Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein.

Sudarat Tharad,Öykü Üzülmez,Boonhiang Promdonkoy,José Toca-Herrera

doi:10.3390/ijms19123819

Abstract

Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein–lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.

Highlights

Bacillus thuringiensis (Bt) is a soil Gramm-negative bacterium
The cholesterol content was varied in the lipid bilayers in order to mimic the cell membrane of insect and mammalian cells
The results have shown that the cholesterol content influences the protein–lipid binding of Cyt2Aa2 protein by increasing its binding rate and changing the surface properties of the final protein:lipid:Chol layer

Summary

Introduction

Bacillus thuringiensis (Bt) is a soil Gramm-negative bacterium. Bt is wildly known because of its insecticidal property [1]. Insecticidal toxins are produced at different stages of cell growth. A vegetative protein (Vip) is initially produced at the vegetative phase, whereas crystal protein (Cry) and cytolytic protein (Cyt) are subsequently found at sporulation phase. The toxins have insecticidal properties, their amino acid sequences are dissimilar [2,3,4]. To improve the efficiency of insecticidal activity, it has been necessary to investigate lipid–protein binding mechanisms

Methods

Results

Conclusion