Mass spectrometry techniques for detection of ligand-dependent changes in the conformational flexibility of cellular retinol-binding protein type I localized by hydrogen/deuterium exchange.
Hydrogen/deuterium exchange, measured by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), was used as a means to probe and map differences in conformational flexibility between the ligand-free and ligand-bound forms of cellular retinol-binding protein type I. Labelled fragments were obtained by digestion of the protein with pepsin. The differences in space-resolved time courses of deuterium incorporation identified regions that exhibit a remarkably higher degree of flexibility in the apo-protein than in the holo-protein. These segments encompass residues that are thought, on the basis of structural homology of the retinol carrier with other members of the intracellular lipid-binding proteins family, to belong to the dynamic portal through which all-trans retinol can access its high-affinity, solvent-shielded, binding site.
Careri
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M
Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Università degli Studi di Parma, Parco Area delle Scienze 17/A, I-43100 Parma, Italy.
Elviri
L
L
Mangia
A
A
Zagnoni
I
I
Torta
F
F
Cavazzini
D
D
Rossi
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GL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054840:Retinol-Binding Proteins, Cellular; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D008024:Ligands; D010446:Peptide Fragments; C518458:Rbp1 protein, rat; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D006859:Hydrogen; D003903:Deuterium