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.
|Titolo:||Reorganization in apo- and holo-beta-lactoglobulin upon protonation of Glu89: molecular dynamics and pKa calculations|
|Autori interni:||GIANAZZA, ELISABETTA|
EBERINI, IVANO (Primo)
BERINGHELLI, TIZIANA (Ultimo)
|Parole Chiave:||Electrostatics; Hydrogen bond; Linear response approximation; Palmitate; Salt bridge; Secondary structure; Tanford transition|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore CHIM/03 - Chimica Generale e Inorganica
|Data di pubblicazione:||2004|
|Digital Object Identifier (DOI):||10.1002/prot.10643|
|Appare nelle tipologie:||01 - Articolo su periodico|
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