A near-native state on the slow refolding pathway of hen lysozyme

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 stat e. This species contains stable structure in the alpha- and beta-domains bu t lacks a functional active site. Although previous experiments have demons trated that the near-native state is populated on the fast refolding pathwa y, its relevance to slow refolding molecules could not be directly determin ed from previous experiments. In this paper, we describe experiments that i nvestigate the effect of added salts on the refolding pathway of lysozyme a t pH 5.2, 20 degrees C. We show, using stopped flow tryptophan fluorescence , inhibitor binding, and circular dichroism (CD), that the rate of formatio n of native lysozyme on the slow refolding track is significantly reduced i n 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 stabili zation and native state formation on the slow refolding pathway become kine tically decoupled such that the near-native state becomes significantly pop ulated. Thus, by changing the energy landscape with the addition of salts n ew insights into the relevance of intermediate states in lysozyme refolding are revealed.