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, 11 (4), 223-32

Fully Automated High-Quality NMR Structure Determination of Small (2)H-enriched Proteins

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Fully Automated High-Quality NMR Structure Determination of Small (2)H-enriched Proteins

Yuefeng Tang et al. J Struct Funct Genomics.

Abstract

Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 Å, an all atom RMSD value of 1.2 Å to the crystal structure for well-defined regions, and RMSD value of 1.1 Å to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with (15)N- and (13)C-edited NOESY data obtained with a perdeuterated (15)N, (13)C-enriched (13)CH(3) methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR.

Figures

Fig. 1
Fig. 1
Summary of backbone and 13Cβ resonances assignments for CspA derived from triple resonance NMR experiments. Red bars and yellow bars underneath the amino acid sequence represent the connectivity established between intra and sequential residues respectively. These data were obtained by analyzing six 2D and 3D NMR spectra, summarized in Table 1. Slowly exchanging backbone amides, used in the conventional structure analysis but not in the CS-Rosetta analysis, identified by 1H/2H exchange measurements, are represented by filled circles. Secondary structures of the β-barrel found in the final structure are indicated by arrows along the amino acid sequence
Fig. 2
Fig. 2
Stereoview of the superimposition of AutoStructure-CNS structure for [1H-13C]-I(δ1)LV, 13C, 15N, 2H-enriched CspA determined by conventional automated analysis methods (blue) with 1mjc (red). a Backbone line representations of the 10 lowest energy conformers obtained from AutoStructure-CNS structure compared with 1mjc. b Ribbon diagram of the lowest energy conformer of AutoStructure-CNS structure versus 1mjc. c The packing of the hydrophobic residues (viz, V9, I21, V30, V32, I37, L45, V51, F53, A64, and V67) for the lowest energy conformer of AutoStructure-CNS structure versus 1mjc. The disordered N-terminal hexaHis expression tag is excluded from the analysis
Fig. 3
Fig. 3
Stereoview of the superimposition of the CS-Rosetta structure for 2H,13C,15N-enriched CspA (blue) with 1mjc (red). a Backbone line representations of the 10 lowest energy conformers obtained from CS-Rosetta structure compared with 1mjc. b Ribbon diagram of the lowest energy conformer of CS-Rosetta structure versus 1mjc. c The packing of the core hydrophobic residues (viz, V9, I21, V30, V32, I37, L45, V51, F53, A64, and V67) for the lowest energy conformer of CS-Rosetta structure versus 1mjc. The disordered N-terminal expression tag is excluded from the analysis

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