title: A near-native state on the slow refolding pathway of hen lysozyme.
abstract: The refolding of four disulfide lysozyme (at pH 5.2, 20 degrees C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the alpha- and beta-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 degrees C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths beta-domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near-native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.
authors: School of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
authors: S K
authors: SK
authors: Kulkarni
authors: A E
authors: AE
authors: Ashcroft
authors: M
authors: M
authors: Carey
authors: D
authors: D
authors: Masselos
authors: C V
authors: CV
authors: Robinson
authors: S E
authors: SE
authors: Radford
mesh_terms: D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D009994:Osmolar Concentration; D017510:Protein Folding; D055672:Static Electricity
chemical_list: D009113:Muramidase