Thermodynamic and structural analysis of interactions between peptide ligands and SEB

Abstract

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB-binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (10(5) M(-1)). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications.

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

“…The binding of SEB to the off‐cell peptide in the peptide‐ELISA was approximately 10‐times greater than the apparent on‐cell K d . The on‐cell versus off‐cell binding affinity differences has also been observed for phage peptides with SEB; high‐affinity samples were identified in phage‐ELISA but did not bind during peptide‐ITC experiments (Dudak et al, ). The specificity difference between on‐cell and off‐cell reagent (Figures , , ) was less disparate than the affinity results (Supplemental Figure 1, Figure 2).…”

“…Selectivity or specificity of a target capture reagent is as important as binding affinity when detecting samples in a complex matrix. Further complicating the isolation of SEB‐selective reagents using phage is the observed specificity difference between the phage‐ELISA (on‐cell) and the SPR analysis of the identical free peptide (off‐cell), even for high‐affinity phage clones (Soykut et al, ; Dudak et al, ). In this study of bacterial display libraries, we will compare the on‐cell selectivity using a SEB concentration‐dependent flow cytometry method with the selectivity of the synthetic peptide using a peptide‐ELISA method (Kogot et al, ).…”

“…SEB‐binding peptides selected from phage display with a consensus sequence of WHK have been used in whole‐cell ELISA, with a SEB detection limit of 1.4 ng/well in a 96‐well plate format without a direct comparison to the free peptide binding (Goldman et al, ). In SEB‐binding peptide studies from phage display using the free peptide, the affinity of the free peptides determined by SPR had binding association constants ( K a ) of 4.2 × 10 5 M −1 ( K d = 2.38 μM) (Soykut et al, ) and ( K a ) 2.3 × 10 5 M −1 ( K d = 4.35 μM) (Dudak et al, ); phage‐ELISA on‐cell was used as a screening method, and no binding constant was reported using phage‐ELISA. In bacterial display, flow cytometry analysis provides a rapid screening tool, similar to the use of phage‐ELISA in phage display.…”

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

“…Protein interactions with other proteins Protein interactions with small ligands (including co‐factors and drugs) Protein/peptide interactions with metals and ions …”

A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins

“…However, this study did not involve an examination of the mechanism of the peptide–SEB interaction. Two years later, Dudak et al reported a thermodynamic and structural analysis of the binding interactions between peptide ligands and SEB [ 125 ]. They studied the core amino acids playing an important role in the binding between the peptides and SEB.…”