Synthesis and Characterization of Cholesteryl Conjugated Lysozyme (CHLysozyme).

Shinji Katsura,Takayuki Furuishi,Etsuo Yonemochi,Haruhisa Ueda

doi:10.3390/molecules25163704

Shinji Katsura, Takayuki Furuishi + Show 2 more

Open Access

https://doi.org/10.3390/molecules25163704

Copy DOI

Abstract

Hydrophobic interaction is important for protein conformation. Conjugation of a hydrophobic group can introduce intermolecular hydrophobic contacts that can be contained within the molecule. It is possible that a strongly folded state can be formed in solution compared with the native state. In this study, we synthesized cholesteryl conjugated lysozyme (CHLysozyme) using lysozyme and cholesterol as the model protein and hydrophobic group, respectively. Cholesteryl conjugation to lysozyme was confirmed by nuclear-magnetic resonance. Differential-scanning calorimetry suggested that CHLysozyme was folded in solution. CHLysozyme secondary structure was similar to lysozyme, although circular dichroism spectra indicated differences to the tertiary structure. Fluorescence measurements revealed a significant increase in the hydrophobic surface of CHLysozyme compared with that of lysozyme; CHLysozyme self-associated by hydrophobic interaction of the conjugated cholesterol but the hydrophobic surface of CHLysozyme decreased with time. The results suggested that hydrophobic interaction changed from intramolecular interaction to an intermolecular interaction. Furthermore, the relative activity of CHLysozyme to lysozyme increased with time. Therefore, CHLysozyme likely forms a folded state with an extended durability of activity. Moreover, lysozyme was denatured in 100% DMSO but the local environment of tryptophan in CHLysozyme was similar to that of a native lysozyme. Thus, this study suggests that protein solution stability and resistance to organic solvents may be improved by conjugation of a hydrophobic group.

Highlights

The chemical modification of proteins for improvement of functionality has become widespread with advances in organic synthetic chemistry and protein engineering [1,2]
We investigated the structure of CHLysozyme using nuclear-magnetic resonance (NMR) spectroscopy, differential-scanning calorimetry (DSC), circular dichroism (CD), and fluorescence measurement
CD measurements suggested that the secondary structure of CHLysozyme was similar to CD measurements suggested that the secondary structure of CHLysozyme was similar to lysozyme (α+β type structure), but that the tertiary structure differed between the two enzymes

Summary

Introduction

The chemical modification of proteins for improvement of functionality has become widespread with advances in organic synthetic chemistry and protein engineering [1,2]. There are several ways to chemically modify proteins, for instance conjugating polyethylene glycol to protein (PEGylated protein) is of value as this enhances the chemical stability, solubility, and blood retention of the protein during clinical use [3,4,5,6,7,8]. The chemical stability (e.g., against thermal stress) of proteins has been improved by modification with polysaccharides, which are hydrophilic groups [9,10,11]. The chemical modification of proteins has mostly been via conjugation with hydrophilic groups, but there are a few reports on chemical modification using hydrophobic groups [12,13]. Chemical modification of hydrophobic groups to water soluble polymers has been reported. Akiyoshi et al used hydrogel biomaterials such as cholesterol-bearing pullulan (CHP) [14], Molecules 2020, 25, 3704; doi:10.3390/molecules25163704 www.mdpi.com/journal/molecules

Methods

Results

Conclusion