title: pH-dependent deformations of the energy landscape of avidin-like proteins investigated by single molecule force spectroscopy.
abstract: Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very high pH values. We compared avidin with streptavidin and a recently developed avidin-based mutant, chimeric avidin. To gain insights of the energy landscape of these interactions we used a single molecule approach and performed the Single Molecule Force Spectroscopy atomic force microscopy technique. There, the ligand (biotin) is covalently coupled to a sharp AFM tip via a distensible hetero-bi-functional crosslinker, whereas the receptor of interest is immobilized on the probe surface. Receptor-ligand complexes are formed and ruptured by repeatedly approaching and withdrawing the tip from the surface. Varying both pulling velocity and pH value, we could determine changes of the energy landscape of the complexes. Our results clearly demonstrate that avidin, streptavidin and chimeric avidin are stable over a wide pH range although we could identify differences at the outer pH range. Taking this into account, they can be used in a broad range of applications, like surface sensors at extreme pH values.
authors.affiliation: Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria. melanie.koehler@jku.at.
authors.forename: Melanie
authors.initials: M
authors.lastname: Köhler
authors.affiliation: Center for Advanced Bioanalysis, Gruberstrasse 40, 4020 Linz, Austria. andreas.karner@cbl.at.
authors.forename: Andreas
authors.initials: A
authors.lastname: Karner
authors.affiliation: Center for Advanced Bioanalysis, Gruberstrasse 40, 4020 Linz, Austria. michael.leitner@cbl.at.
authors.forename: Michael
authors.initials: M
authors.lastname: Leitner
authors.affiliation: Institute of Biomedical Technology, University of Tampere, FI-33014 Tampere, Finland. vesa.hytonen@uta.fi.
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.affiliation: Institute of Biomedical Technology, University of Tampere, FI-33014 Tampere, Finland. markku.kulomaa@uta.fi.
authors.forename: Markku
authors.initials: M
authors.lastname: Kulomaa
authors.affiliation: Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria. peter.hinterdorfer@jku.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
authors.affiliation: Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria. andreas.ebner@jku.at.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebner
mesh_terms: D001360:Avidin / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry; D007700:Kinetics; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D055767:Immobilized Proteins; D001360:Avidin; D001710:Biotin; D019809:Streptavidin