The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: Comparison with the X-ray structure

The structure and dynamics of rat ape-cellular retinol binding protein II ( apo-CRBP II) in solution has been determined by multidimensional NMR analys is of uniformly enriched recombinant rat C-13, N-15-apo-CRBP II and 15N-apo -CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/residue. The average root -mean-square deviation of the backbone atoms for the final 24 structures re lative to their mean structure is 1.06 Angstrom. Although the average solut ion structure is very similar to the crystal structure, it differs at the p utative entrance to the binding cavity, which is formed by the helix-turn-h elix motif, the beta C-beta D turn and the beta E-beta F turn. The mean coo rdinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chai n of F58, located on the beta C-beta D turn, is reoriented such that it int eracts with L37 and no longer blocks entry into the ligand-binding pocket. Residues 28-35, which form the second helix of the helix-turn-helix motif i n the crystal structure, do not exhibit a helical conformation in the solut ion structure. The solution structure of apo-CRBP II exhibits discrete regi ons of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the beta E-beta F turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide H-1 exchange rates and N-15 relaxation studies. These studies indicate that fluctuations in protei n conformation occur on the ys to ms timescale in these regions of the prot ein. Some of these exchange processes can be directly observed in the three -dimensional N-15-resolved NOESY spectrum. These results suggest that in so lution, apo-CRBP II undergoes conformational changes on the ys to ms time-s cale which result in increased access to the binding cavity. (C) 1999 Acade mic Press.