Mechanism of ligand-induced folding of a natively unfolded helixless variant of rabbit I-BABP.
Substitution of the helix-turn-helix capping motif (residues 9-35) of rabbit I-BABP with a flexible Gly-Gly-Ser-Gly linker results in the loss of stabilizing hydrophobic contacts and renders the beta-clamshell structure of this steroidal bile acid transport protein unfolded. However, in the presence of a bile acid ligand, we observe strong coupling between binding and folding, resulting in an enthalpy-driven high-affinity interaction (K(A) approximately 4 x 10(5) M(-1)) that "rescues" the native state. We investigate the mechanism of induced folding using fluorescence stopped-flow kinetic measurements to distinguish between conformational selection and induced-fit models. We observe both ligand-dependent and -independent kinetic phases which, together with their relative amplitudes, we attribute to an induced-fit "fly casting" type of model in which transient encounter complexes between the ligand and the extended polypeptide chain may act as nucleation sites for folding. An initial fast ligand-dependent kinetic process appears to be consistent with formation of a hydrophobically collapsed intermediate state which slowly rearranges to a nativelike beta-clamshell structure. We show that the intermediate forms at a rate 1000 times slower than the rate of ligand association with wild-type I-BABP, reflecting the large configurational entropic barrier to the coupled binding and folding steps of Deltaalpha-I-BABP. We have provided mechanistic insights into how natively disordered states, now commonly identified in biology, may fold on binding a target substrate or ligand.
Rea
Anita M
AM
School of Chemistry, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK.
Thurston
Victoria
V
Searle
Mark S
MS
D000818:Animals; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005768:Gastrointestinal Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D019077:Helix-Turn-Helix Motifs / Q000235:genetics*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary / Q000235:genetics*; D011817:Rabbits; D040681:Structural Homology, Protein
D050556:Fatty Acid-Binding Proteins; D005768:Gastrointestinal Hormones; D008024:Ligands; D010455:Peptides; C057275:fatty acid-binding protein 6