X-ray scattering studies on structure of an enzyme protein in solution

Pohang University of Science & Technology
â—‹Kyeong Sik Jin Sangwoo Jin Moonhor Ree

To understand the conformational behavior of a protein, it is necessary to define not only the structure of its native state but also that of various denatured states. Recent studies have revealed the biological significance of denatured states in processes such as aggregation, chaperone binding, and transport across membrane. A variety of denatured states have also been identified, differing in their overall dimensions and the extent of residual secondary and tertiary structures. In particular, pepsin is a good model for the study of conformational behavior under various conditions because detailed information can be obtainable on the secondary structure, enzymatic properties, and zymogen activation. In the present study, we carried out solution X-ray scattering (SAXS) experiments on porcine pepsin, which is a gastric aspartic proteinase that plays an integral role in the digestive process of vertebrates, in order to obtain detailed information on the overall structure of porcine pepsin at the native state and its structural changes in solutions of various pH values. The solution SAXS data were analyzed in detail, providing important information on the structure and variations with pH conditions. From the SAXS profiles and determined parameters, structural models of the porcine pepsin were reconstructed, which was made inside the search volume of maximum diameter Dmax calculated from the p(r) function. The reconstructed models were obtained without imposing any restrictions on the symmetry and anisometry of pepsin molecule. Under several pH conditions, the reconstructed models reveal various conformational states, when compare to the crystal structure. The structural differences between solution and crystal structure of pepsin can be account for the inherent conformations of the flexible subdomain under carefully controlled specific pH conditions.