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Quantitative Characterization of Configurational Space Sampled by HIV‐1 Nucleocapsid Using Solution NMR, X‐ray Scattering and Protein Engineering
Author(s) -
Deshmukh Lalit,
Schwieters Charles D.,
Grishaev Alexander,
Clore G. Marius
Publication year - 2016
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.201600212
Subject(s) - characterization (materials science) , space (punctuation) , scattering , chemistry , crystallography , x ray , materials science , physics , analytical chemistry (journal) , nanotechnology , optics , organic chemistry , computer science , operating system
Nucleic‐acid‐related events in the HIV‐1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X‐ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N‐ and C‐termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full‐length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand‐bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function.