Hierarchical Map of Protein Unfolding and Refolding at Thermal Equilibrium Revealed by Wide-Angle X-ray Scattering

Abstract

Hierarchical features of the thermal unfolding-refolding structural transition of hen egg white lysozyme (HEWL) have been studied in the temperature range from 13 to 84 degrees C by using high-resolution wide-angle X-ray scattering (WAXS) measurements at a third-generation synchrotron source. We have gathered high-statistic WAXS data of the reversible unfolding-refolding process of HEWL in the q range from approximately 0.05 to approximately 3 A(-1) [q = (4pi/lambda) sin(theta/2), where theta is the scattering angle and lambda the wavelength]. This measured q range corresponds to the spatial distance from approximately 2 to approximately 125 A, which covers all hierarchical structures of a small globular protein such as HEWL, namely, tertiary, domain, and secondary structures. Because of this, we have found that the pH dependence of the thermal structural transition of HEWL is well characterized by the various hierarchical levels and the transition concurrence among them. In this report, we present a new hierarchical map depiction of unfolding-refolding transitions. Using scattering with various ranges of q values, we determine the molar ratio of native-like protein structure defined by the data in each range, thus producing a map of the amount of native-like structure as a function of the hierarchical level or resolution. This map can visualize a detailed feature of the unfolding-refolding transition of a protein depending on various structural hierarchical levels; however, the exact meaning of the map will await sharpening by additional works.

Collapse of the hydration shell of a protein prior to thermal unfolding

“…The beam size at the sample position was~0.1 mm 2 . Other details of the WAXS measurements are given in the previous reports (Hirai et al, 2002(Hirai et al, , 2004.…”

“…To obtain the net scattering from the proteins the following equation was used (Hirai et al, 2002(Hirai et al, , 2004.…”

“…2A shows the pH dependence of the HSD and radius of gyration R g of 0.01 g ml À1 HEWL, and Fig. 2B shows the WAXS data used for the calculation (Hirai et al, 2004). As shown in Fig.…”

“…Recently we demonstrated that the wide-angle X-ray scattering (WAXS) method using high-intensity X-rays from a third-generation synchrotron-radiation source enables us to observe directly the whole hierarchical structure of proteins including quaternary, tertiary, domain and secondary structures in solution (Hirai et al, 2002), and we showed that the structures of proteins in solutions can be compared with crystallographic structures by using a program such as CRYSOL (Svergun et al, 1995). By using SR-WAXS we successfully analysed the details of the reversible unfolding-refolding process of HEWL covering all the hierarchical structures from tertiary to secondary structure (over the real-space distance from~2.5 tõ 125 Å ), and showed that the pH dependence of the thermal structural transition of HEWL is characterized by its hierarchical structures and the cooperativity between them (Hirai et al, 2004). In that report we presented a new hierarchical map of unfoldingrefolding transitions by determining the molar ratios of the nativestructure protein for each hierarchical level at an intermediate transition state, which would be comparable to the fraction of native contacts used in a 'folding funnel' representation (Dill & Chan, 1997;Dinner et al, 2000).…”

“…We have found that the initial stage of HEWL unfolding accompanies a collapse of the hydration shell. In the present analyses we have used the WAXS data reported previously (Hirai et al, 2004). We will also discuss the feasibility and limitation of the present analysis.…”