Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo-and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most

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... vant difference between the NMR structure ensembles of apo-and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligandbinding site. These comprise ␣-helix II, the subsequent linker to ␤-strand B, the hairpin turn between ␤-strands C and D, and the ␤E-␤F turn. The internal backbone dynamics, obtained from 15 N relaxation data (T 1 , T 2 , and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the ␤C-␤D and ␤E-␤F turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment. 1 The abbreviations used are: i-LBP, intracellular lipid-binding protein; CRBP, cellular retinol-binding protein type I; CRBP-II and CRBP-III, cellular retinol-binding protein type II and type III, respectively; I-FABP, intestinal fatty acid-binding protein; CRABP-I and CRABP-II, cellular retinoic acid-binding protein type I and type II, respectively; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser enhancement and exchange spectroscopy; HSQC, heteronuclear singlequantum coherence; HTQC, heteronuclear triple-quantum coherence; NOE, nuclear Overhauser effect; r.m.s.d., root mean square deviation.