A 200 nanoseconds all-atom simulation of the pH-dependent EF loop transition in bovine β-lactoglobulin. The role of the orientation of the E89 side chain.

In silico molecular dynamics (MD) using crystallographic and NMR data was used to simulate the effects of the protonation state of E89 on the pH-dependent conformational rearrangement of the EF loop, also known as the Tanford transition, in a series of apo-β-lactoglobulin (BLG) structures. Compared to existing studies these simulations were carried out over a much longer time scale (200 ns where the stability of the transition can be evaluated) and used an explicit water model. We considered eight different entries from the Brookhaven Protein Data Bank (PDB) separated into two groups. We observed that fixing the protonation state of E89 prompts the transition of the EF loop only when its side chain is oriented under the loop and into the entrance of the interior cavity. The motion of the EF loop occurs mostly as a step-function and its timing varies greatly from ∼ 20 ns to ∼170 ns from the beginning of the simulation. Once the transition is completed, the protein appears to reach a stable conformation as in a true two-state transition. We also observed novel findings. When the transition occurs, the hydrogen bond between E89 and S116 is replaced with a salt bridge with Lys residues in the βC-CD loop-βD motif. This electrostatic interaction causes the distortion of this motif as well as the protrusion of the GH loop into the aperture of the cavity with the result of limiting the increase of its contour area despite the rotation of the EF loop.Communicated by Ramaswamy H. Sarma.