Cooperative folding of the isolated alpha-helical domain of hen egg-white lysozyme.
Proteins in the alpha-lactalbumin and c-type lysozyme family have been studied extensively as model systems in protein folding. Early formation of the alpha-helical domain is observed in both alpha-lactalbumin and c-type lysozyme; however, the details of the kinetic folding pathways are significantly different. The major folding intermediate of hen egg-white lysozyme has a cooperatively formed tertiary structure, whereas the intermediate of alpha-lactalbumin exhibits the characteristics of a molten globule. In this study, we have designed and constructed an isolated alpha-helical domain of hen egg-white lysozyme, called Lyso-alpha, as a model of the lysozyme folding intermediate that is stable at equilibrium. Disulfide-exchange studies show that under native conditions, the cysteine residues in Lyso-alpha prefer to form the same set of disulfide bonds as in the alpha-helical domain of full-length lysozyme. Under denaturing conditions, formation of the nearest-neighbor disulfide bonds is strongly preferred. In contrast to the isolated alpha-helical domain of alpha-lactalbumin, Lyso-alpha with two native disulfide bonds exhibits a well-defined tertiary structure, as indicated by cooperative thermal unfolding and a well-dispersed NMR spectrum. Thus, the determinants for formation of the cooperative side-chain interactions are located mainly in the alpha-helical domain. Our studies suggest that the difference in kinetic folding pathways between alpha-lactalbumin and lysozyme can be explained by the difference in packing density between secondary structural elements and support the hypothesis that the structured regions in a protein folding intermediate may correspond to regions that can fold independently.
Bai
P
P
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA.
Peng
Z
Z
D000818:Animals; D002645:Chickens / Q000235:genetics; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004529:Egg White; D004795:Enzyme Stability; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D004220:Disulfides; D010446:Peptide Fragments; D007768:Lactalbumin; D009113:Muramidase; D003545:Cysteine