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Protein Science (2001), 10:1056-1066.
Copyright © 2001 The Protein Society

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Yawen Bai,1, John Chung, H. Jane Dyson and Peter E. Wright

Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA

Reprint requests to: H. Jane Dyson or Peter E. Wright, Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA; e-mail: wright{at}scripps.edu or dyson{at}scripps.edu; fax:(858) 784-9822.

Plastocyanin is a predominantly ß-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The 3JHN,H{alpha} coupling constants, 15N-edited proton–proton nuclear Overhauser effects (NOEs), and 15N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to ß-strands in the folded protein exhibit small upfield shifts of the 13C{alpha} and 13CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the ß region of ({phi},{psi}) space. This is further supported by the presence of strong sequential d{alpha}N(i, i + 1) NOEs throughout the sequence. The few dNN(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the 13C{alpha}, 13CO, and 1H{alpha} resonances relative to random coil values and by the decreased values of the 3JHN,H{alpha} coupling constants. The 15N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form ß-strands in the native protein spontaneously populate the ß-region of ({phi},{psi}) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.

Keywords: NMR; denatured state; protein folding; ß-sheet protein


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