Rienstra Chad M, Hohwy Morten, Mueller Leonard J, Jaroniec Christopher P, Reif Bernd, Griffin Robert G
Department of Chemistry, Center for Magnetic Resonance, Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
J Am Chem Soc. 2002 Oct 9;124(40):11908-22. doi: 10.1021/ja020802p.
We demonstrate constraint of peptide backbone and side-chain conformation with 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift, magic-angle spinning NMR experiments. In these experiments, polarization is transferred from (15)N[i] by ramped SPECIFIC cross polarization to the (13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1] resonances and evolves coherently under the correlated (1)H-(15)N and (1)H-(13)C dipolar couplings. The resulting set of frequency-labeled (15)N(1)H-(13)C(1)H dipolar spectra depend strongly upon the molecular torsion angles phi[i], chi1[i], and psi[i - 1]. To interpret the data with high precision, we considered the effects of weakly coupled protons and differential relaxation of proton coherences via an average Liouvillian theory formalism for multispin clusters and employed average Hamiltonian theory to describe the transfer of (15)N polarization to three coupled (13)C spins ((13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1]). Degeneracies in the conformational solution space were minimized by combining data from multiple (15)N(1)H-(13)C(1)H line shapes and analogous data from other 3D (1)H-(13)C(alpha)-(13)C(beta)-(1)H (chi1), (15)N-(13)C(alpha)-(13)C'-(15)N (psi), and (1)H-(15)N[i]-(15)N[i + 1]-(1)H (phi, psi) experiments. The method is demonstrated here with studies of the uniformly (13)C,(15)N-labeled solid tripeptide N-formyl-Met-Leu-Phe-OH, where the combined data constrains a total of eight torsion angles (three phi, three chi1, and two psi): phi(Met) = -146 degrees, psi(Met) = 159 degrees, chi1(Met) = -85 degrees, phi(Leu) = -90 degrees, psi(Leu) = -40 degrees, chi1(Leu) = -59 degrees, phi(Phe) = -166 degrees, and chi1(Phe) = 56 degrees. The high sensitivity and dynamic range of the 3D experiments and the data analysis methods provided here will permit immediate application to larger peptides and proteins when sufficient resolution is available in the (15)N-(13)C chemical shift correlation spectra.
我们通过三维（1）H - （15）N - （13）C - （1）H偶极化学位移、魔角旋转核磁共振实验证明了肽主链和侧链构象的限制。在这些实验中，极化通过斜坡式特定交叉极化从（15）N[i]转移到（13）Cα[i]、（13）Cβ[i]和（13）Cα[i - 1]共振，并在相关的（1）H - （15）N和（1）H - （13）C偶极耦合下相干演化。由此产生的一组频率标记的（15）N1H - （13）C1H偶极光谱强烈依赖于分子扭转角phi[i]、chi1[i]和psi[i - 1]。为了高精度解释数据，我们通过多自旋簇的平均刘维尔理论形式考虑了弱耦合质子的影响和质子相干的差异弛豫，并采用平均哈密顿理论来描述（15）N极化向三个耦合的（13）C自旋（（13）Cα[i]、（13）Cβ[i]和（13）Cα[i - 1]）的转移。通过结合来自多个（15）N1H - （13）C1H线形的数据以及来自其他三维（1）H - （13）Cα - （13）Cβ - （1）H（chi1）、（15）N - （13）Cα - （13）C' - （15）N（psi）和（1）H - （15）N[i] - （15）N[i + 1] - （1）H（phi，psi）实验的类似数据，构象解空间中的简并性被最小化。本文用均匀（13）C、（15）N标记的固体三肽N - 甲酰基 - 蛋氨酸 - 亮氨酸 - 苯丙氨酸 - OH的研究证明了该方法，其中组合数据约束了总共八个扭转角（三个phi、三个chi1和两个psi）：phi（Met） = -146°，psi（Met） = 159°，chi1（Met） = -85°，phi（Leu） = -90°，psi（Leu） = -40°，chi1（Leu） = -59°，phi（Phe） = -166°，chi1（Phe） = 56°。当（15）N - （13）C化学位移相关光谱具有足够分辨率时，本文提供的三维实验的高灵敏度和动态范围以及数据分析方法将允许立即应用于更大的肽和蛋白质。
