A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule.
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.
Pardon
E
E
Interdisciplinary Research Center, K. U. Leuven, Kortrijk, Belgium.
Haezebrouck
P
P
De Baetselier
A
A
Hooke
S D
SD
Fancourt
K T
KT
Desmet
J
J
Dobson
C M
CM
Van Dael
H
H
Joniau
M
M
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
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D007768:Lactalbumin; D009113:Muramidase; D002118:Calcium