Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR

被引：29

作者：

Klein, Alexander ^{[1
,2
]}

Rovo, Petra ^{[1
]}

Sakhrani, Varun V. ^{[3
]}

Wang, Yangyang ^{[3
]}

Holmes, Jacob B. ^{[3
]}

Liu, Viktoriia ^{[3
]}

Skowronek, Patricia ^{[1
]}

Kukuk, Laura ^{[2
]}

Vasa, Suresh K. ^{[1
,2
]}

Guentert, Peter ^{[4
,5
,6
]}

Mueller, Leonard J. ^{[3
]}

Linser, Rasmus ^{[1
,2
]}

机构：

[1] Ludwig Maximilians Univ Munchen, Dept Chem & Pharm, D-81377 Munich, Germany

[2] TU Dortmund Univ, Dept Chem & Chem Biol, D-44227 Dortmund, Germany

[3] Univ Calif Riverside, Dept Chem, Riverside, CA 92521 USA

[4] Goethe Univ, Inst Biophys Chem, D-60438 Frankfurt, Germany

[5] Eidgenoss TH ETH Zurich, Lab Phys Chem, CH-8093 Zurich, Switzerland

[6] Tokyo Metropolitan Univ, Dept Chem, Tokyo 1920397, Japan

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2022年 / 119卷 / 04期

关键词：

solid-state NMR; NMR crystallography; tryptophan synthase; PLP-dependent enzymes; tautomerism; ANGLE-SPINNING NMR; H-1-H-1 DISTANCE RESTRAINTS; FULLY PROTONATED PROTEINS; BACKBONE ASSIGNMENT; RESONANCE ASSIGNMENT; PYRIDOXAL-PHOSPHATE; SCHIFF-BASE; SPECTROSCOPY; CRYSTALLOGRAPHY; DYNAMICS;

D O I：

10.1073/pnas.2114690119

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the beta-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the beta-subunit from solution.

引用

页数：9