NMR characterization of residual structure in the denatured state of protein L

TitleNMR characterization of residual structure in the denatured state of protein L
Publication TypeJournal Article
Year of Publication2000
AuthorsYi, Q., Scalley-Kim M. L., Alm E. J., & Baker D.
JournalJournal of molecular biology
Volume299
Issue5
Pagination1341-51
Date Published2000 Jun 23
ISSN0022-2836
KeywordsAmino Acid Sequence, Bacterial Proteins, Computer Simulation, DNA-Binding Proteins, Guanidine, Models, Molecular, Molecular Sequence Data, Nitrogen Oxides, Nuclear Magnetic Resonance, Biomolecular, Primary Publication, Protein Conformation, Protein Denaturation, Protons, Spin Labels, Thermodynamics
Abstract

Triple-resonance NMR experiments were used to assign the (13)C(alpha), (13)C(beta), (15)N and NH resonances for all the residues in the denatured state of a destabilized protein L variant in 2 M guanidine. The chemical shifts of most resonances were very close to their random coil values. Significant deviations were observed for G22, L38 and K39; increasing the denaturant concentration shifted the chemical shifts of these residues towards theory random coil values. Medium-range nuclear Overhauser enhancements were detected in segments corresponding to the turn between the first two strands, the end of the second strand through the turn between the second strand and the helix, and the turn between the helix and the third strand in 3D H(1), N(15)-HSQC-NOESY-HSQC experiments on perdeuterated samples. Longer-range interactions were probed by measuring the paramagnetic relaxation enhancement produced by nitroxide spin labels introduced via cysteine residues at five sites around the molecule. Damped oscillations in the magnitude of the paramagnetic relaxation enhancement as a function of distance along the sequence suggested native-like chain reversals in the same three turn regions. The more extensive interactions within the region corresponding to the first beta-turn than in the region corresponding to the second beta-turn suggests that the asymmetry in the folding reaction evident in previous studies of the protein L folding transition state is already established in the denatured state.

Alternate JournalJ. Mol. Biol.
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