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

Abstract

The effects of amino acid substitutions in the N-terminus of bovine recombinant alpha-lactalbumin (including enzymatic removal of the N-terminal methionine and deletion of Glu-1) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC). Wild-type recombinant alpha-lactalbumin has a lower thermostability and calcium affinity compared to the native protein, while the properties of wild-type protein 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-terminal methionine in its place, shows 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. It was concluded that the N-terminus of the protein dramatically affects both stability and function as manifested in calcium affinity. Proteins 1999;37:65-72.

The Additional Methionine Residue at the N-Terminus of Bacterially Expressed Human Interleukin-2 Affects the Interaction between the N- and C-Termini

“…There has been compelling evidence that the additional methionine at the N-terminus of R-lactalbumin destabilizes the native-state folding (18)(19)(20). Similar destabilizing effects have been reported for recombinant hen egg-white lysozyme (21,22) and apomyoglobin (23).…”

Soybean peptide aggregates improved calcium binding capacity

“…Kim and Lim (2004) also found that Lys (7.12%) also took up a high percentage in calcium binding peptides from cheese whey protein. Bovine a-LA is known to have strong calciumbinding sites with Asp and Lys as binding residues (Anderson, Brooks, & Berliner, 1997;Veprintsev et al, 1999;Noyelle & van Dael, 2002). The results of this experiment are similar to those in the report of Tamura, Oku, and Hosoya (1982) who stated that Asp, Glu, Leu and Lys were abundant in the two materials, which showed calcium-binding ability in milk protein.…”

The alphabet of intrinsic disorder

“…120 Therefore, important glutamic acid residues responsible for calcium coordination can be found in various members of the major Ca 2+ -binding proteins, such as EF-hand domains, EGF-like domains, γ-carboxyl glutamic acid (GLA)-rich domains, cadherin domains, Ca 2+ -dependent (C)-type lectin-like domains and Ca 2+ -binding pockets of family C G-protein-coupled receptors. 120 A particularly intriguing role was described for the N-terminal glutamic acid residues in the canonical Ca 2+ -protein, α-lactabumin, 121 which is frequently used as a model protein in folding studies and in studies on the effect of calcium binding on protein structure, stability and folding. For example, In nudix hydrolases (which is a family of Mg 2+ -requiring enzymes that catalyze the hydrolysis of nucleoside diphosphates linked to other moieties) there is a specific motif, Nudix box (GX 5 EX 7 REUXEEXGU, where U is a bulky hydrophobic residue), that forms a loop-α helix-loop structural motif that functions as a common Mg 2+ -binding and catalytic site.…”