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

Molecules

Melanie Köhler³  Andreas Karner²  Michael Leitner²  Vesa P. Hytönen¹  Markku Kulomaa¹  Peter Hinterdorfer³ 

[1] Institute of Biomedical Technology, University of Tampere, FI-33014 Tampere, Finland; E-Mails:;Center for Advanced Bioanalysis, Gruberstrasse 40, 4020 Linz, Austria; E-Mails:;Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria; E-Mails:

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

DOI  :  10.3390/molecules190812531

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.