Fine tuning the N-terminus of a calcium binding protein: alpha-lactalbumin

The effects of amino acid substitutions in the N-terminus of bovine recombi nant ol-lactalbumin (including enzymatic removal of the N-terminal methioni ne and deletion of Glu-l) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC), Wild-type recombinant alpha-lactalbumin has a lower thermostability and calcium affi nity compared to the native protein, while the properties of wild-type prot ein with the N-terminal methionine enzymatically removed are similar to the native protein. Taken together, the fluorescence, CD, and DSC results show that recombinant wild type alpha-lactalbumin in the absence of calcium ion is in a type of molten globule state. The delta-E1 mutant, where the Glu(1 ) residue of the native sequence is genetically removed, leaving an N-termi nal methionine in its place, shows almost one order of magnitude higher aff inity for calcium and higher thermostability (both in the absence and prese nce of calcium) than the native protein isolated from milk. It was conclude d that the N-terminus of the protein dramatically affects both stability an d function as manifested in calcium affinity, (C) 1999 Wiley-Liss, Inc.