Sequence-specific resonance assignment of soluble nonglobular proteins by 7D APSY-NMR spectroscopy.
Based on sequence-specific resonance assignments, NMR is the method of choice for obtaining atomic-resolution experimental data on soluble nonglobular proteins. So far, however, NMR assignment of unfolded polypeptides in solution has been a time-consuming task, mainly due to the small chemical shift dispersion, which has limited practical applications of the NMR approach. This paper presents an efficient, fully automated method for sequence-specific backbone and beta-carbon NMR assignment of soluble nonglobular proteins with sizes up to at least 150 residues. The procedure is based on new APSY (automated projection spectroscopy) experiments which benefit from the short effective rotational correlation times in soluble nonglobular polypeptides to record five- to seven-dimensional NMR data sets, which reliably resolves chemical shift degeneracies. Fully automated sequence-specific resonance assignments of the backbone nuclei and C(beta) are described for the uniformly (13)C,(15)N-labeled urea-denatured 148-residue outer membrane protein X (OmpX) from E. coli. The method is generally applicable to systems with similar spectroscopic properties as unfolded OmpX, and we anticipate that this paper may open the door for extensive atomic-resolution studies of chemical denaturant-unfolded proteins, as well as some classes of functional nonglobular polypeptides in solution.
Hiller
Sebastian
S
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
Wasmer
Christian
C
Wider
Gerhard
G
Wüthrich
Kurt
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006867:Hydrolases / Q000737:chemistry*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D020539:Sequence Analysis, Protein / Q000379:methods*; D014508:Urea / Q000737:chemistry
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C482905:OmpX protein, E coli; D014508:Urea; D006867:Hydrolases