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

Citation
Yw. Bai et al., Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions, PROTEIN SCI, 10(5), 2001, pp. 1056-1066
Citations number
63
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
10
Issue
5
Year of publication
2001
Pages
1056 - 1066
Database
ISI
SICI code
0961-8368(200105)10:5<1056:SADCOA>2.0.ZU;2-Y
Abstract
Plastocyanin is a predominantly beta -sheet protein containing a type I cop per center. The conformational ensemble of a denatured state of apo-plastoc yanin formed in solution under conditions of low salt and neutral pH has be en investigated by multidimensional heteronuclear NMR spectroscopy. Chemica l shift assignments were obtained by using three-dimensional triple-resonan ce NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,H alpha) coupling constants, N-1 5-edited proton-proton nuclear Overhauser effects (NOEs), and N-15 relaxati on parameters were also measured for the purpose of structural and dynamic characterization Most of the residues corresponding to beta -strands in the folded protein exhibit small upfield shifts of the C-13(alpha) and (CO)-C- 13 resonances relative to random coil values, suggesting a slight preferenc e for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i +1 ) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that ar e observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, b etween residues 59 and 63, was found to populate a nonnative helical confor mation in the unfolded state, as indicated by the shift of the C-13(alpha), (CO)-C-13, and H-1(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,H alpha) coupling constants. The N-15 relaxation parameters indicate restriction of motions on a nanosecond times cale 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 shor t helix in the native folded protein. The results are consistent with earli er NMR studies of peptide fragments of plastocyanin and confirm that the re gions of the sequence that form beta -strands in the native protein spontan eously populate the beta -region of (phi,psi) space under folding condition s, even in the absence of stabilizing tertiary interactions. We conclude th at 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.