title: Two-Photon Fluorescence Anisotropy Imaging to Elucidate the Dynamics and the Stability of Immobilized Proteins.
abstract: Time/spatial-resolved fluorescence determines anisotropy values of supported-fluorescent proteins through different immobilization chemistries, evidencing some of the molecular mechanisms that drive the stabilization of proteins at the interfaces with solid surfaces. Fluorescence anisotropy imaging provides a normalized protein mobility parameter that serves as a guide to study the effect of different immobilization parameters (length and flexibility of the spacer arm and multivalency of the protein-support interaction) on the final stability of the supported proteins. Proteins in a more constrained environment correspond to the most thermostable ones, as was shown by thermal inactivation studies. This work contributes to explain the experimental evidence found with conventional methods based on observable measurements; thus this advanced characterization technique provides reliable molecular information about the immobilized proteins with sub-micrometer spatial resolution. Such information has been very useful for fabricating highly stable heterogeneous biocatalysts with high interest in industrial developments.
authors.affiliation: Enzymatic Engineering Group, Instituto de Catálisis y Petroleoquímica, CSIC , c/Marie Curie 2, 28049 Madrid, Spain.
authors.forename: Alejandro H
authors.initials: AH
authors.lastname: Orrego
authors.affiliation: Fluorescence Molecular Biophysics Group, Instituto Química Física "Rocasolano", CSIC , Serrano 119, 28006 Madrid, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: García
authors.affiliation: Crystallography and Structural Biology Group, Instituto Química Física "Rocasolano", CSIC , Serrano 119, 28006 Madrid, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Mancheño
authors.affiliation: Enzymatic Engineering Group, Instituto de Catálisis y Petroleoquímica, CSIC , c/Marie Curie 2, 28049 Madrid, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Guisán
authors.affiliation: Fluorescence Molecular Biophysics Group, Instituto Química Física "Rocasolano", CSIC , Serrano 119, 28006 Madrid, Spain.
authors.forename: M Pilar
authors.initials: MP
authors.lastname: Lillo
authors.affiliation: Heterogeneus Biocatalysis Group, CIC BiomaGUNE , Pase Miramon 182, 20009 San Sebastian-Donostia, Spain.|IKERBASQUE, Basque Foundation for Science , Bilbao, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: López-Gallego
mesh_terms: D005454:Fluorescence Polarization / Q000379:methods*; D055767:Immobilized Proteins / Q000737:chemistry*; D017785:Photons; D011506:Proteins / Q000737:chemistry*
chemical_list: D055767:Immobilized Proteins; D011506:Proteins