title: Engineering fluorogen activating proteins into self-assembling materials.
abstract: We present herein characteristics of a conjugate in which dL5, a fluorogen activating protein (FAP), and AEAEAKAK, an amphiphilic peptide, are combined to form a solid-phase fluorescence detection platform. The FAP dL5 is a covalently linked dimer of two identical light chain variable fragments which activates the fluorescence of the fluorogen malachite green (MG). The amphiphilic peptide of sequence AEAEAKAK is a building block of stimuli-responsive materials that undergoes sol-gel phase transition at high ionic strengths. We hypothesize that the novel bifunctional protein containing both the FAP and the amphiphile, termed dL5_EAK coassembles with the self-assembling peptide [AEAEAKAK]2 (EAK16-II) to form an insoluble membrane composite whereby the fluorescence enhancement function of the FAP domain remains intact. Denaturing polyacrylamide electrophoresis indicated that greater than 78% of dL5_EAK incorporates into the EAK16-II membrane. Conversely, less than 32% of dL5 without the EAK sequence associates with the insoluble fraction of EAK16-II in buffers. Membranes containing dL5_EAK and EAK16-II exhibited at least 4-fold higher fluorescence intensity compared to mixtures containing dL5 and EAK16-II. Scanning electron microscopy revealed the presence of particulates, presumably FAPs, scattered on the membrane fibrils. The evidence suggests a system of materials that can be developed into in situ forming local sensors by immobilizing dL5 into coacervate, on which MG can be detected. It is envisioned that dL5 membranes can be established in diseased locales to monitor infiltration and migration of inflammatory cells marked with antibodies conjugated to MG.
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA. matsaun@andrew.cmu.edu
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Saunders
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Christopher
authors.initials: C
authors.lastname: Szent-Gyorgyi
authors.forename: Yi
authors.initials: Y
authors.lastname: Wen
authors.forename: Zachary
authors.initials: Z
authors.lastname: Drennen
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waggoner
authors.forename: Wilson S
authors.initials: WS
authors.lastname: Meng
mesh_terms: D000595:Amino Acid Sequence; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis*; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012394:Rosaniline Dyes / Q000032:analysis*; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: C098329:EAK16 peptide; D005456:Fluorescent Dyes; D007147:Immunoglobulin Light Chains; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; D057127:Single-Chain Antibodies; C005095:malachite green