title: Fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer from cyan to yellow fluorescent protein validates a novel method to cluster proteins on solid surfaces.
abstract: A novel method to distribute proteins on solid surfaces is proposed. Proteins microencapsulated in the water pool of reverse micelles were used to coat a solid surface with well-individualized round spots of 1 to 3 microm in diameter. The number of spots per unit area can be increased through the concentration of reverse micelles, and networks of spots were obtained at high concentrations of large reverse micelles. Moreover, depending on the pool size of the water reverse micelles, proteins can be deposited far from each other or in close proximity within the range of 50 to 70 A. This proximity obtained with small reverse micelles was proved through fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer (FLIM-FRET) measurements for the most relevant FRET pair in cell biology studies, the cyan and yellow fluorescent proteins. This novel procedure has several advantages and reveals the potential for study of protein-protein interactions on solid surfaces and for developing novel biomaterials and molecular devices based on biorecognition elements.
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Tecnico, Av Rovisco Pais, Lisbon, 1049-001, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Madeira
authors.forename: Nídia
authors.initials: N
authors.lastname: Estrela
authors.forename: José A B
authors.initials: JA
authors.lastname: Ferreira
authors.forename: Suzana M
authors.initials: SM
authors.lastname: Andrade
authors.forename: Silvia M B
authors.initials: SM
authors.lastname: Costa
authors.forename: Eduardo P
authors.initials: EP
authors.lastname: Melo
mesh_terms: D000327:Adsorption; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry*; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry*; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins