title: NMR-based approach to measure the free energy of transmembrane helix-helix interactions.
abstract: Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Str. Miklukho-Maklaya, 16/10, Moscow 117997, Russian Federation. Electronic address: mineev@nmr.ru.
authors.forename: Konstantin S
authors.initials: KS
authors.lastname: Mineev
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Lesovoy
authors.forename: Dinara R
authors.initials: DR
authors.lastname: Usmanova
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Goncharuk
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Shulepko
authors.forename: Ekaterina N
authors.initials: EN
authors.lastname: Lyukmanova
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
authors.forename: Eduard V
authors.initials: EV
authors.lastname: Bocharov
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Arseniev
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008823:Micelles; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013816:Thermodynamics; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D003902:Detergents; D008823:Micelles; D009842:Oligopeptides; D011994:Recombinant Proteins; D012996:Solutions; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: CECF; CMC; DPC; Detergent micelle; Dimerization; FGFR3; FGFR3tm; FRET; Forster resonance energy transfer; Free energy; HSQC; LPR; NMR; NMR spectroscopy; SDS-PAGE; TCEP; TFE; TM; TM domain of FGFR3; TM domain of VEGFR2 bearing V(769)E substitution; TMD; TROSY; TSP; Transmembrane helix; VEGFR2; VEGFR2tm; continuous exchange cell-free; critical micelle concentration; dodecylphosphocholine; fibroblast growth factor receptor 3; heteronuclear single quantum coherence; lipid to protein ratio; nuclear magnetic resonance; sodium dodecyl sulfate polyacrylamide gel electrophoresis; transmembrane; transmembrane domain; transverse relaxation optimized spectroscopy; trifluoroethanol; trimethylsilyl propanoic acid; tris(2-carboxyethyl)phosphine; vascular endothelium growth factor receptor 2