Complementary Analysis of Thermal Transition Multiplicity of Hen Egg-White Lysozyme at Low pH Using X-ray Scattering and Scanning Calorimetry

Abstract

In this present paper we present a new method of small-angle X-ray scattering (SAXS) data analysis (called the TMA method) to examine multiplicity of structural transition of denaturation processes of proteins. The TMA method gives us a molar fraction of spatial-conformational-state and an indication of deviation from the two-state structural transition hypothesis in a structural transition process. Using this method we have successfully compared a spatial-conformational-state transition observed by SAXS with a thermodynamic-microstate transition observed by differential scanning calorimetry (DSC) in the thermal denaturation process of hen egg-white lysozyme (HEWL) at the pH range of 2.4−3.1. The TMA method is able to dissect spatial-conformational-state transition multiplicity depending on structural hierarchy. We have found that the thermal structural transition of HEWL is well-characterized mostly in terms of two different spatial-conformational-state transitions in the tertiary and intramolecular structures. Upon heating, the transition-midpoint temperature of the spatial-conformational-state transition of the tertiary structure mostly agrees with that of DSC, while the transition-midpoint temperature of the intramolecular structure greatly differs from that of DSC. The tertiary structural transition essentially obeys the two-state transition feature with a sharp critical temperature in comparison with the intramolecular structural transition. When the pH is lowered from 3.1 to 2.4, the former transition tends to shows a maximum deviation from the two-state transition feature around the transition temperature. On the other hand, the intramolecular structural transition proceeds continuously with a relatively large deviation in the whole temperature range measured at all pH values. The present results obtained by the TMA method essentially agree with our previous report using standard SAXS analyses in HEWL system at different pH range of 7.0−1.2, and would afford us further aspects to discuss experimental evidences with theoretical models of protein folding, such as nucleation-collapse models.