pH-Dependent Deformations of the Energy Landscape of Avidin-like Proteins Investigated by Single Molecule Force SpectroscopyReport as inadecuate




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1

Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria

2

Center for Advanced Bioanalysis, Gruberstrasse 40, 4020 Linz, Austria

3

Institute of Biomedical Technology, University of Tampere, FI-33014 Tampere, Finland

4

Fimlab Laboratories Ltd., FI-33101 Tampere, Finland





*

Author to whom correspondence should be addressed.



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. View Full-Text

Keywords: avidin mutant; avidin-biotin; force spectroscopy; molecular recognition; pH dependence; single molecules; biophysics avidin mutant; avidin-biotin; force spectroscopy; molecular recognition; pH dependence; single molecules; biophysics





Author: Melanie Köhler 1, Andreas Karner 2, Michael Leitner 2, Vesa P. Hytönen 3,4, Markku Kulomaa 3, Peter Hinterdorfer 1,2 and Andreas Ebner 1,*

Source: http://mdpi.com/



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