∗ values against two interatomic distances, r C–N1 and r C–N2 . When the intramolecular C–N distance ( r C–N1 ) of the particular carbon nucleus is substantially shorter than the intermolecular one ( r C–N2 ), C–N distances within a single molecule were obtained with an accuracy of ±0.06xa0A as in the cases of C O, C α and C β carbon nuclei. C–N distances between the molecule in question and the nearest neighboring molecules can be also obtained, although accuracy was lower. On the contrary, it was difficult to determine the interatomic distances in the same molecule when the intermolecular dipolar contribution is larger than the intramolecular one as in the case of C δ carbon nucleus.