Proteomic Exploration of Membrane Curvature Sensors Using a Series of Spherical Supported Lipid Bilayers.

Membrane curvature-sensing (MCS) proteins recognize and regulate the morphologies of biological membranes. As these proteins lack characteristic sequence motifs in their primary structure, they are not instantly recognizable by genomic databases. Overcoming this technological challenge toward the agile identification of new proteins can promote the elucidation of membrane morphological regulation. Here, for the selective identification of MCS proteins, comparative proteomic analysis was performed using different sizes of the spherical supported lipid bilayer (SSLB), which consists of spherical SiO2 particles covered with a lipid bilayer. Because of the presence of SiO2 core, the curvature of the surrounding membrane is well-controlled and stable even on a micron scale. To prove this concept, known membrane curvature-sensing protein domains, Bin/Amphiphysin/Rvs (BAR) and Epsin N-terminal homology (ENTH), were evaluated by performing a binding assay using SSLBs, and the preferential binding to the highly curved membrane was confirmed. Peripheral membrane proteins obtained from normal human dermal fibroblast (NHDF) and human breast cancer (MDA-MB-231) cells were used in shotgun proteomic analysis, and 786 and 949 proteins were identified from SSLBs as lipid membrane binders, respectively. Statistical quantitative analyses of proteins detected from each SSLB with a different size revealed 118 candidate proteins, including 23 proteins unique to MDA-MB-231 cells, as membrane curvature sensors, including some previously reported curvature sensors. Functional clustering analysis based on the KEGG orthology database revealed that the protein-binding property to specific high or low membrane curvature correlated with their functions. Further investigation of candidate proteins will lead to the identification of new MCS proteins as well as cancer biomarkers.

A bioinspired peptide matrix for the detection of 2,4,6-trinitrotoluene (TNT).

A novel peptide-based three-dimensional probe called "peptide matrix," inspired by the antibody paratope region, was fabricated on a surface plasmon resonance (SPR) sensor chip to enhance the sensitivity of detecting the explosive 2,4,6-trinitrotoluene (TNT). Although peptide aptamer is an attractive candidate for a molecular recognition probe because of its ease of synthesis and chemical stability, it still has difficulty in applying to highly sensitive (i.e. parts-per-billion (ppb) or sub-ppb level) detections. Thus, we developed the concept of peptide matrix structure, which is constructed by consecutive disulfide bond formation between a large number of peptide fragments. This robust three-dimensional structure displays multiple binding sites which can efficiently associate with each TNT molecule. The peptide matrix lowered the dissociation constant (KD) by two orders of magnitude compared to the linear peptide aptamer, estimating KD as 10.1 nM, which is the lowest concentration reported by using peptide-based TNT probe. Furthermore, the concentration limit of detection of peptide matrix modified SPR sensor was 0.62 ppb, and hence comparable to single-chain variable fragment (scFv)-based TNT sensors. To our knowledge, this is the first report demonstrating peptide matrix fabrication and its application for small explosive molecule detection. This peptide matrix-based approach, which has the advantage of simple synthesis and high sensitivity, will be applicable to many other small-molecule label-free detections.

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 µM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.