Cross-saturation and transferred cross-saturation experiments.
Structural analyses of protein-protein interactions are required to reveal their functional mechanisms, and accurate protein-protein complex models, based on experimental results, are the starting points for drug development. In addition, structural information about proteins under physiologically relevant conditions is crucially important for understanding biological events. However, for proteins such as those embedded in lipid bilayers and transiently complexed with their effectors under physiological conditions, structural analyses by conventional methods are generally difficult, due to their large molecular weights and inhomogeneity. We have developed the cross-saturation (CS) method, which is an nuclear magnetic resonance measurement technique for the precise identification of the interfaces of protein-protein complexes. In addition, we have developed an extended version of the CS method, termed transferred cross-saturation (TCS), which enables the identification of the residues of protein ligands in close proximity to huge (>150 kDa) and heterogeneous complexes under fast exchange conditions (>0.1 s(-1)). Here, we discuss the outline, basic theory, and practical considerations of the CS and TCS methods. In addition, we will review the recent progress in the construction of models of protein-protein complexes, based on CS and TCS experiments, and applications of TCS to in situ analyses of biologically and medically important proteins in physiologically relevant states.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Takumi
T
Ueda
Molecular Profiling Research Center, National Institute of Advanced Industrial Science and Technology, Aomi, Koto-ku, Tokyo 135-0064, Japan.
Koh
K
Takeuchi
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Noritaka
N
Nishida
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Pavlos
P
Stampoulis
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Yutaka
Y
Kofuku
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Masanori
M
Osawa
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Ichio
I
Shimada
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
D000596:Amino Acids; D011506:Proteins