Thermodynamic and molecular dynamic insights into how fusion influences peptide-tag recognition of an antibody.

To understand the effect of protein fusion on the recognition of a peptide-tag by an antibody, we fused a CCR5-derived peptide-tag (pep1) to GFP and investigated its recognition by an anti-pep1 antibody, 4B08. First, to characterize the thermodynamic properties associated with the pep1-4B08 binding, isothermal titration calorimetry experiments were conducted. It was found that pep1 fused to the C-terminus of GFP (GFP-CT) enhanced the enthalpic gain by 2.1 kcal mol-1 and the entropic loss only by 0.9 kcal mol-1, resulting in an 8-fold increase in the binding affinity compared to the unfused pep1. On the other hand, pep1 fused to the N-terminus of GFP (GFP-NT) enhanced the enthalpic gain by 3.0 kcal mol-1 and the entropic loss by 3.2 kcal mol-1, leading to no significant enhancement of the binding affinity. To gain deeper insights, molecular dynamics simulations of GFP-NT, GFP-CT, and pep1 were performed. The results showed that the location of the fusion point sensitively affects the interaction energy, the solvent accessible surface area, and the fluctuation of pep1 in the unbound state, which explains the difference in the experimental thermodynamic properties.

A Collaborative Study on the Classification of Silicone Oil Droplets and Protein Particles Using Flow Imaging Method.

In this study, we conducted a collaborative study on the classification between silicone oil droplets and protein particles detected using the flow imaging (FI) method toward proposing a standardized classifier/model. We compared four approaches, including a classification filter composed of particle characteristic parameters, principal component analysis, decision tree, and convolutional neural network in the performance of the developed classifier/model. Finally, the points to be considered were summarized for measurement using the FI method, and for establishing the classifier/model using machine learning to differentiate silicone oil droplets and protein particles.

Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody.

Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody-antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2 kcal mol-1 of free energy. Collectively, our results suggest that antibodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role.

Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible Peptide, Strengthening the Binding Affinity for an Antibody.

Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein-protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.