title: Fine tuning the N-terminus of a calcium binding protein: alpha-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.
authors.affiliation: Institute for Biological Instrumentation, Russian Academy of Science, Pushchino, Russia.
authors.forename: D B
authors.initials: DB
authors.lastname: Veprintsev
authors.forename: M
authors.initials: M
authors.lastname: Narayan
authors.forename: S E
authors.initials: SE
authors.lastname: Permyakov
authors.forename: V N
authors.initials: VN
authors.lastname: Uversky
authors.forename: C L
authors.initials: CL
authors.lastname: Brooks
authors.forename: A M
authors.initials: AM
authors.lastname: Cherskaya
authors.forename: E A
authors.initials: EA
authors.lastname: Permyakov
authors.forename: L J
authors.initials: LJ
authors.lastname: Berliner
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D017136:Ion Transport; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D007768:Lactalbumin; D002118:Calcium