Investigation of the pH stability of avidins and newly developed avidin mutants with atomic force microscopy based on single molecule sensors

Full text: The great stability of several avidin proteins over a wide pH range, particularly when combined with biotin, has been studied qualitatively in the last fifty years. In the present study, a more detailed investigation is made by performing molecular recognition studies, using AFM force spectroscopy. The applied measuring principle enables the investigation of forces and dynamics of the interaction between the proteins and a corresponding ligand, during a pH treatment and with different loading rates. Therefore, the ligand (biotin) is coupled via a bifunctional PEG-crosslinker on the outer AFM tip apex, whereas the receptor is immobilized on the probe surface. By repeatedly approaching and withdrawing of the tip in z-direction, receptor-ligand complexes are formed and released. If this experiment is repeated at different pulling speeds (loading rates) and pH values, the energy landscape and the pH stability of the receptors can be examined. The measurements have been clearly shown that the three examined proteins are stable over a wide pH range. Moreover chimeric avidin does not offer the pH stability on single molecule level as expected. All in all, the three proteins open the possibility for more applications, like for e.g. surface sensors, which are exposed extremes of pHs. (author)