Direct detection of heat and cold denaturation for partial unfolding of a protein.
Direct detection of heat and cold denaturation for partial unfolding of a protein.
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DOI：
10.1021/ja016144a
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发表时间：
2001-08
影响因子：
15
通讯作者：
Carma J. Nelson;Michael J. LaConte;B. Bowler
中科院分区：
文献类型：
--
作者：
Carma J. Nelson;Michael J. LaConte;B. Bowler
Partially unfolded states of proteins are an important focus of research in the investigation of protein folding due to their presumed role as intermediates in the folding of proteins from the denatured state to the native state. 1 Interest in such states has intensified of late because of the apparent role of partially unfolded proteins in aggregation in protein misfolding diseases. 2 It is difficult to significantly populate partially unfolded states of proteins near physiological pH. Normally, to stabilize such states, extremes of pH must be used, as for the molten globule state which is stabilized at low pH and high salt3 or the alkaline conformer of cytochrome c. 4 Partially unfolded states of proteins have also been observed indirectly using NMR-detected hydrogen/deuterium (H/D) exchange experiments. 5 Here, we describe direct detection of heat and cold denaturation for partial unfolding of yeast iso-1-ferricytochrome c, at pH 7.5. Recently, we have been developing methods to couple a favorable equilibrium to the unfavorable partial unfolding of a protein to allow substantial population of partially unfolded states of proteins. 6 In particular, we have been using the free energy provided by heme ligand exchange reactions to drive partial unfolding, as outlined below (Scheme 1) for cytochrome c. When ligand L: is histidine, the methionine f histidine ligand exchange for Fe (III) heme can provide∼ 5 kcal/mol of stabilization energy based on data from heme-peptide model systems. 7 In previous work, we have demonstrated that mutation of lysine 73 to histidine in the least stable substructure of cytochrome c5a stabilizes an equilibrium folding intermediate during guanidine hydrochloride (gdnHCl) unfolding of iso-1-cytochrome c at pH 7.5. 6, 8 In the present investigation, we determine the temperature dependence of the population of this partially unfolded state. Since this partial unfolding involves loss of the Met 80 heme ligand (Scheme 1), the conformational transition can be monitored using the absorbance band at 695 nm, which is characteristic of heme-Met 80 ligation. 9 In Figure 1, the absorbance at 695 nm, A695, is highest near 15 C. The native (Met 80 ligated heme) state is lost in favor of the partially unfolded (His 73 ligated heme) state10 either by increasing or decreasing the temperature, indicating heat and cold denaturation. The behavior of the A695 band as a function of temperature is similar in the presence of 0.1 to 0.5 M gdnHCl, except that the magnitude of A695 becomes progressively smaller as the [gdnHCl] increases (data not shown). The A695 data as a function of temperature and [gdnHCl] were converted into free energy of partial unfolding, ΔGobs, using standard methods (Figure 2). 11 The A695 data used to generate the ΔGobs versus T data in Figure 2 were collected with cyclic temperature scans as in Figure 1. Inspection of Figure 2 shows that the partial unfolding reaction is reversible. Since ΔGobs is∼ 0 for partial unfolding of His 73 iso-1-cytochrome c, ΔGobs can be obtained over a broad temperature range, allowing the curvature due to the heat capacity increment, ΔCp, to be observed directly. Normally, long extrapolations of temperature-dependent gdnHCl or urea denaturation data are required to obtain ΔCp from (1)(a) Brockwell, DJ; Smith, DA; Radford, SE Curr. Opin. Struct. Biol. 2000, 10, 16-25.(b) Roder, H.; Colón, W. Curr. Opin. Struct. Biol. 1997, 7, 15-28.(2)(a) Dobson, CM Trends Biochem. Sci. 1999, 24, 329-332.(b) Fink,
