Looking into live cells with in-cell NMR spectroscopy.

In-cell NMR spectroscopy has gained recent popularity since it provides means to analyze the conformational and functional properties of proteins inside living cells and at atomic resolution. High-resolution in-cell NMR spectroscopy was originally established in bacterial cells and based on a rationale that relies on protein over-expression and sample analysis within the same cellular environment. Here, we review in-cell NMR approaches in Xenopus laevis oocytes and evaluate potential future applications in other eukaryotic cell types.

[1]  B. Szwergold.  NMR spectroscopy of cells. , 1992, Annual review of physiology.

[2]  E. Komives,et al.  Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation , 1999, Journal of biomolecular NMR.

[3]  G. Wagner,et al.  Investigation of protein motions via relaxation measurements. , 1994, Methods in enzymology.

[4]  J. Wieruszeski,et al.  In vivo detection of the cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum by high-resolution magic angle spinning NMR. , 2001, Journal of magnetic resonance.

[5]  A. Petros,et al.  A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells. , 1992, Biochemistry.

[6]  J. Ruderman,et al.  Using Xenopus oocyte extracts to study signal transduction. , 2006, Methods in molecular biology.

[7]  Volker Dötsch,et al.  In‐Cell NMR Spectroscopy , 2005, Methods in enzymology.

[8]  A. Fosberry,et al.  Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli , 2003, Molecular microbiology.

[9]  A. Murray,et al.  Cell cycle extracts. , 1991, Methods in cell biology.

[10]  Andrew L. Lee,et al.  Protein dynamics in living cells. , 2005, Biochemistry.

[11]  S. Chou,et al.  Preparation of amino‐acid‐type selective isotope labeling of protein expressed in Pichia pastoris , 2005, Proteins.

[12]  Jeffrey W. Peng,et al.  [20] Investigation of protein motions via relaxation measurements , 1994 .

[13]  Vladimir N Uversky,et al.  What does it mean to be natively unfolded? , 2002, European journal of biochemistry.

[14]  Gunnar P. H. Dietz,et al.  Delivery of bioactive molecules into the cell: the Trojan horse approach , 2004, Molecular and Cellular Neuroscience.

[15]  Gary J. Pielak,et al.  Macromolecular Crowding in the Escherichia coli Periplasm Maintains α-Synuclein Disorder , 2006 .

[16]  L. Spicer,et al.  Multidimensional NMR spectroscopy for protein characterization and assignment inside cells. , 2005, Journal of the American Chemical Society.

[17]  R. Lyon,et al.  Monitoring intracellular metabolism by nuclear magnetic resonance. , 1989, Methods in enzymology.

[18]  L. Iakoucheva,et al.  Intrinsic Disorder and Protein Function , 2002 .

[19]  E. Zuiderweg,et al.  Mapping protein-protein interactions in solution by NMR spectroscopy. , 2002, Biochemistry.

[20]  Julie E Bryant.  In-cell protein dynamics. , 2006, Molecular bioSystems.

[21]  S. Cavagnero,et al.  NMR spectroscopic filtration of polypeptides and proteins in complex mixtures , 2004, Journal of biomolecular NMR.

[22]  A. Palmer,et al.  Nmr probes of molecular dynamics: overview and comparison with other techniques. , 2001, Annual review of biophysics and biomolecular structure.

[23]  A. Fink.  Natively unfolded proteins. , 2005, Current opinion in structural biology.

[24]  V. Dötsch,et al.  High-resolution macromolecular NMR spectroscopy inside living cells. , 2001, Journal of the American Chemical Society.

[25]  C. Brown,et al.  Intrinsic protein disorder in complete genomes. , 2000, Genome informatics. Workshop on Genome Informatics.

[26]  Shigeyuki Yokoyama,et al.  Protein expression systems for structural genomics and proteomics. , 2003, Current opinion in chemical biology.

[27]  D. Jones,et al.  Nuclear Magnetic Resonance Spectroscopy , 1978 .

[28]  G. Pielak,et al.  Macromolecular crowding in the Escherichia coli periplasm maintains alpha-synuclein disorder. , 2006, Journal of molecular biology.

[29]  G. Wagner,et al.  egg extracts and intact oocytes Xenopus laevisFrom the Cover : Quantitative NMR analysis of the protein G B 1 domain in , 2006 .

[30]  W. Franke,et al.  Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos , 1991, The Journal of cell biology.

[31]  E. Zuiderweg,et al.  Characterizing semilocal motions in proteins by NMR relaxation studies. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Yin Li,et al.  Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide. , 2002, Biochemical and biophysical research communications.

[33]  Andrew W. Murray,et al.  Chapter 30 Cell Cycle Extracts , 1991 .

[34]  J. Ferrell,et al.  Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy , 2006, Nature Protocols.

[35]  B. Chait,et al.  Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures , 1996, Journal of biomolecular NMR.

[36]  Gerhard Wagner,et al.  Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes , 2006, Proceedings of the National Academy of Sciences.

[37]  L. Kay,et al.  New developments in isotope labeling strategies for protein solution NMR spectroscopy. , 2000, Current opinion in structural biology.

[38]  G. Pielak,et al.  FlgM gains structure in living cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Shirakawa,et al.  In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes , 2006, Journal of biomolecular NMR.

[40]  A. Pickford,et al.  Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris. , 2004, Methods in molecular biology.

[41]  A. Prochiantz,et al.  Trojan peptides: the penetratin system for intracellular delivery. , 1998, Trends in cell biology.

[42]  Alexander Shekhtman,et al.  Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR) , 2006, Nature Methods.

[43]  K. Wüthrich,et al.  Automated Protein NMR Structure Determination in Crude Cell-Extract , 2006, Journal of biomolecular NMR.

[44]  B. Kay,et al.  Xenopus laevis : practical uses in cell and molecular biology , 1991 .

[45]  P. Ortiz de Montellano,et al.  Methyl groups as probes for proteins and complexes in in-cell NMR experiments. , 2004, Journal of the American Chemical Society.

[46]  W. Jahnke,et al.  Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells , 2005, Journal of biomolecular NMR.

[47]  J Li,et al.  Conformation and function of the N-linked glycan in the adhesion domain of human CD2 , 1995, Science.

[48]  H. Dyson,et al.  Intrinsically unstructured proteins and their functions , 2005, Nature Reviews Molecular Cell Biology.

[49]  Michael Fejtl,et al.  The roboocyte: automated cDNA/mRNA injection and subsequent TEVC recording on Xenopus oocytes in 96-well microtiter plates. , 2003, Receptors & channels.

[50]  W. Jahnke,et al.  Amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies , 2003, Journal of biomolecular NMR.