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%) and structurally very similar, A-LBP and M-FABP display distinct ligand binding characteristics. Since ligand binding may be influenced by intrinsic protein dynamical properties, we have characterized the backbone 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-free spectral density functions and reduced spectral density mapping. The dynamics of methyl-containing side chains were characterized by measurements 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 residues and H-2 relaxation data were obtained for 42 methyl groups. For M-FABP. N-15 relaxation data were obtained for 111 residues and H-2 relaxation data were obtained for 53 methyl groups. The intrinsic flexibilities of these two proteins are compared, with particular emphasis placed on binding pocket residues. There are a number of distinct dynamical differences among corresponding residues between the two proteins. In particular, many residues display greater backbone picosecond to nanosecond and/or microsecond to millisecond time scale mobility in A-LBP relative to M-FABP, including F57, K58, and most residues in alpha-helix 2 (residues 28-35). Variations in the dynamics of this region may play a role in ligand selectivity. The side chains lining the fatty acid 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 information provides a necessary benchmark for determining dynamical changes induced by ligand binding and may ultimately lead to an enhanced understanding of ligand affinity and selectivity among fatty acid-binding proteins.