Partially folded states of equine lysozyme. Structural characterization and significance for protein folding.
Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly dependent on the state of calcium binding. Differential scanning calorimetry shows the two transitions to be particularly well resolved in the calcium-free protein, where the first transition occurs with a midpoint at 44 degrees C at pH 4.5 or in 0.8 M GdnHCl at pH 7.5, 25 degrees C, and the second occurs near 70 degrees C at pH 4.5 or in 3.7 M GdnHCl at pH 7.5, 25 degrees C. In the presence of calcium, the first transition takes place with a midpoint of 55 degrees C or in excess of 2.5 M GdnHCl, but the parameters for the second transition remain unchanged. Fluorescence emission and UV difference absorption spectroscopy suggest that the first transition generates an intermediate state in which sequestration of some aromatic side chains from solvent has occurred whereas the second represents denaturation to a highly unfolded state. CD and 1H NMR results indicate that the intermediate state possesses extensive secondary and tertiary structure, although the latter is substantially disordered.(ABSTRACT TRUNCATED AT 250 WORDS)
Van Dael
H
H
Interdisciplinary Research Centre, K.U. Leuven Campus Kortrijk, Belgium.
Haezebrouck
P
P
Morozova
L
L
Arico-Muendel
C
C
Dobson
C M
CM
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D005260:Female; D019791:Guanidine; D006146:Guanidines; D006736:Horses; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008892:Milk / Q000201:enzymology; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D009113:Muramidase; D019791:Guanidine