The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: comparison with the X-ray structure 1 1Edited by P. E. Wright

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13 C, 15 N-apo-CRBP II and 15 N-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 relative to their mean structure is 1.06 A ˚ . Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the bC-bD turn and the bE-bF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the bC-bD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligandbinding pocket. Residues 28-35, which form the second helix of the helixturn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the bE-bF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1 H exchange rates and 15 N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the ms to ms timescale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15 N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the ms to ms time-scale which result in increased access to the binding cavity. # 1999 Academic Press