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Characterization of the Molten Globule Conformation of V26A Ubiquitin by Far-UV Circular Dichroic Spectroscopy and Amide Hydrogen/Deuterium Exchange

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Characterization of the Molten Globule Conformation of V26A Ubiquitin by Far-UV Circular Dichroic Spectroscopy and Amide Hydrogen/Deuterium Exchange Characterization of the molten globule conformation of V26A ubiquitin by far-UV circular dichroic spectroscopy and amide hydrogen/deuterium exchange Soon-Ho Park* Department of Biochemistry and Molecular Biology, College of Dentistry and Research Institute of Oral Sciences, Kangnung National University, Gangneung, Korea The molten globular conformation of V26A ubiquitin (valine (14, 15). Unfortunately, due to the highly cooperative nature to alanine mutation at residue 26) was studied by nuclear mag- of protein folding process in the condition that favors the na- netic resonance spectroscopy in conjunction with amide hy- tive conformation, these intermediate states were transiently drogen/deuterium exchange. Most of the amide protons that populated so that the detailed structural analysis was not are involved in the native secondary structures were observed feasible. Interestingly, it has been observed that some proteins to be protected in the molten globule state with the protection form a conformation that was neither the native nor unfolded factors from 1.2 to 6.7. These protection factors are about 2 to random coil state in a solution that mildly destabilizes the na- 6 orders of magnitude smaller than those of the native state. tive conformation (4, 16). This conformation was named as the These observations indicate that V26A molten globule has na- molten globule, since it had a globular shape without tightly tive-like backbone structure with marginal stability. The com- packed side-chains. Although well-defined tertiary structures parison of amide protection factors of V26A ubiquitin molten were absent, significant amount of native-like secondary struc- globule state with those of initial collapsed state of the wild- tures were observed in the molten globule state. Furthermore, type ubiquitin suggests that V26A ubiquitin molten globule the molten globule state has been shown to have similar con- state is located close to unfolded state in the folding reaction formational aspects with the transiently populated partially coordinate. It is considered that V26A ubiquitin molten glob- folded state observed in the kinetic folding experiments (11, ule is useful model to study early events in protein folding 17-20). Therefore, it is considered useful to study the con- reaction. [BMB reports 2008; 41(1): 35-40] formational properties of the molten globule to expand our un- derstanding of the protein folding problem. Among the small globular proteins, ubiquitin has been INTRODUCTION widely used as a model to study the protein folding process (8, 10, 21-25). Wild-type ubiquitin has been shown to form a The mechanism by which a protein acquires the native three- loosely collapsed conformation in an acidic solution contain- dimensional structure, the protein folding problem, is not ing 60% (v/v) methanol. This conformational state was named clearly understood yet. It was considered that a protein folds as the alcohol form (26). Although the alcohol form was wide- through progressively more structured ensemble intermediate ly studied to understand ubiquitin folding reaction, there were states to a unique native structure (1-4). Thus, it is thought that some doubts whether this conformation was true folding inter- the analysis of conformation of these ensemble intermediate mediate in aqueous condition (27). Recently, a mutant ubiq- states would provide valuable information to understand pro- uitin with valine to alanine mutation at residue position 26 tein folding problem. The ensemble folding intermediate states (V26A ubiquitin) was observed to form neither the native nor were observed in the early phase of folding reaction explored fully unfolded random coil conformation in solution at pH 2 by the kinetic folding measurements for several proteins, in- (25). The far-UV circular dichroic (CD) spectrum indicated that cluding cytochrome c (5), B1 domain of protein G (6, 7), ubiq- significant amount of secondary structures were present in the uitin variants (8-10), α-lactalbumin (11-13), and apomyoglobin acid-denatured V26A ubiquitin, while the near-UV CD spec- trum of V26A ubiquitin indicated the absence of tightly *Corresponding Author. Tel: 82-33-640-2458; Fax: 82-33-642-6410; packed tertiary structure. Acid-denatured V26A ubiquitin ob- E-mail: [email protected] served to bind to the hydrophobic dye, 8-anilinonaph- thalene-1-sulfonic acid (ANS), indicating that the presence of Received 2 July 2007, Accepted 8 August 2007 loosely collapsed hydrophobic cluster. The urea-induced un- folding of V26A ubiquitin at acidic solution was observed to Keywords: Amide hydrogen/deuterium exchange, Molten globule, Ubiquitin be less cooperative than that observed in neutral pH. All these http://bmbreports.org BMB reports 35 Ubiquitin molten globule Soon-Ho Park observations strongly indicated that V26A ubiquitin formed a spectively, which is consistent with that of the native wild-type molten globule in the solution at pH 2. In this report, (a) the ubiquitin (9). The far-UV CD spectrum for the molten globule conformations of alcohol form and molten globule form of form of V26A ubiqutin at pH 2 shows strong negative molar V26A ubiquitin were compared by using far-UV CD spectro- ellipticity near 200 nm with a shoulder at 220 nm, features scopy, and (b) the conformational properties of V26A ubiquitin typical to the peptide backbone containing some random coil molten globule were studied by using nuclear magnetic reso- conformation (29). The observed molar ellipticity value of nance (NMR) spectroscopy in conjunction with amide hydro- 4,400 deg cm2 dmol1 at 220 nm for V26A ubiquitin molten gen/deuterium (H/D) exchange. globule was slightly less negative than that observed for the native state at neutral pH. The far-UV CD spectra of V26A RESULTS AND DISCUSSION ubiquitin at pH 2 became more negative by the addition of methanol. Furthermore, the negative band at 208 nm became Wild-type ubiquitin has been shown to form a partially folded conspicuous so that the far-UV CD spectrum of the alcohol conformation in a solution containing 60% (v/v) methanol at form of V26A ubiquitin in 60% methanol at pH 2 had two pH 2 (26-28). This conformation was named as the alcohol negative bands near 208 and 220 nm, which are the spectral form of ubiquitin and considered to be an intermediate state features typical to the α-helical conformation (29). The molar that may appear during the ubiquitin folding reaction. ellipticitiy values at 208 and 220 nm for the alcohol form of Recently, partially folded molten globule conformation was V26A ubiquitin in 60% methanol at pH 2 were 17,000 and observed for hydrophobic core residue variant ubiquitin 13,000 deg cm2 dmol1, respectively. The far-UV CD spec- (V26A ubiquitin) in the aqueous solution at pH 2 (25). This trum for the alcohol form of V26A ubiquitin appeared to be molten globule form of V26A ubiquitin was also considered to nearly superimposed to that of the wild-type ubiquitin (dotted be an intermediate state of ubiquitin folding reaction. To com- line in Fig. 1), indicating that the alcohol form of V26A ubiq- pare the conformational properties of these states and to find uitin has the same backbone conformation as that of the wild- their role in the folding process, the polypeptide backbone type ubiquitin. structures of the alcohol form of wild-type and V26A ubiquitin, It has been known that the molar ellipticity value at 220 nm and the molten globule form of V26A ubiquitin were meas- reflects the content of secondary structures in a polypeptide ured by far-UV CD spectroscopy. The far-UV CD spectrum of backbone (30). The more negative molar ellipticity value rep- the native V26A ubiquitin in neutral pH (Fig. 1, dashed line) resents the more secondary structures in the polypeptide shows two negative bands at 208 and 220 nm with molar el- backbone. The observed far-UV CD spectrum suggests that lipticity values of 6,500 and 5,000 deg cm2 dmol1, re- there still some secondary structure remains, albeit disrupted as compare to the native state, in the molten globule form of V26A ubiquitin. The content of secondary structures in the molten globule form of V26A ubiquitin appears to be less than those of the native state V26A ubiquitin. Based on the spectral features and molar ellipticity value at 220 nm, the content of secondary structures, especially α-helical conformation, in the alcohol form of V26A ubiquitin appears to be more than those in the native state V26A ubiquitin. This observation suggests that the regions of polypeptide backbone which are not α-heli- cal structure in the native state assume α-helical conformation in the presence of methanol. Therefore, it can be concluded that the alcohol form of V26A ubiquitin contains nonnative α-helical backbone structures. If the alcohol form of V26A ubiquitin is ever occurred during the folding process, the non- native α-helical structures should be unfolded prior to the for- mation of the native conformation. It is considered that the al- cohol form of V26A ubiquitin would be more like off-pathway intermediate that is trapped in a local energy minimum. Furthermore, no such highly α-helical folding intermediate was observed in the folding kinetics experiment for the wild- Fig. 1. Far-UV CD spectra. Solid lines represent the far-UV CD type ubiquitin measured by stopped-flow CD device (23). All spectra of V26A ubiquitin at pH 2 with 0%, 20%, 40%, and 60% methanol, reading downward at 220 nm. Dashed line repre- these observations suggest that alcohol form of ubiquitin may sents the native state far-UV CD spectrum of V26A ubiquitin at not be a proper model to study ubiquitin folding process. By pH 5. Dotted line represents the far-UV CD spectrum of wild contrast, the molten globule form of V26A ubiquitin appeared type ubiquitin in 60% (v/v) methanol at pH 2.
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