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.
Mizuguchi
Mineyuki
M
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Kroon
Gerard J
GJ
Wright
Peter E
PE
Dyson
H Jane
HJ
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