Chemically Denatured Structures of Porcine Pepsin using Small-Angle X-ray Scattering.

Yecheol Rho,Kyeong Sik Jin,Byoungseok Min,Jun Ha Kim

doi:10.3390/polym11122104

Abstract

Porcine pepsin is a gastric aspartic proteinase that reportedly plays a pivotal role in the digestive process of many vertebrates. We have investigated the three-dimensional (3D) structure and conformational transition of porcine pepsin in solution over a wide range of denaturant urea concentrations (0–10 M) using Raman spectroscopy and small-angle X-ray scattering. Furthermore, 3D GASBOR ab initio structural models, which provide an adequate conformational description of pepsin under varying denatured conditions, were successfully constructed. It was shown that pepsin molecules retain native conformation at 0–5 M urea, undergo partial denaturation at 6 M urea, and display a strongly unfolded conformation at 7–10 M urea. According to the resulting GASBOR solution models, we identified an intermediate pepsin conformation that was dominant during the early stage of denaturation. We believe that the structural evidence presented here provides useful insights into the relationship between enzymatic activity and conformation of porcine pepsin at different states of denaturation.

Highlights

The biological functions of proteins require the correct folding and sufficient stability.protein stability is strongly influenced by the type of solvent, ionic strength, and protein concentration
To obtain more information on the 3D structure of porcine pepsin and its conformational stability, and because of the importance of establishing the relationship between the structure and enzymatic activity of proteins, we investigated the effect of the denaturing agent urea on the structure of porcine pepsin using Raman spectroscopy and small-angle X-ray scattering (SAXS)
The protein concentration of the samples was determined based on the absorbance at 280 nm using a the buffer solution in the absence of urea; we observed a single distinct peak with no other oligomers theoretical extinction coefficient calculated based on the amino acid sequence of the protein

Summary

Introduction

The biological functions of proteins require the correct folding and sufficient stability.protein stability is strongly influenced by the type of solvent, ionic strength, and protein concentration. Protein folding is the process by which a peptide chain assumes its functional shape or conformation. By coiling and folding into a specific three-dimensional (3D) shape, they can perform their biological functions. The reversal of this process is known as denaturation, whereby a native protein loses its functional conformation and becomes an amorphous and non-functional amino acid chain [1]. Studying the structural dynamics of denatured proteins can provide insights into the physical and chemical principles that govern protein folding. Recent studies have revealed the biological significance of denatured states in processes such as aggregation [5,6,7], chaperone binding [8,9], and membrane transport [10,11]

Methods

Results

Conclusion