Automated protein fold determination using a minimal NMR constraint strategy |
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| Authors: | Zheng Deyou Huang Yuanpeng J Moseley Hunter N B Xiao Rong Aramini James Swapna G V T Montelione Gaetano T |
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| Affiliation: | Center for Advanced Biotechnology and Medicine (CABM), Northeast Structural Genomics Consortium, and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA. |
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| Abstract: | Determination of precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium-accuracy fold information can often provide key clues about protein evolution and biochemical function(s). In this article we describe a largely automatic strategy for rapid determination of medium-accuracy protein backbone structures. Our strategy derives from ideas originally introduced by other groups for determining medium-accuracy NMR structures of large proteins using deuterated, (13)C-, (15)N-enriched protein samples with selective protonation of side-chain methyl groups ((13)CH(3)). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side-chain (15)N, H(N) resonances, and side-chain (13)CH(3) methyl resonances. These assignments are determined automatically by the program AutoAssign using backbone triple resonance NMR data, together with Spin System Type Assignment Constraints (STACs) derived from side-chain triple-resonance experiments. The program AutoStructure then derives conformational constraints using these chemical shifts, amide (1)H/(2)H exchange, nuclear Overhauser effect spectroscopy (NOESY), and residual dipolar coupling data. The total time required for collecting such NMR data can potentially be as short as a few days. Here we demonstrate an integrated set of NMR software which can process these NMR spectra, carry out resonance assignments, interpret NOESY data, and generate medium-accuracy structures within a few days. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A. |
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| Keywords: | Automated NMR data analysis deuteration minimal NMR constraints selective labeling structural genomics AP antiphase IP in‐phase IPAP in‐phase/antiphase RMSD root‐mean‐square deviation RDC residual dipolar coupling STAC Spin System Type Assignment Constraints STACi intraresidue STAC constraint STACs sequential STAC constraint |
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