투고논문

Abstract Conventional structural biology has largely relied on purified proteins studied under artificial in vitro conditions, which cannot fully recapitulate the crowded, heterogeneous, and dynamic intracellular environment. In-cell NMR spectroscopy overcomes this limitation by enabling atomic-resolution observation of protein structures, dynamics, and interactions directly inside living cells. This review summarizes recent advances in in-cell NMR methodologies, focusing on strategies for preparing isotope-labeled target proteins in different cellular contexts. We discuss in situ expression approaches in prokaryotic, insect, and mammalian cells, as well as exogenous delivery methods for mammalian in-cell NMR, including electroporation, cell-penetrating peptides, and reversible membrane permeabilization using streptolysin O. We further highlight representative applications in the characterization of intrinsically disordered proteins, protein maturation, and intracellular interactions. Future integration with bioreactor-based sample systems, microfluidics, cryo-electron tomography, and AI-assisted structural modeling is expected to expand the scope and impact of in-cell NMR. By preserving the native cellular context of proteins, in-cell NMR offers a powerful platform for understanding biomolecular function and pharmacology inside living cells.

Keywords In-cell NMR; Escherichia coli; mammalian cells; insect cells; cell-penetrating peptides; streptolysin O; protein delivery; isotope labeling

* Address correspondence to: Ji-Hun Kim, College of Pharmacy, Chungbuk National University, Cheongju 28160, Republic of Korea, Tel: 82-43-249-1343; E-mail: nmrjhkim@chungbuk.ac.kr; Soo Jae Lee, College of Pharmacy, Chungbuk National University, Cheongju 28160, Republic of Korea, Tel: 82-43-261-2816; E-mail: sjlee@chungbuk.ac.kr