The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.

CEITEC Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic

Center for Integrated Protein Science Munich at Chair of Biomolecular NMR Department Chemie Technische Universität München Lichtenbergstr 4 85747 Garching Germany

Institute of Structural Biology Helmholtz Zentrum München Ingolstädter Landstr 1 85764 Neuherberg Germany

See more in PubMed

Breeze AL. Isotope-filtered NMR methods for the study of biomolecular structure and interactions. Prog Nucl Magn Reson Spectrosc. 2000;36:323–372. doi: 10.1016/S0079-6565(00)00020-0. DOI

Caplan AJ, Tsai J, Casey PJ, Douglas MG. Farnesylation of YDJ1p is required for function at elevated growth temperatures in Saccharomyces cerevisiae. J Biol Chem. 1992;267:18890–18895. PubMed

Crespi HL, Katz JJ. High resolution proton magnetic resonance studies of fully deuterated and isotope hybrid proteins. Nature. 1969;224:560–562. doi: 10.1038/224560a0. PubMed DOI

Crespi HL, Rosenberg RM, Katz JJ. Proton magnetic resonance of proteins fully deuterated except for 1H-leucine side chains. Science. 1968;161:795–796. doi: 10.1126/science.161.3843.795. PubMed DOI

Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6:277–293. doi: 10.1007/BF00197809. PubMed DOI

Emmanouilidis L, Schutz U, Tripsianes K, Madl T, Radke J, Rucktaschel R, Wilmanns M, Schliebs W, Erdmann R, Sattler M. Allosteric modulation of peroxisomal membrane protein recognition by farnesylation of the peroxisomal import receptor PEX19. Nat Commun. 2017;8:14635. doi: 10.1038/ncomms14635. PubMed DOI PMC

Gans P, Hamelin O, Sounier R, Ayala I, Dura MA, Amero CD, Noirclerc-Savoye M, Franzetti B, Plevin MJ, Boisbouvier J. Stereospecific isotopic labeling of methyl groups for NMR spectroscopic studies of high-molecular-weight proteins. Angew Chem Int Ed Engl. 2010;49:1958–1962. doi: 10.1002/anie.200905660. PubMed DOI

Goddard TD, Kneller DG (1996) Sparky 3

Guntert P. Automated structure determination from NMR spectra. Eur Biophys J. 2009;38:129–143. doi: 10.1007/s00249-008-0367-z. PubMed DOI

Hyberts SG, Arthanari H, Wagner G. Applications of non-uniform sampling and processing. Top Curr Chem. 2012;316:125–148. doi: 10.1007/128_2011_187. PubMed DOI PMC

Kerfah R, Plevin MJ, Sounier R, Gans P, Boisbouvier J. Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins. Curr Opin Struct Biol. 2015;32:113–122. doi: 10.1016/j.sbi.2015.03.009. PubMed DOI

Lacabanne D, Meier BH, Bockmann A (2017) Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy. J Biomol NMR PubMed

LeMaster DM. Deuteration in protein proton magnetic resonance. Methods Enzymol. 1989;177:23–43. doi: 10.1016/0076-6879(89)77004-X. PubMed DOI

Manne V, Roberts D, Tobin A, O’Rourke E, De Virgilio M, Meyers C, Ahmed N, Kurz B, Resh M, Kung HF, et al. Identification and preliminary characterization of protein-cysteine farnesyltransferase. Proc Natl Acad Sci USA. 1990;87:7541–7545. doi: 10.1073/pnas.87.19.7541. PubMed DOI PMC

McTaggart SJ. Isoprenylated proteins. Cell Mol Life Sci. 2006;63:255–267. doi: 10.1007/s00018-005-5298-6. PubMed DOI PMC

Metzler WJ, Wittekind M, Goldfarb V, Mueller L, Farmer BT. Incorporation of 1H/13C/15N-{Ile, Leu, Val} into a perdeuterated, 15N-labeled protein: potential in structure determination of large proteins by NMR. J Am Chem Soc. 1996;118:6800–6801. doi: 10.1021/ja9604875. DOI

Miura GI, Treisman JE. Lipid modification of secreted signaling proteins. Cell Cycle. 2006;5:1184–1188. doi: 10.4161/cc.5.11.2804. PubMed DOI PMC

Novelli G, D’Apice MR. Protein farnesylation and disease. J Inherit Metab Dis. 2012;35:917–926. doi: 10.1007/s10545-011-9445-y. PubMed DOI

Rucktäschel R, Thoms S, Sidorovitch V, Halbach A, Pechlivanis M, Volkmer R, Alexandrov K, Kuhlmann J, Rottensteiner H, Erdmann R. Farnesylation of pex19p is required for its structural integrity and function in peroxisome biogenesis. J Biol Chem. 2009;284:20885–20896. doi: 10.1074/jbc.M109.016584. PubMed DOI PMC

Sattler M, Schleuchter J, Griesinger C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog NMR Spectrosc. 1999;34:93–158. doi: 10.1016/S0079-6565(98)00025-9. DOI

Schueller N, Holton SJ, Fodor K, Milewski M, Konarev P, Stanley WA, Wolf J, Erdmann R, Schliebs W, Song YH, Wilmanns M. The peroxisomal receptor Pex19p forms a helical mPTS recognition domain. EMBO J. 2010;29:2491–2500. doi: 10.1038/emboj.2010.115. PubMed DOI PMC

Sorek N, Bloch D, Yalovsky S. Protein lipid modifications in signaling and subcellular targeting. Curr Opin Plant Biol. 2009;12:714–720. doi: 10.1016/j.pbi.2009.09.003. PubMed DOI

Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev. 2001;81:153–208. doi: 10.1152/physrev.2001.81.1.153. PubMed DOI

Tugarinov V, Hwang PM, Kay LE. Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins. Annu Rev Biochem. 2004;73:107–146. doi: 10.1146/annurev.biochem.73.011303.074004. PubMed DOI

Tugarinov V, Kanelis V, Kay LE. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc. 2006;1:749–754. doi: 10.1038/nprot.2006.101. PubMed DOI

Umetsu M, Wang ZY, Kobayashi M, Nozawa T. Interaction of photosynthetic pigments with various organic solvents. Magnetic circular dichroism approach and application to chlorosomes. Biochim Biophys Acta. 1999;1410:19–31. doi: 10.1016/S0005-2728(98)00170-4. PubMed DOI

Vuister GW, Kim S-J, Wu C, Bax A. 2D and 3D NMR study of phenylalanine residues in proteins by reverse isotopic labeling. J Am Chem Soc. 1994;116:9206–9210. doi: 10.1021/ja00099a041. DOI