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, 13C-, 15N-enriched protein samples with selective protonation of side-chain methyl groups (13CH3). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side-chain 15N, HN resonances, and side-chain 13CH3 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 1H/2H 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.
Original language | English (US) |
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Pages (from-to) | 1232-1246 |
Number of pages | 15 |
Journal | Protein Science |
Volume | 12 |
Issue number | 6 |
DOIs | |
State | Published - Jun 1 2003 |
Externally published | Yes |
Keywords
- Automated NMR data analysis
- Deuteration
- Minimal NMR constraints
- Selective labeling
- Structural genomics
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology