Once upon a time, when one of the editors (Yutaka Ito) was an undergraduate student and had just started a solution NMR study of human H-Ras protein, there was a graduate student in the same laboratory arguing that he would not believe in the biological meaning of the results obtained for proteins which were overexpressed in E. coli, purified, concentrated into the millimolar range, and incubated during long NMR experiments (a series of 2D ¹H NMR experiments at that time). He may have been an “in-cell chauvinist.” Luckily the bet for a dinner resulted in a win by demonstrating the neurite outgrowth in PC12 cells induced by a microinjection of an oncogenic Ras protein sample, which had experienced more than a week of NMR experiments at 37 °C. Strictly speaking the experiment using PC12 cells only showed that the Ras protein retained its activity even after the NMR experiments, but not the claimed appropriateness of the NMR data. Nevertheless, at that time, it was probably the best we could do for connecting the proteins providing the NMR signals in vitro with their biological behaviours in cells. However, at the present time, we are able to measure high-resolution NMR spectra of proteins in living cells as a result of technological advances in NMR hardware and methodology.

In-cell NMR, heteronuclear multidimensional NMR of biomacromolecules in living cells, was first reported in 2001 using E. coli recombinant expression systems, with the expectation of it becoming a major tool for investigating protein behaviour, structure, dynamics, and binding events, etc. in natural environments, thus, ultimately providing information for medical and pharmaceutical applications, e.g. screening of drug candidates. Almost two decades later, the scope of in-cell NMR has extended from bacteria to eukaryotic cells, particularly cultured human cells, and various intracellular biological events such as structural changes, post-translational modifications, ligand bindings and protein–protein interactions have been analysed. Considering the methodological maturations as the only existing tool for observing the intracellular behaviour of biological macromolecules at atomic resolution, in-cell NMR is going to become even more attractive for molecular and cell biologists. So far, only in-cell NMR can bridge the gap between the information obtained from in vitro biophysical/biochemical characterisation of target molecules and their intracellular behaviour.

In-cell NMR Spectroscopy: From Molecular Sciences to Cell Biology provides detailed descriptions covering the background of in-cell NMR, methods on cell biological techniques and NMR spectroscopy, as well as applications, and future perspectives. The brief history of in-cell NMR studies is described in Part I: (Chapter 1) In-cell NMR spectroscopy: from 2001 to now. In Part II: Host cells for in-cell NMR studies, methods for sample preparations using bacterial cells (Chapter 2) and eukaryotic cells (Chapters 3 and 4) are introduced. Part III: Basic in-cell NMR techniques focuses on procedures for structural analyses (Chapters 5 and 6), the bioreactor system (Chapter 7), and the use of ¹⁹F nuclei (Chapter 8). Solid-state NMR approaches (Chapter 9) and in-cell EPR studies (Chapter 10) are also described in this part. Part IV: In-cell NMR applications overviews in-cell NMR studies targeted for protein physical properties (Chapters 11 and 12) and maturation processes (Chapter 13). Molecular dynamics approaches (in combination with in vitro/in-cell NMR studies) toward protein behaviours under intracellular crowding environments are also described in Chapter 14. As a perspective to medical and pharmaceutical applications, Chapter 15 overviews the present situation of in-cell NMR-assisted drug-screening. The final chapter, Chapter 16, reviews recent advances and applications of in-cell NMR to nucleic acids.

Despite the interest in in-cell NMR, to the best of our knowledge, no informative books specifically focused on in-cell NMR have been published yet. We are confident that we have collected a large majority of the latest and biologically important in-cell NMR applications from leading in-cell NMR groups with a global spread. We hope that In-cell NMR Spectroscopy: From Molecular Sciences to Cell Biology will be helpful for students and researchers with an interest in in-cell NMR spectroscopy.

Yutaka Ito

Volker Dötsch

Masahiro Shirakawa