title: A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule.
abstract: In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin. In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C. The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry. Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme. One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein. From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range. These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e. stabilization through Ca2+ binding and molten globule behavior.
authors.affiliation: Interdisciplinary Research Center, K. U. Leuven, Kortrijk, Belgium.
authors.forename: E
authors.initials: E
authors.lastname: Pardon
authors.forename: P
authors.initials: P
authors.lastname: Haezebrouck
authors.forename: A
authors.initials: A
authors.lastname: De Baetselier
authors.forename: S D
authors.initials: SD
authors.lastname: Hooke
authors.forename: K T
authors.initials: KT
authors.lastname: Fancourt
authors.forename: J
authors.initials: J
authors.lastname: Desmet
authors.forename: C M
authors.initials: CM
authors.lastname: Dobson
authors.forename: H
authors.initials: H
authors.lastname: Van Dael
authors.forename: M
authors.initials: M
authors.lastname: Joniau
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002417:Cattle; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002135:Calcium-Binding Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D007768:Lactalbumin; D009113:Muramidase; D002118:Calcium