TY - JOUR
T1 - Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements
AU - Kodama, Yuya
AU - Reese, Michael L.
AU - Shimba, Nobuhisa
AU - Ono, Katsuki
AU - Kanamori, Eiji
AU - Dötsch, Volker
AU - Noguchi, Shuji
AU - Fukunishi, Yoshifumi
AU - Suzuki, Ei ichiro
AU - Shimada, Ichio
AU - Takahashi, Hideo
N1 - Funding Information:
We thank Dr. T. Kohno for the genes of YUH and yeast Ub and Dr. T. Onizuka, Y. Mizukoshi, and Y. Tokunaga for technical advice. This study was supported by grants from New Energy and Industrial Technology Development Organizations.
PY - 2011/6
Y1 - 2011/6
N2 - Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an 15N-labeled amino acid and several amino acids 13C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω1-TROSY-type backbone 1H-15N and aromatic 1H-13C shift correlations together with single quantum methyl 1H-13C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development.
AB - Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an 15N-labeled amino acid and several amino acids 13C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω1-TROSY-type backbone 1H-15N and aromatic 1H-13C shift correlations together with single quantum methyl 1H-13C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development.
KW - Assignment free
KW - NMR
KW - Protein-protein docking
KW - Protein-protein interaction
KW - TROSY
KW - Time-sharing measurements
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U2 - 10.1016/j.jsb.2011.04.001
DO - 10.1016/j.jsb.2011.04.001
M3 - Article
C2 - 21501688
AN - SCOPUS:79955655057
SN - 1047-8477
VL - 174
SP - 434
EP - 442
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -