Abstract
In this review, we discuss how time-resolved Fourier transform infrared (FTIR) spectroscopy is used to understand how GTP hydrolysis is catalyzed by small GTPases and their cognate GTPase-activating proteins (GAPs). By interaction with small GTPases, GAPs regulate important signal transduction pathways and transport mechanisms in cells. The GTPase reaction terminates signaling and controls transport. Dysfunctions of GTP hydrolysis in these proteins are linked to serious diseases including cancer. Using FTIR, we resolved both the intrinsic and GAP-catalyzed GTPase reaction of the small GTPase Ras with high spatiotemporal resolution and atomic detail. This provided detailed insight into the order of events and how the active site is completed for catalysis. Comparisons of Ras with other small GTPases revealed conservation and variation in the catalytic mechanisms. The approach was extended to more nearly physiological conditions at a membrane. Interactions of membrane-anchored GTPases and their extraction from the membrane are studied using the attenuated total reflection (ATR) technique.
About the authors
Carsten Kötting obtained a PhD in physical organic chemistry from the chemistry department of Ruhr-University Bochum, Germany in 1999, working on matrix isolation of organic intermediates. Afterwards he joined as a Feodor Lynen fellow the Zewail group at Caltech, were he did femtochemistry of organic intermediates. After two years he returned to Ruhr-University but joined the biophysics institute of Klaus Gerwert. He finished his habilitation in chemical biology working on time resolved FTIR spectroscopy of GTPases in 2009. His current research interests include besides the details of the reaction mechanisms of GTPases, the extension of vibrational spectroscopy to larger systems using Raman microscopy and the development of chemically modified surfaces for protein immobilization on ATR crystals.
Klaus Gerwert is head of the Department of Biophysics at RUB. He is internationally recognized for his contributions in protein-dynamics with very high spatiotemporal resolution using a combination of time-resolved vibrational spectroscopy (FTIR and Raman) and biomolecular simulations (QM/MM). He elaborated the role of proteinbound water molecules in proton-transfer in proteins, especially in microbial rhodopsins like bacteriorhodopsin and channelrhodopsin. Furthermore, he contributed to the detailed understanding of the catalysis of G-proteins by their respective G-activating proteins like Ras and Rab. Recently his approach is extended to markerfree vibrational imaging in cells and tissue for their application in diagnostics. K. Gerwert graduated in biophysical chemistry (Freiburg 1985), was research scientist at the Max-Planck-Institute Dortmund (1986–1989), became Heisenberg-fellow at Scripps, USA, and MPI Dortmund (1990–1993). Since 1993 he is a full professor at the Ruhr-University Bochum (chair of biophysics). Since 2004 spokesman of SFB642 and since 2010 of PURE. Since 2009 Max-Planck fellow at the Max-Planck- CAS Partner-Institute in Shanghai (2009–2013 as director in a dual appointment). He received several awards and is a member of the NRW academy of science.
Acknowledgments
We acknowledge the Deutsche Forschungsgemeinschaft (grant no. SFB 642) for its financial support. We thank past and present members of the Bochum GTPase group and our collaborators for all their contributions and Konstantin Gavriljuk for manuscript proofreading. Special thanks are given to Roger S. Goody and Alfred Wittinghofer for an excellent and very fruitful collaboration over the years.
References
Ahearn, I.M., Haigis, K., Bar-Sagi, D., and Philips, M.R. (2011). Regulating the regulator: post-translational modification of RAS. Nat. Rev. Mol. Cell Biol. 13, 39–51.10.1038/nrm3255Search in Google Scholar
Ataka, K., Kottke, T., and Heberle, J. (2010). Thinner, smaller, faster: IR techniques to probe the functionality of biological and biomimetic systems. Angew. Chem. Int. Ed. 49, 5416–5424.10.1002/anie.200907114Search in Google Scholar
Bader, B., Kuhn, K., Owen, D.J., Waldmann, H., Wittinghofer, A., and Kuhlmann, J. (2000). Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nat. Lond. 403, 223–226.10.1038/35003249Search in Google Scholar
Barth, A. and Zscherp, C. (2002). What vibrations tell about proteins. Q. Rev. Biophys. 35, 369–430.10.1017/S0033583502003815Search in Google Scholar
Barth, A., Corrie, E.T.J., Gradwell, M.J., Maeda, Y., Mäntele, W., Meier, T., and D.R. Trentham. (1997). Time-resolved infrared spectroscopy of intermediates and products from photolysis of 1-(2-nitrophenyl)ethyl phosphates: reaction of the 2-nitrosoacetophenone byproduct with Thiols. J. Am. Chem. Soc. 119, 4149–4159.10.1021/ja964430uSearch in Google Scholar
Brucker, S., Gerwert, K., and Kötting, C. (2010). Tyr39 of Ran preserves the Ran.GTP gradient by inhibiting GTP hydrolysis. J. Mol. Biol. 401, 1–6.10.1016/j.jmb.2010.05.068Search in Google Scholar
Cepus, V., Ulbrich, C., Allin, C., Troullier, A., and Gerwert, K. (1998). Fourier transform infrared photolysis studies of caged compounds. Methods Enzymol. 291, 223–245.10.1016/S0076-6879(98)91015-1Search in Google Scholar
Chakrabarti, P.P., Daumke, O., Suveyzdis, Y., Kötting, C., Gerwert, K., and Wittinghofer, A. (2007). Insight into catalysis of a unique GTPase reaction by a combined biochemical and FTIR approach. J. Mol. Biol. 367, 983–995.10.1016/j.jmb.2006.11.022Search in Google Scholar PubMed
Cherfils, J. and Zeghouf, M. (2013). Regulation of Small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269–309.10.1152/physrev.00003.2012Search in Google Scholar PubMed
Cox, A.D. and Der, C.J. (2010). Ras history – the saga continues. Small GTPases 1, 2–27.10.4161/sgtp.1.1.12178Search in Google Scholar PubMed PubMed Central
Dannenberg, J.J. (2006). Enthalpies of hydration of N-methylacetamide by one, two, and three waters and the effect upon the C:O stretching frequency. An ab initio DFT study. J. Phys. Chem. A 110, 5798–5802.10.1021/jp060452jSearch in Google Scholar
Daumke, O., Weyand, M., Chakrabarti, P.P., Vetter, I.R., and Wittinghofer, A. (2004). The GTPase-activating protein Rap1GAP uses a catalytic asparagine. Nature 429, 197–201.10.1038/nature02505Search in Google Scholar
Deng, H., Wang, J.H., Callender, R., and Ray, W.J. (1998). Relationship between bond stretching frequencies and internal bonding for [O-16(4)]- and [O-18(4)]phosphates in aqueous solution. J. Phys. Chem. B 102, 3617–3623.10.1021/jp973314qSearch in Google Scholar
Engelhard, M., Gerwert, K., Hess, B., and Siebert, F. (1985). Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation of static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane. Biochemistry (Mosc) 24, 400–407.10.1021/bi00323a024Search in Google Scholar
Frasa, M.A.M., Koessmeier, K.T., Ahmadian, M.R., and Braga, V.M.M. (2012). Illuminating the functional and structural repertoire of human TBC/RABGAPs. Nat. Rev. Mol. Cell Biol. 13, 67–73.10.1038/nrm3267Search in Google Scholar
Gavriljuk, K., Gazdag, E.-M., Itzen, A., Kötting, C., Goody, R.S., and Gerwert, K. (2012). Catalytic mechanism of a mammalian RabRabGAP complex in atomic detail. Proc. Natl. Acad. Sci. USA 109, 21348–21353.10.1073/pnas.1214431110Search in Google Scholar
Gavriljuk, K., Itzen, A., Goody, R.S., Gerwert, K., and Kötting, C. (2013). Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy. Proc. Natl. Acad. Sci. USA 110, 13380–13385.10.1073/pnas.1307655110Search in Google Scholar
Gelb, M.H. (1997). Protein prenylation, et cetera – signal transduction in two dimensions. Science 275, 1750–1750.10.1126/science.275.5307.1750Search in Google Scholar
Gerwert, K. (1988). Intramolecular protein dynamics study with time-resolved Fourier-transform IR-difference spectroscopy. Berichte Bunsen-Ges 92, 978–982.10.1002/bbpc.198800244Search in Google Scholar
Gerwert, K. (1993). Molecular reaction mechanisms of proteins as monitored by time-resolved FTIR spectroscopy. Curr. Opin. Struct. Biol. i, 769–773.10.1016/0959-440X(93)90062-PSearch in Google Scholar
Gerwert, K. and Kötting, C. (2010). Fourier transform infrared (FTIR) spectroscopy. Encycl. Life Sci. (Chichester: John Wiley & Sons, Ltd.). DOI: 10.1002/9780470015902.a0003112.pub210.1002/9780470015902.a0003112.pub2Search in Google Scholar
Gerwert, K. and Siebert, F. (1986). Evidence for light-induced 13-cis, 14-s-cis, isomerization in bacteriorhodopsin obtained by FTIR difference spectroscopy using isotopically labelled retinals. EMBO J. 5, 805–811.10.1002/j.1460-2075.1986.tb04285.xSearch in Google Scholar PubMed PubMed Central
Gerwert, K., Hess, B., Soppa, J., and Oesterhelt, D. (1989). Role of aspartate-96 in proton translocation by bacteriorhodopsin. Proc. Natl. Acad. Sci. USA 86, 4943–4947.10.1073/pnas.86.13.4943Search in Google Scholar PubMed PubMed Central
Gerwert, K., Souvignier, G., and Hess, B. (1990). Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy. Proc. Natl. Acad. Sci. USA 87, 9774–9778.10.1073/pnas.87.24.9774Search in Google Scholar PubMed PubMed Central
Goormaghtigh, E., Gasper, R., Benard, A., Goldsztein, A., and Raussens, V. (2009). Protein secondary structure content in solution, films and tissues: redundancy and complementarity of the information content in circular dichroism, transmission and ATR FTIR spectra. Biochim. Biophys. Acta 1794, 1332–1343.10.1016/j.bbapap.2009.06.007Search in Google Scholar PubMed
Grigorenko, B.L., Nemukhin, A.V., Shadrina, M.S., Topol, I.A., and Burt, S.K. (2007). Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations. Proteins Struct Funct Bioinform 66, 456–466.10.1002/prot.21228Search in Google Scholar PubMed
Gruschus, J.M., Byrd, R.A., and Randazzo, P.A. (2014). The importance of seeing surface (effects). Structure 22, 363–365.10.1016/j.str.2014.02.005Search in Google Scholar PubMed PubMed Central
Güldenhaupt, J., Adigüzel, Y., Kuhlmann, J., Waldmann, H., Kötting, C., and Gerwert, K. (2008). Secondary structure of lipidated Ras bound to a lipid bilayer. FEBS J. 275, 5910–5918.10.1111/j.1742-4658.2008.06720.xSearch in Google Scholar PubMed
Güldenhaupt, J., Rudack, T., Bachler, P., Mann, D., Triola, G., Waldmann, H., Kötting, C., and Gerwert, K. (2012). N-Ras forms dimers at POPC membranes. Biophys. J. 103, 1585–1593.10.1016/j.bpj.2012.08.043Search in Google Scholar PubMed PubMed Central
Ham, S. and Cho, M. (2003). Amide I modes in the N-methylacetamide dimer and glycine dipeptide analog: diagonal force constants. J. Chem. Phys. 118, 6915.10.1063/1.1559681Search in Google Scholar
Henis, Y.I., Hancock, J.F., and Prior, I.A. (2009). Ras acylation, compartmentalization and signaling nanoclusters (review). Mol. Membr. Biol. 26, 80–92.10.1080/09687680802649582Search in Google Scholar
Hering, J.A. and Haris, P.I. (2009). FTIR Spectroscopy for Analysis of Protein Secondary Structure (Amsterdam; IOS Press).Search in Google Scholar
Hessling, B., Souvignier, G., and Gerwert, K. (1993). A model-independent approach to assigning bacteriorhodopsin’s intramolecular reactions to photocycle intermediates. Biophys. J. 65, 1929–1941.10.1016/S0006-3495(93)81264-5Search in Google Scholar
Hodges-Loaiza, H.B., Parker, L.E., and Cox, A.D. (2011). Prenylation and Phosphorylation of Ras Superfamily Small GTPases. The Enzymes (Elsevier: Amsterdam, The Netherlands), pp. 43–69.10.1016/B978-0-12-415922-8.00003-3Search in Google Scholar
Jacobsen, R.L., Johnson, R.D., Irikura, K.K., and Kacker, R.N. (2013). Anharmonic vibrational frequency calculations are not worthwhile for small basis sets. J. Chem. Theory Comput. 9, 951–954.10.1021/ct300293aSearch in Google Scholar PubMed
Jamali, T., Jamali, Y., Mehrbod, M., and Mofrad, M.R.K. (2011). Nuclear pore complex. Int. Rev. Cell Mol. Biol. 287, 233–286.10.1016/B978-0-12-386043-9.00006-2Search in Google Scholar PubMed
Khan, A.R. and Ménétrey, J. (2013). Structural biology of Arf and Rab GTPases’ effector recruitment and specificity. Structure 21, 1284–1297.10.1016/j.str.2013.06.016Search in Google Scholar PubMed
Klán, P., Šolomek, T., Bochet, C.G., Blanc, A., Givens, R., Rubina, M., Popik, V., Kostikov, A., and Wirz, J. (2013). Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy. Chem. Rev. 113, 119–191.10.1021/cr300177kSearch in Google Scholar PubMed PubMed Central
Kötting, C. and Gerwert, K. (2004). Time-resolved FTIR studies provide activation free energy, activation enthalpy and activation entropy for GTPase reactions. Chem. Phys. 307, 227–232.10.1016/j.chemphys.2004.06.051Search in Google Scholar
Kötting, C. and Gerwert, K. (2005a). Proteins in action monitored by time-resolved FTIR spectroscopy. ChemPhysChem 6, 881–888.10.1002/cphc.200400504Search in Google Scholar PubMed
Kötting, C. and Gerwert, K. (2005b). Monitoring protein-protein interactions by time-resolved FTIR difference spectroscopy. In: Protein-Protein Interact, 2nd Ed, E. Golemis and P. Adams, eds. (Cold Spring Harbor, NY, USA: Cold Spring Harb Lab Press) pp. 279–299.10.1007/978-1-62703-398-5_11Search in Google Scholar PubMed
Kötting, C. and Gerwert, K. (2013). The dynamics of the catalytic site in small GTPases, variations on a common motif. FEBS Lett. 587, 2025–2027.10.1016/j.febslet.2013.05.021Search in Google Scholar PubMed
Kötting, C., Blessenohl, M., Suveyzdis, Y., Goody, R.S., Wittinghofer, A., and Gerwert, K. (2006). A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein. Proc. Natl. Acad. Sci. USA 103, 13911–13916.10.1073/pnas.0604128103Search in Google Scholar PubMed PubMed Central
Kötting, C., Kallenbach, A., Suveyzdis, Y., Eichholz, C., and Gerwert, K. (2007). Surface change of Ras enabling effector binding monitored in real time at atomic resolution. ChemBioChem 8, 781–787.10.1002/cbic.200600552Search in Google Scholar PubMed
Kötting, C., Kallenbach, A., Suveyzdis, Y., Wittinghofer, A., and Gerwert, K. (2008). The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy. Proc. Natl. Acad. Sci. USA 105, 6260–6265.10.1073/pnas.0712095105Search in Google Scholar PubMed PubMed Central
Kötting, C., Suveyzdis, Y., Bojja, R.S., Metzler-Nolte, N., and Gerwert, K. (2010). Label-free screening of drug-protein interactions by time-resolved Fourier transform infrared spectroscopic assays exemplified by Ras interactions. Appl. Spectrosc. 64, 967–972.10.1366/000370210792434341Search in Google Scholar PubMed
Kötting, C., Güldenhaupt, J., and Gerwert, K. (2012). Time-resolved FTIR spectroscopy for monitoring protein dynamics exemplified by functional studies of Ras protein bound to a lipid bilayer. Chem. Phys. 396, 72–83.10.1016/j.chemphys.2011.08.007Search in Google Scholar
Kupzig, S., Deaconescu, D., Bouyoucef, D., Walker, S.A., Liu, Q., Polte, C.L., Daumke, O., Ishizaki, T., Lockyer, P.J., Wittinghofer, A., et al. (2006). GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins. J. Biol. Chem. 281, 9891–9900.10.1074/jbc.M512802200Search in Google Scholar PubMed PubMed Central
Ligeti, E., Welti, S., and Scheffzek, K. (2012). Inhibition and termination of physiological responses by GTPase activating proteins. Physiol. Rev. 92, 237–272.10.1152/physrev.00045.2010Search in Google Scholar PubMed
Lin, W.-C., Iversen, L., Tu, H.-L., Rhodes, C., Christensen, S.M., Iwig, J.S., Hansen, S.D., Huang, W.Y., and Groves, J.T. (2014). H-Ras forms dimers on membrane surfaces via a protein-protein interface. Proc. Natl. Acad. Sci. USA 111, 2996–3001.10.1073/pnas.1321155111Search in Google Scholar PubMed PubMed Central
Marshall, C.B., Ho, J., Buerger, C., Plevin, M.J., Li, G.Y., Li, Z., Ikura, M., and Stambolic, V. (2009). Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR. Sci. Signal 2, ra3.Search in Google Scholar
Nottingham, R.M. and Pfeffer, S.R. (2014). Mutant enzymes challenge all assumptions. eLife 3, e02171.10.7554/eLife.02171Search in Google Scholar PubMed PubMed Central
Pan, X.J., Eathiraj, S., Munson, M., and Lambright, D.G. (2006). TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442, 303–306.10.1038/nature04847Search in Google Scholar PubMed
Phillips, R.A., Hunter, J.L., Eccleston, J.F., and Webb, M.R. (2003). The Mechanism of Ras GTPase activation by neurofibromin. Biochemistry (Mosc) 42, 3956–3965.10.1021/bi027316zSearch in Google Scholar PubMed
Pinkerneil, P., Güldenhaupt, J., Gerwert, K., and Kötting, C. (2012). Surface-attached polyhistidine-Tag proteins characterized by FTIR difference spectroscopy. ChemPhysChem 13, 2649–2653.10.1002/cphc.201200358Search in Google Scholar PubMed PubMed Central
Prakash, P. and Gorfe, A.A. (2014). Overview of simulation studies on the enzymatic activity and conformational dynamics of the GTPase Ras. Mol. Simul. 40, 839–847.10.1080/08927022.2014.895000Search in Google Scholar PubMed PubMed Central
Rocks, O., Peyker, A., and Kahms, M., Verveer, P.J., Koerner, C., Lumbierres, M., Kuhlmann, J., Waldmann, H., Wittinghofer, A., and Bastiaens, P.I. (2005). An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752.10.1126/science.1105654Search in Google Scholar PubMed
Rudack, T., Xia, F., Schlitter, J., Kötting, C., and Gerwert, K. (2012a). The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations. Biophys. J. 103, 293–302.10.1016/j.bpj.2012.06.015Search in Google Scholar PubMed PubMed Central
Rudack, T., Xia, F., Schlitter, J., Kötting, C., and Gerwert, K. (2012b). Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations. Proc. Natl. Acad. Sci. USA 109, 15295–15300.10.1073/pnas.1204333109Search in Google Scholar PubMed PubMed Central
Santos, E. (2014). Dimerization opens new avenues into Ras signaling research. Sci. Signal 7, pe12.Search in Google Scholar
Schartner, J., Güldenhaupt, J., Mei, B., Rögner, M., Muhler, M., Gerwert, K., and Kötting, C. (2013). Universal method for protein immobilization on chemically functionalized germanium investigated by ATR-FTIR difference spectroscopy. J. Am. Chem. Soc. 135, 4079–4087.10.1021/ja400253pSearch in Google Scholar PubMed
Schlichting, I., Rapp, G., John, J., Wittinghofer, A., Pai, E.F., and Goody, R.S. (1989). Biochemical and crystallographic characterization of a complex of C-Ha-Ras P21 and caged Gtp with flash-photolysis – (time-resolved structure). Proc. Natl. Acad. Sci. USA 86, 7687–7690.10.1073/pnas.86.20.7687Search in Google Scholar PubMed PubMed Central
Scrima, A., Thomas, C., Deaconescu, D., and Wittinghofer, A. (2008). The Rap–RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues. EMBO J. 27, 1145–1153.10.1038/emboj.2008.30Search in Google Scholar PubMed PubMed Central
Siebert, F. and Hildebrandt, P. (2008). Vibrational Spectroscopy in Life Science (Weinheim: Wiley-VCH).10.1002/9783527621347Search in Google Scholar
Siebert, F., Maentele, W., and Gerwert, K. (1983). Fourier-transform infrared spectroscopy applied to rhodopsin. The problem of the protonation state of the retinylidene Schiff base reinvestigated. Eur. J. Biochem. 136, 119–127.10.1111/j.1432-1033.1983.tb07714.xSearch in Google Scholar PubMed
Sit, S.-T. and Manser, E. (2011). Rho GTPases and their role in organizing the actin cytoskeleton. J. Cell Sci. 124, 679–683.10.1242/jcs.064964Search in Google Scholar PubMed
Sot, B., Kötting, C., Deaconescu, D., Suveyzdis, Y., Gerwert, K., and Wittinghofer, A. (2010). Unravelling the mechanism of dual-specificity GAPs. EMBO J. 29, 1205–1214.10.1038/emboj.2010.20Search in Google Scholar PubMed PubMed Central
Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 10, 513–525.10.1038/nrm2728Search in Google Scholar PubMed
Stieglitz, B., Bee, C., Schwarz, D., Yildiz, O., Moshnikova, A., Khokhlatchev, A., and Herrmann, C. (2008). Novel type of Ras effector interaction established between tumour suppressor NORE1A and Ras switch II. EMBO J. 27, 1995–2005.10.1038/emboj.2008.125Search in Google Scholar PubMed PubMed Central
Vetter, I.R. and Wittinghofer, A. (2001). Signal transduction – the guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304.10.1126/science.1062023Search in Google Scholar PubMed
Wang, Y., Pascoe, H.G., Brautigam, C.A., He, H., and Zhang, X. (2013). Structural basis for activation and non-canonical catalysis of the Rap GTPase activating protein domain of plexin. eLife 2, e01279.10.7554/eLife.01279.020Search in Google Scholar
Warscheid, B., Brucker, S., Kallenbach, A., Meyer, H.E., Gerwert, K., and Kötting, C. (2008). Systematic approach to group-specific isotopic labeling of proteins for vibrational spectroscopy. Vib. Spectrosc. 48, 28–36.10.1016/j.vibspec.2007.11.003Search in Google Scholar
Wittinghofer, A. and Vetter, I.R. (2011). Structure-function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem. 80, 943–971.10.1146/annurev-biochem-062708-134043Search in Google Scholar PubMed
Wu, Y.-W., Oesterlin, L.K., Tan, K.-T., Waldmann, H., Alexandrov, K, and Goody, R.S. (2010). Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic protein probes. Nat. Chem. Biol. 6, 534–540.10.1038/nchembio.386Search in Google Scholar PubMed
Xia, F., Rudack, T., Cui, Q., Kötting, C., and Gerwert, K. (2012). Detailed structure of the H2PO4--guanosine diphosphate intermediate in Ras-GAP decoded from FTIR experiments by biomolecular simulations. J. Am. Chem. Soc. 134, 20041–20044.10.1021/ja310496eSearch in Google Scholar PubMed
©2015 by De Gruyter
