BACKBONE AND SIDE-CHAIN DYNAMICS OF UNCOMPLEXED HUMAN ADIPOCYTE AND MUSCLE FATTY-ACID-BINDING PROTEINS

Adipocyte lipid-binding protein (A-LBP) and muscle fatty acid-binding protein (M-FABP) are members of a family of small (similar to 15 kDa) cytosolic proteins that are involved in the metabolism of fatty acids and other lipid-soluble molecules. Although highly homologous (65%) an d structurally very similar, A-LBP and M-FABP display distinct ligand binding characteristics. Since ligand binding may be influenced by int rinsic protein dynamical properties, we have characterized the backbon e and side chain dynamics of uncomplexed (apo) human A-LBP and M-FABP, Backbone dynamics were characterized by measurements of N-15 T-1 and T-2 values and {H-1}-N-15 NOEs. These data were analyzed using model-f ree spectral density functions and reduced spectral density mapping. T he dynamics of methyl-containing side chains were characterized by mea surements of H-2 T-1 and T-1 rho relaxation times of (CH2H)-C-13-H-1-H -2 groups. The H-2 relaxation data were analyzed using the model-free approach. For A-LBP, N-15 relaxation data were obtained for 111 residu es and H-2 relaxation data were obtained for 42 methyl groups. For M-F ABP. N-15 relaxation data were obtained for 111 residues and H-2 relax ation data were obtained for 53 methyl groups. The intrinsic flexibili ties of these two proteins are compared, with particular emphasis plac ed on binding pocket residues. There are a number of distinct dynamica l differences among corresponding residues between the two proteins. I n particular, many residues display greater backbone picosecond to nan osecond and/or microsecond to millisecond time scale mobility in A-LBP relative to M-FABP, including F57, K58, and most residues in alpha-he lix 2 (residues 28-35). Variations in the dynamics of this region may play a role in ligand selectivity. The side chains lining the fatty ac id binding pocket display a wide range of motional restriction in both proteins. Side chains showing distinct dynamical differences between the two proteins include those of residues 20, 29, and 51. This inform ation provides a necessary benchmark for determining dynamical changes induced by ligand binding and may ultimately lead to an enhanced unde rstanding of ligand affinity and selectivity among fatty acid-binding proteins.