Electrostatics in beta-lactoglobulin (BLG) has already been explored: the Tanford transition involving a carboxylic side-chain with an anomalous pKa value of 7.3 was reported in 1959. Here we report on electrophoretic, spectroscopic, and computational studies aimed at clarifying at atomic level the electrostatics of folded and unfolded BLG with a detailed characterization of the specific amino acids involved. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility in DGGE is not just the result of changes in gyration radius upon unfolding. ETC runs across the 3.5-9 pH range in the presence of urea concentrations between 0 and 8 M suggest that more than one residue in the protein may have anomalous pKa values in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114 due mainly to changes in global and local desolvation. For His161 the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C-terminus, the end-effect associated to the solvated form can also influence its pKa value. DGGE evidence also indicates a shift of the isoelectric point of BLG-B from 4.8 (no urea) to 5.2 (8 M urea). Interestingly, at 4M urea – that is, below the Cm required for macroscopic structural changes (5.5-6 M urea) - the value of pI is already increased to 5, suggesting that relevant structural transitions occur prior to the denaturation process. This may offer a further clue to explaining the apparently contradictory features of BLG, which shows remarkable stability to pH extremes and denaturants as a whole but also shows remarkable flexibility in the interactions between secondary structure elements upon physical and chemical denaturation.
Electrostatics of folded and unfolded bovine beta-lactoglobulin / I. Eberini, C. Sensi, A. Barbiroli, F. Bonomi, S. Iametti, M. Galliano, E. Gianazza. ((Intervento presentato al 36. convegno FEBS congress tenutosi a Torino nel 2011.
|Titolo:||Electrostatics of folded and unfolded bovine beta-lactoglobulin|
EBERINI, IVANO (Primo)
SENSI, CRISTINA (Secondo)
GIANAZZA, ELISABETTA (Ultimo)
|Data di pubblicazione:||25-giu-2011|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||Electrostatics of folded and unfolded bovine beta-lactoglobulin / I. Eberini, C. Sensi, A. Barbiroli, F. Bonomi, S. Iametti, M. Galliano, E. Gianazza. ((Intervento presentato al 36. convegno FEBS congress tenutosi a Torino nel 2011.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|