The alphabet of intrinsic disorder: II. Various roles of glutamic acid in ordered and intrinsically disordered proteins

Uversky, Vladimir N

α-lactabumin was shown to possess significantly different thermal and structural stability in its calcium-bound and calciumfree apo-forms, 122 with the apo-protein possessing molten globule-like properties at slightly elevated temperatures. 123, 124 This strong dependence of the α-lactabumin structural properties on metal-binding is determined by the simple fact that in the apo-form, many acidic side chains have unfavorable chargecharge interactions, with 11 residues (Glu1, Glu7, Glu11, Asp63, Asp64, Asp78, Asp82, Asp83, Asp84, Asp87 and Asp88) possessing significantly unfavorable charge-charge repultion. 125 Although calcium binding has the most pronounced effect on residues directly involved in cation coordination (Asp82, Asp87 and Asp88) and strongly affects the other two residues in the Ca 2+ -binding loop, Asp83 and Asp84, Ca 2+ binding has relatively minor effects on residues more distant from the Ca 2+ -binding site (Glu1, Glu7, Glu11, Asp63 and Asp64), which mostly preserve unfavorable electrostatic interactions seen in the apo-form. 125 It was also shown that the mutation-induced neutralization of unfavorable charge-charge interactions in the N-terminus (residues 1-11 of which are characterized by a high proportion of negatively charged residues that cluster on the surface of the native protein) results in stabilization of both the apo-and Ca 2+bound protein. 125 Unexpectedly, the ΔGlu1 mutant, where the Glu1 residue was removed, leaving an N-terminal methionine in its place, possessed almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. 121 This unique tuning of the α-lactabumin structure and calcium binding suggested that the N-terminal region of this protein might have a direct effect on the calcium-binding loop (and perhaps other regions of the structure). 121