Determination of the Three-Dimensional Structure of Crystalline Leu-Enkephalin Dihydrate Based on Six Sets of Accurately Determined Interatomic Distances from ¹³C-REDOR NMR and the Conformation-Dependent ¹³C Chemical Shifts

Abstract

We have determined the three-dimensional structure of [13C,15N]-labeled Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) dihydrate (crystallized from aqueous methanol) on the basis of six sets of accurately determined 13C···15N interatomic distances by rotational echo double resonance (REDOR) and some additional constraints from 13C chemical shifts. This compound has not yet been refined by X-ray diffraction. Six kinds of [13C,15N]-doubly-labeled samples, in which the doubly-labeled positions are four-bonds apart (four samples) and five-bonds apart (two samples), were chemically synthesized. These labeled peptides (100%) and an isotopically diluted one with unlabeled samples (60% or 30%) were crystallized from aqueous methanol solution. 13C or 15N chemical shifts were carefully evaluated prior to and after every REDOR experiment in order to check that the crystalline polymorphs under consideration were not modified either by loss of or by freezing of motion of solvent molecules in the crystals. Accurate and precise interatomic distances (±0.10 Å) were obtained from REDOR factors of infinite dilution, which were extrapolated from the data of 100% and 30% isotopically diluted samples to eliminate dipolar contributions from the labeled nuclei of neighboring molecules in the crystals. These distance data were converted to a possible set of local torsion angles (φ i and ψ i ) in a peptide unit of the respective amino acid residue of interest using standard bond lengths and angles in a sequential manner. It turned out that a unique set of the torsion angles corresponding to the most appropriate three-dimensional structure was determined with reference to some additional constraints from the conformation-dependent displacements of 13C chemical shifts of certain peptide units. The three-dimensional structure thus obtained was finally subject to a calculation for energy minimization in order to ensure that the conformation obtained was at least at one of local minima. Finally, the biological consequence of the peptide structure thus determined is discussed.

Recoupling of Heteronuclear Dipolar Interactions with Rotational-Echo Double-Resonance at High Magic-Angle Spinning Frequencies

“…Therefore, for REDOR experiments where RF pulses occupy a significant fraction of the rotor period, the dipolar coupling constant, b IS , can be calculated from the experimentally determined effective coupling as b IS ϭ b IS eff (1 Ϫ 2 )/cos((/2)). For the strong 13 C-15 N coupling in [2-13 C, 15 N]glycine, only the ϭ 0.1 experiment ( r / 2 ϭ 5.0 kHz, rf / 2 ϭ 50 kHz) deviates slightly from theory. However, for r / 2 Յ 5.0 kHz we expect the accuracy and precision of the fit for the strong dipolar coupling to be somewhat compromised, due to the limitation in the number of points that can be acquired on the ⌬S/S 0 curve.…”

“…4a and 4b we show experimental REDOR ⌬S/S 0 curves and simulations for [1-13 C, 15 N]glycine (b IS ϳ 200 Hz) and [2-13 C, 15 N]glycine (b IS ϳ 900 Hz), respectively, recorded at spinning frequencies r / 2 ϭ 5-15 kHz and recoupling RF fields rf / 2 ϭ 25-50 kHz (i.e., 0.1 Յ Յ 0.606). The lower fields were used to create experimental conditions, where RF pulses occupy a significant fraction of the rotor period.…”

“…6 we compare RE-DOR reference (S 0 ) curves for the C ␣ resonance in [1,2-13 C, 15 N]glycine recorded at r /2 ϭ 7.576-16.667 kHz, where the coherent evolution under the CЈ-C ␣ J coupling was removed as described previously (21). It is clear that for the CH 2 group in glycine the combination of fast MAS and two-pulse phase-modulated (TPPM) 1 H decoupling (34) significantly attenuates the dephasing of the 13 C spin-echo; the echo intensity at 20 ms of evolution increases from approximately 0.25 to 0.6 as the spinning frequency is increased from 7.576 to 16.667 kHz.…”

“…Eqs. [9], [10] and [14], [15]) with numerical simulations. The REDOR (S 0 Ϫ S)/S 0 ϭ ⌬S/S 0 curves (S 0 and S represent the reference and dipolar dephasing experiments, respectively) were calculated for b IS ϭ 190 Hz (corresponding to a C-N distance of 2.53 Å) and ϭ 0.1-0.66.…”

Structural diversity of amyloid fibril formed in human calcitonin as revealed by site‐directed 13C solid‐state NMR spectroscopy

“…field. 30,31 The xy four-pulse sequence for irradiation of 15 N nuclei was used to compensate the error of flip angle, offresonance effect and fluctuation of the r.f. field.…”