Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid-binding protein.

Horváth, Gergő and Biczók, László and Majer, Zsuzsa and Kovács, Mihály and Micsonai, András and Kardos, József and Tőke, Orsolya (2017) Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid-binding protein. FEBS JOURNAL, 284 (21). pp. 3637-3661. ISSN 1742-464X

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Abstract

Human ileal bile acid-binding protein (I-BABP) has a key role in the intracellular transport and metabolic targeting of bile salts. Similar to other members of the family of intracellular lipid-binding proteins (iLBPs), disorder-order transitions and local unfolding processes are thought to mediate ligand entry and release in human I-BABP. To gain insight into the stability of various protein regions, the temperature response of human I-BABP was investigated using NMR, CD and fluorescence spectroscopy, as well as molecular dynamics (MD) simulations. A joint analysis of NMR thermal melting and relaxation dispersion data indicates a complex pattern of internal dynamics with a dominating single barrier and a likely presence of rapidly exchanging conformational substates on both sides of the barrier. Moreover, our residue-specific analysis uncovers a partially unfolded U* state in which part of the helical region with three proximate beta-strands contains a substantial amount of residual structure, whereas several segments of the C-terminal half exhibit a high susceptibility to temperature elevation. Cluster analysis of atomic temperature responses indicates a thermodynamic coupling between distant protein sites including the bottom of the beta-barrel, the E-F region and part of the helical cap. MD simulations up to 1 mus show correlated motions in the same protein regions and together with the NMR data suggest a role for the highly dynamic D-E turn and E-F region in the initiation of unfolding. The response of human I-BABP to temperature elevation is discussed in the context of the folding/unfolding behaviour of different members of the iLBP family.

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