High-Resolution 3D Structure Determination of Kaliotoxin by Solid-State NMR Spectroscopy

Jegannath Korukottu,Vinesh Vijayan,Olaf Pongs,Markus Zweckstetter,Adam Lange,Marc Baldus,Stefan Becker,Robert Schneider,Jianren Lu

doi:10.1371/journal.pone.0002359

Abstract

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.

Highlights

Structural characterization of membrane proteins and many other biological systems by X-ray crystallography or solution NMR spectroscopy is difficult because of problems with crystallization, solubility or molecular size
Significant advances, have been made to construct three-dimensional (3D) molecular structures from solid-state NMR data obtained under Magic Angle Spinning (MAS)[1] conditions[2,3,4]
These efforts resulted in high-resolution 3D conformations for small peptides[5,6,7,8] and the determination of medium-resolution backbone structures for a few solid-phase proteins.[9,10,11,12]

Summary

Introduction

Structural characterization of membrane proteins and many other biological systems by X-ray crystallography or solution NMR spectroscopy is difficult because of problems with crystallization, solubility or molecular size. Significant advances, have been made to construct three-dimensional (3D) molecular structures from solid-state NMR data obtained under Magic Angle Spinning (MAS)[1] conditions[2,3,4] These efforts resulted in high-resolution 3D conformations for small peptides[5,6,7,8] and the determination of medium-resolution backbone structures for a few solid-phase proteins.[9,10,11,12]. Unless sample orientation provides a direct route to monitor molecular structure under MAS conditions[13,14], the collection of medium and long-range distance constraints is most crucial These correlations are closely related to molecular structure, can be measured in high spectral resolution and lead to unequivocal assignments of structure-relevant correlations. Two strategies have been developed in this direction: (i) measurement of 13C-13C distances on 13C block-labeled protein microcrystals[9] and (ii) extraction of 1H-1H-distance restraints from 13C,13C- and 15N,13C-encoded 1H/1H mixing experiments on a uniformly 13C/15N-labeled sample[11]

Methods

Results

Conclusion