6 research outputs found
Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of <i>Bacillus thuringiensis</i> Cytolytic Protein
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
Effect of the Concentration of Cytolytic Protein Cyt2Aa2 on the Binding Mechanism on Lipid Bilayers Studied by QCM‑D and AFM
Bacillus thuringiensis is known
by its insecticidal property. The insecticidal proteins are produced
at different growth stages, including the cytolytic protein (Cyt2Aa2),
which is a bioinsecticide and an antimicrobial protein. However, the
binding mechanism (and the interaction) of Cyt2Aa2 on lipid bilayers
is still unclear. In this work, we have used quartz crystal microbalance
with dissipation (QCM-D) and atomic force microscopy (AFM) to investigate
the interaction between Cyt2Aa2 protein and (cholesterol-)lipid bilayers.
We have found that the binding mechanism is concentration dependent.
While at 10 μg/mL, Cyt2Aa2 binds slowly on the lipid bilayer
forming a compliance protein/lipid layer with aggregates, at higher
protein concentrations (100 μg/mL), the binding is fast, and
the protein/lipid layer is more rigid including holes (of about a
lipid bilayer thickness) in its structure. Our study suggests that
the protein/lipid bilayer binding mechanism seems to be carpet-like
at low protein concentrations and pore forming-like at high protein
concentrations
Specific domain V reduction of beta‑2‑glycoprotein I induces protein flexibility and alters pathogenic antibody binding
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder
antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and
comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide
bond at domain V has been associated with different cysteine redox states. The role of this disulfide
bond in conformational dynamics of this protein has not been investigated so far. Here, we report
on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine;
TCEP) of β2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry
analyses confirming majority targeting to the fifth domain of β2GPI. Atomic force microscopy images
suggested that reduced β2GPI shows a slightly higher proportion of open conformation and is more
flexible compared to the untreated protein as confirmed by modelling studies. We have determined a
strong increase in the binding of pathogenic APS autoantibodies to reduced β2GPI as demonstrated
by ELISA. Our study is relevant for understanding the effect of β2GPI reduction on the protein
structure and its implications for antibody binding in APS patients