Dipolar chemical shift correlation spectroscopy for homonuclear carbon distance measurements in proteins in the solid state: application to structure determination and refinement.
High-resolution solid-state NMR spectroscopy has become a promising tool for protein structure determination. Here, we describe a new dipolar-chemical shift correlation experiment for the measurement of homonuclear 13C-13C distances in uniformly 13C,15N-labeled proteins and demonstrate its suitability for protein structure determination and refinement. The experiments were carried out on the beta1 immunoglobulin binding domain of protein G (GB1). Both intraresidue and interresidue distances between carbonyl atoms and atoms in the aliphatic side chains were collected using a three-dimensional chemical shift correlation spectroscopy experiment that uses homogeneously broadened rotational resonance recoupling for carbon mixing. A steady-state approximation for the polarization transfer function was employed in data analysis, and a total of 100 intramolecular distances were determined, all in the range 2.5-5.5 A. An additional 41 dipolar contacts were detected, but the corresponding distances could not be accurately quantified. Additional distance and torsional restraints were derived from the proton-driven spin diffusion measurements and from the chemical shift analysis, respectively. Using all these restraints, it was possible to refine the structure of GB1 to a root-mean square deviation of 0.8 A. The approach is of general applicability for peptides and small proteins and can be easily incorporated into a structure determination and refinement protocol.
Peng
Xiaohu
X
Department of Physics, Department of Molecular and Cellular Biology, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada.
Libich
David
D
Janik
Rafal
R
Harauz
George
G
Ladizhansky
Vladimir
V
D002247:Carbon Isotopes; D015394:Molecular Structure; D009419:Nerve Tissue Proteins / Q000737:chemistry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
D002247:Carbon Isotopes; C029152:G-substrate; D009419:Nerve Tissue Proteins; D009587:Nitrogen Isotopes; D011506:Proteins