Folding of a beta-sheet protein monitored by real-time NMR spectroscopy.
At low ionic strength, apoplastocyanin forms an unfolded state under non-denaturing conditions. The refolding of this state is sufficiently slow to allow real-time NMR experiments to be performed. Folding of apoplastocyanin, initiated by the addition of salt and followed by real-time 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy, is highly cooperative. A concomitant increase in the intensity of both sequential and long-range nuclear Overhauser effects (NOEs) between backbone amide protons in successive acquisitions of 1H-15N HSQC-NOESY-HSQC spectra provides the first direct observation of the development of structure-specific NOEs as a protein folds. Our results show that the local and long-range interactions in the native apoplastocyanin are formed simultaneously, consistent with highly cooperative formation of the native structure.
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Mineyuki
M
Mizuguchi
Gerard J
GJ
Kroon
Peter E
PE
Wright
H Jane
HJ
Dyson
D001059:Apoproteins / Q000737:chemistry*; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010970:Plastocyanin / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
D001059:Apoproteins; D011994:Recombinant Proteins; C040778:apoplastocyanin; D010970:Plastocyanin