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Title: Dynamics of liver fatty acid binding protein
Authors: LONG DONG
Keywords: NMR spectroscopy, spin relaxation, molecular dynamics simulation, liver fatty acid binding protein, protein-ligand interaction, buffer interference
Issue Date: 11-Jan-2010
Citation: LONG DONG (2010-01-11). Dynamics of liver fatty acid binding protein. ScholarBank@NUS Repository.
Abstract: Over a decade, scientists have been attempting to know more about the conformational dynamics of fatty acid binding proteins (FABPs), in order to answer the puzzling question ? how ligands could access the internalized binding site(s) of FABPs. Despite numerous efforts made in this field, the appreciation of this question is still relatively poor nowadays. In the current study, we continued the effort to explore the dynamical properties of liver fatty acid binding protein (LFABP) using NMR spectroscopy and MD simulation techniques, aiming at advancing our knowledge on this interesting topic. The microsecond to millisecond timescale dynamics of FABPs was historically hypothesized to represent a dynamical equilibrium between the ?open? and ?closed? states, regulating the ligand entry/exit processes. Despite the potential significance, the validity of this hypothesis has not yet been demonstrated. In the current study, the slow dynamics of LFABP was quantitatively characterized using relaxation dispersion NMR spectroscopy, which shows that LFABP is indeed highly flexible on the millisecond timescales. In order to further examine the hypothetical role of the millisecond dynamics of LFABP, the potential correlation between slow dynamics and ligand entry/exit processes was modeled and evaluated by analyzing the kinetic rates of LFABP-ANS interaction. The experimental result demonstrates that the intrinsic millisecond dynamics of LFABP, somewhat disappointedly, does not represent a critical conformational reorganization required for ligand entry due to the contradiction of timescales, but implies that it may represent a dynamical equilibrium between the apo-state and a state resembling the singly-bound conformation. Analysis of the kinetic rates of the ligand association shows that the ligand-entry related dynamics could occur on the microsecond or sub-microsecond timescales, which is much faster than previously assumed. Despite fast advancement of experimental techniques for exploring protein dynamics, direct visualization of ligand entry/exit processes which potentially involves multiple transient steps is still formidable nowadays. In silico simulation, thus, provides a good alternative way to investigate such dynamical details. However, the ligand exit/entry is a slow event which could hardly be accessed by standard MD simulations. In order to overcome this problem, random expulsion simulation, which accelerates ligand motions with a randomly oriented external force, was applied to investigate the ligand dissociation processes. Different ligand egress routes were identified for LFABP in this work, which furthered our understanding on the protein-ligand interplay. Future mechanistic studies on the ligand release and uptake would benefit from the experimental and computational studies shown in this thesis. As a fortuitous discovery during our experimental studies, the millisecond timescale dynamics of LFABP was found to be perturbed by the presence of buffer agents. Although not being our initial aim, we characterized the amplitude of such buffer perturbation to the slow motions of LFABP. This case study offers an example of how the biophysical properties of proteins could be influenced by buffer molecules, which would deserve the attention of scientists in the in vitro manipulation of protein molecules.
Appears in Collections:Ph.D Theses (Open)

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