High-Resolution Solid-State NMR Structure of a 17.6 kDa Protein

摘要

Abstract: The use of pseudocontact shifts arising from paramagnetic metal ions in a microcrystalline proteinsample is proposed as a strategy to obtain unambiguous signal assignments in solid-state NMR spectrnenabling distance extraction for protein structure calculation. With this strategy, 777 unambiguous (28nsequential, 217 medium-range, and 279 long-range) distance restraints could be obtained from PDSDnDARR, CHHC, and the recently introduced PAR and PAIN-CP solid-state experiments for the cobalt(IInsubstituted catalytic domain of matrix metalloproteinase 12 (159 amino acids, 17.6 kDa). The obtainenstructure is a high resolution one, with backbone rmsd of 1.0 0.2 Å, and is in good agreement with thnX-ray structure (rmsd to X-ray 1.3 Å). The proposed strategy, which may be generalized for nonmetallonproteins with the use of paramagnetic tags, represents a significant step ahead in protein structurndetermination using solid-state NMR.