4:15 PM - 4:30 PM
▼ [19p-222-10] Detection of intermediate transient states of the streptavidin-biotin bond by atomic force microscopy
Keywords:single-molecule force spectroscopy, atomic force microscopy, transient states
The streptavidin-biotin system is considered one of the strongest noncovalent interactions occurring in nature, which ultimately originates from at least five hydrogen bond interactions as suggested by its chemical structure and as revealed by its crystallographic images. Therefore, we can expect the presence of several intermediate states along its intermolecular energy potential landscape. Through experimental studies, two states were revealed during the course of bond breakage. However, results from computer simulation studies suggested the presence of at least three intermediate states between the system’s bound and unbound states. To explain this discrepancy, along with providing experimental information on the simulation results, we performed single-molecule force spectroscopic measurements of the streptavidin-biotin system using atomic force microscopy (AFM). We employed a quasi-static process of slowly loading force onto the bond (loading rate = 20 pN/s) to provide a meaningful description of the system in near-equilibrium through thermodynamic assumptions. This, together with utilizing a fast sampling rate for AFM detection (20 microsec/data point), several transient states of the system were clearly resolved allowing the generation of a free energy landscape that illustrates the several intermediate metastable states of the system. Our findings show that the results from several experimental and simulation conditions are just bits of information from the stochastic behavior of this receptor-ligand system, that is, several intermediate metastable states are present where the system can possibly reside during the recognition process.