Noritaka Nishida1, Yutaka Ito2, Ichio Shimada3. 1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2. Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0373, Japan. Electronic address: ito-yutaka@tmu.ac.jp. 3. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: shimada@iw-nmr.f.u-tokyo.ac.jp.
Abstract
BACKGROUND: Accumulating evidence from the experimental and computational studies indicated that the functional properties of proteins are different between in vitro and living cells, raising the necessity to examine the protein structure under the native intracellular milieu. To gain structural information of the proteins inside the living cells at an atomic resolution, in-cell NMR method has been developed for the past two decades. SCOPE OF REVIEW: In this review, we will overview the recent progress in the methodological developments and the biological applications of in-cell NMR, and discuss the advances and challenges in this filed. MAJOR CONCLUSIONS: A number of methods were developed to enrich the isotope-labeled proteins inside the cells, enabling the in-cell NMR observation of bacterial cells as well as eukaryotic cells. In-cell NMR has been applied to various biological systems, including de novo structure determinations, protein/protein or protein/drug interactions, and monitoring of chemical reactions exerted by the endogenous enzymes. The bioreactor system, in which the cells in the NMR tube are perfused by fresh culture medium, enabled the long-term in-cell NMR measurements, and the real-time observations of intracellular responses upon external stimuli. GENERAL SIGNIFICANCE: In-cell NMR has become a unique technology for its ability to obtain the function-related structural information of the target proteins under the physiological or pathological cellular environments, which cannot be reconstituted in vitro.
BACKGROUND: Accumulating evidence from the experimental and computational studies indicated that the functional properties of proteins are different between in vitro and living cells, raising the necessity to examine the protein structure under the native intracellular milieu. To gain structural information of the proteins inside the living cells at an atomic resolution, in-cell NMR method has been developed for the past two decades. SCOPE OF REVIEW: In this review, we will overview the recent progress in the methodological developments and the biological applications of in-cell NMR, and discuss the advances and challenges in this filed. MAJOR CONCLUSIONS: A number of methods were developed to enrich the isotope-labeled proteins inside the cells, enabling the in-cell NMR observation of bacterial cells as well as eukaryotic cells. In-cell NMR has been applied to various biological systems, including de novo structure determinations, protein/protein or protein/drug interactions, and monitoring of chemical reactions exerted by the endogenous enzymes. The bioreactor system, in which the cells in the NMR tube are perfused by fresh culture medium, enabled the long-term in-cell NMR measurements, and the real-time observations of intracellular responses upon external stimuli. GENERAL SIGNIFICANCE: In-cell NMR has become a unique technology for its ability to obtain the function-related structural information of the target proteins under the physiological or pathological cellular environments, which cannot be reconstituted in vitro.