A three-disulphide derivative of hen lysozyme. Structure, dynamics and stability.
A three-disulphide derivative of hen egg-white lysozyme was made by selective reduction and carboxymethylation of one of the four original disulphide bridges. N-Terminal sequencing and two-dimensional 1H-n.m.r. spectroscopy revealed that the disulphide bridge linking cysteine residues 6 and 127 had been modified and that the three remaining disulphide bonds were native-like in nature. Analysis of COSY and NOESY spectra indicated that the three-disulphide lysozyme (CM6.127-lysozyme retains the same secondary and tertiary structure as its four-disulphide counterpart; its stability to pH and temperature is, however, dramatically decreased. N.m.r. spectroscopy was used to characterize the thermal folding and unfolding transition of CM6.127-lysozyme. Not only is the transition still a highly co-operative event, but the enthalpy change associated with folding and unfolding resembles that of intact lysozyme when their differences in thermal stability are taken into consideration. The significance of these results in terms of the folding process of lysozyme is discussed. By contrast with authentic lysozyme, CM6.127-lysozyme was found to exist in an unfolded state at pH 2 at room temperature. N.m.r. spectroscopy and c.d. were used to characterize this state. Unlike their homologous relative, alpha-lactalbumin, which exists in a partially folded molten globule state under these conditions, only residual non-native-like structure persists in the acid-unfolded state of CM6.127-lysozyme. These results indicate that the difference in folding behaviour of lysozyme and alpha-lactalbumin cannot be accounted for simply by their differences in thermal stability.
Radford
S E
SE
Inorganic Chemistry Laboratory, University of Oxford, U.K.
Woolfson
D N
DN
Martin
S R
SR
Lowe
G
G
Dobson
C M
CM
D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D009113:Muramidase / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D013438:Sulfhydryl Compounds; D009113:Muramidase