Reorganization in apo‐ and holo‐β‐lactoglobulin upon protonation of Glu89: Molecular dynamics and pK_a calculations

Abstract

Molecular dynamics (MD) simulations starting from crystallographic data allowed us to directly account for the effects of the protonation state of Glu89 on the conformational stability of apo- and holo-beta-lactoglobulin (BLG). In apo-BLG simulations starting from the protonated crystal structure, we observe a long-lived H-bond interaction between the protonated Glu89 and Ser116. This interaction, sequestering the proton from the aqueous medium, explains a pK(half) value evaluated at pH 7.3 by continuum electrostatics/Monte Carlo computation on MD data, using linear response approximation. A very large root-mean-square deviation (RMSD; 5.11 A) is observed for the EF loop between protonated and unprotonated apo-BLG. This results from a quite different orientation of the EF loop that acts either as a closed or as an open lid above the protein calyx. Proton exchange by Glu89 in apo- but not in holo-BLG is associated with a reorganization energy of 4.7 kcal/mol. A 3-ns MD simulation starting from the crystal structure of protonated apo-BLG, but considering the Glu89 as unprotonated, shows the progressive opening of the lid giving rise to the Tanford transition. In both holo-BLG forms, the lid is most probably held in place by hydrophobic interactions of amino acid side-chains of the EF loop with the palmitate hydrocarbon tail.

NMR Dynamic Studies Suggest that Allosteric Activation Regulates Ligand Binding in Chicken Liver Bile Acid-binding Protein

“…Nevertheless, it is worthwhile examining molecular dynamics trajectories to understand possible conformational trends. In this line it is worth mentioning that two molecular dynamics simulations of bovine ␤-lactoglobulin (which belongs to the same superfamily of cl-BABP) were able to sample a pH-driven transition in even shorter simulation times (34,38).…”

Free‐Energy Simulations and Experiments Reveal Long‐Range Electrostatic Interactions and Substrate‐Assisted Specificity in an Aminoacyl‐tRNA Synthetase

“…[40][41][42] The free energy to add a proton to the His448 side chain is obtained from the expression: [42] …”

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

“…This result suggests that the water molecules play a relevant role in the cL-BABP binding processes, differently from other proteins of the calycins superfamily, such as BLG, which has no water molecules inside its binding site, either in the apo-or in the holo-form (Qin et al, 1998;Adams et al, 2006). The absence of internal water molecules in BLG was reported not only in all crystallographic forms of BLG, but also in MD simulations (Eberini et al, 2004). The behavior of the water molecules in BABP cannot be generalized to other LBP family members; in fact there are some FABP members in which the waters seem to be relevant for protein structure stabilization and for ligand binding, but this is not true for other members of the same family.…”