NMR in-cell

The current state of in-cell NMR has been recently reviewed [5, 9], and it appears that genuine non-invasive NMR measurements of any protein of interest in any cell type will be achievable only in the mid-term future. However, the... 

Serber Z, Selenko P, Hansel R, Reckel S, Lohr F, Ferrell JE, Wagner G, Dotsch V (2006) Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy. Nat Protoc 1 2701-2709... 

Burz DS, Dutta K, Cowbum D, Shekhtman A (2006) Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Nat Methods 3 91-93... 

Sakakibara D, Sasaki A, Ikeya T, Hamatsu J, Hanashima T, Mishima M, Yoshimasu M, Hayashi N, Mikawa T, Walchli M, Smith BO, Shirakawa M, Guntert P, Ito Y (2009) Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458 102-105... 

Reckel S, Hansel R, Lohr F, Dotsch V (2007) In-cell NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 51 91-101... 

Selenko P, Wagner G (2007) Looking into live cells with in-cell NMR spectroscopy. J Struct Biol 158 244-253... 

Investigation of Proteins in Living Bacteria with In-Cell NMR Experiments... 

Abstract In recent years NMR methods have been developed that enable the observation of proteins inside living bacterial cells. Because of the sensitivity of the chemical shift to environmental changes these in-ceU NMR experiments can be used to study protein conformation, molecular interaction or dynamics in a protein s natmal surrounding. Detection of proteins in the bacterial cytoplasm relies on labeling of the protein of interest with NMR active isotopes. This review describes different labeling techniques based on either imiform i5n or labeling as well as amino acid specific labeling schemes. In addition potential applications of these in-cell NMR experiments and their limitations are discussed. 

Keywords Drug interaction In-cell NMR Isotope labeling Protein conformation... 

Fig.l Potential applications for in-cell NMR experiments. Changes in the chemical environment of a protein s nuclei caused for example by post-translational modifications (A), conformational changes (B) or binding events (C) can be detected by differences in chemical shifts in in-cell NMR experiments. Schematic HSQC spectra indicating the sensitivity of the chemical shift to the changes described above are shown next to each cell. Reprinted with permission from Serber and Dotsch, 2001... 

If substantial conformational differences between the in vivo and the in vitro states of the protein exist the in vitro assignments of the chemical shifts might not be transferable to the in-cell NMR spectra, making a new chemical shift assignment necessary. One of the main difficulties of measuring multi-dimensional NMR spectra of proteins inside hving cells is that the life time of the cells or of the protein inside the cells is often smaller than the measurement time for the average multi-dimensional NMR experiment. 

Recently, however, NMR techniques have been developed that can be used to considerably accelerate the measurement of large three-dimensional data sets [31,32], These reduced-dimensionality techniques have already been successfully used in in-cell NMR applications [16]. In the future these techniques might enable the complete backbone assignment of a protein in its natural environment and might enable detailed investigation of different folding states. 

On the basis of these limitations, in-cell NMR experiments are most useful for the investigation of the folding state of proteins, the binding of small molecules that are highly abundant or are added externally or whenever a catalytic relationship between the interaction partner and the protein of... 

Beyond bacteria, mammalian cells undergoing membrane rupture due to transfection with plasmid DNA have been shown to exhibit increased levels of phosphocholine by H HRMAS NMR.118 In general, this new area of research, which has been coined in-cell NMR spectroscopy, is not limited to HRMAS studies,119 but clearly there is an important role for HRMAS in the study of these heterogeneous living systems. 

In addition to the in vitro studies of well-defined systems that have been discussed here, NMR spectroscopy can also be applied to living systems or complex substance mixtures (146). This broad applicability is an advantage of NMR spectroscopy over X-ray crystallography. Early in vivo NMR studies that mostly identify small metabolite molecules by P or H ID experiments have led to different applications. In-cell NMR uses isotopic labeling combined with two or higher dimensional NMR experiments for structural studies of the phosphorylation state of a given protein in the cellular environment or of intrinsically unstructured proteins. In-cell NMR applications in prokaryotic systems cover structural and functional studies (i.e., protein-protein interactions, protein dynamics, automated structure determination, and de novo resonance assignments (147-149)). 

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