Molecular Dynamics Simulations of Laser-Induced and pH-Induced Unfolding in β-Lactoglobulin at Different Hydration Levels

Abstract

Computational molecular dynamics simulations were used to estimate the unfolding effects of a single electron positioned appropriately in a globular protein of interest, β-lactoglobulin (BLG). Two sets of unfolding simulations were conducted: movement of the EF-loop in BLG, which is involved in the Tanford transition and a photo-induced electron transfer event between a non-covalently bound, dye molecule, meso-tetrakis p-sulfanatophenyl porphyrin, (TSPP) and BLG.The first simulations solvated BLG at several hydration levels for two different crystal structures: 3BLG at pH 7 (closed loop) and 2BLG at pH 9 (open loop), which became the reference initial and final structures to determine the accuracy of our simulations. Movement of the EF-loop was induced by placing an excess charge on the carboxyl group of glutamic acid residue GLU 89. After 10 ns, favorable structures were submitted to a residue based, coarse-graining algorithm to enable simulating 0.1 ms of the loop motion. The goal was three-fold: to allow the crystallized form to relax in an aqueous environment, to determine the minimal number of explicit water molecules that are sufficient to retain the thermodynamics of the system, and to verify the charge placement algorithm.For the second phase, the location of the electron transfer event was determined by the most probable binding site of the ligand that correlated with resonance Raman spectra and docking calculations of the TSPP-BLG complex. Residues within 3 A were examined for alignment with the ligand and electron affinity. Candidate sites became the origin of an excess electron on the protein, and a separate computation was conducted with coarse-graining methods applied to favorable trajectories as described above. The unfolding determined by these simulations was compared to previously collected circular dichroism data of TSPP-BLG complexes.