title: Mass spectrometry and hydrogen/deuterium exchange measurements of alcohol-induced structural changes in cellular retinol-binding protein type I.
abstract: To bind and release its ligand, cellular retinol-binding protein type I (CRBP) needs to undergo conformational and dynamic changes to connect the inner, solvent-shielded cavity, where retinol is found to bind, and the outside medium. Retinol dissociation in vitro is favoured by water/alcohol mixtures whose moderately low dielectric constants mimic a property characteristic of the membrane microenvironment where this process occurs in vivo. Apo- and holo-CRBP, in either water/methanol or water/trifluoroethanol (TFE) mixtures, were analyzed at equilibrium by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) to identify the alcohol-induced species. The questions were asked whether the presence of alcohols affects protein dynamics, as reflected by hydrogen/deuterium (H/D) exchange monitored by continuous-labelling experiments, and to which extent retinol dissociation influences the process. With increasing methanol, at pH near neutrality, apo-CRBP exhibits a progressively more compact conformation, resulting in reduced H/D exchange with respect to the native protein in water. Retinol dissociation from the holo-protein did not promote hydrogen replacement. Similarly, in the presence of the low TFE concentration sufficient to cause retinol dissociation, the hydrogen exchange of the resulting apo-protein was not exalted. However, in contrast with the alkanol, higher TFE concentrations induced a transition of apo-CRBP to a new alpha-helix conformation capable of exchanging all available hydrogen atoms.
authors.affiliation: Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Viale GP Usberti 23/A, I-43100 Parma, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Torta
authors.forename: Lisa
authors.initials: L
authors.lastname: Elviri
authors.forename: Maria
authors.initials: M
authors.lastname: Careri
authors.forename: Alessandro
authors.initials: A
authors.lastname: Mangia
authors.forename: Davide
authors.initials: D
authors.lastname: Cavazzini
authors.forename: Gian Luigi
authors.initials: GL
authors.lastname: Rossi
mesh_terms: D041961:Deuterium Exchange Measurement / Q000379:methods*; D000431:Ethanol / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D054840:Retinol-Binding Proteins, Cellular / Q000737:chemistry* / Q000648:ultrastructure*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D054840:Retinol-Binding Proteins, Cellular; D000431:Ethanol