Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells

Abstract

Background: The serotonin transporter is the site of action of antidepressants and amphetamines.Results: Single molecular force spectroscopy allowed for mapping the energy landscape involved in MFZ2-12/SERT binding.Conclusion: Our data indicate that the outer vestibule imposes a barrier on the entry of MFZ2-12 into the SERT substrate binding site.Significance: Our results provide a useful framework for a further exploration of antidepressant binding.

Direct measurement and modulation of single-molecule coordinative bonding forces in a transition metal complex

“…4e) 40,41 . On the one hand, the discrepancy could be in part owing to high uncertainty in the estimates of parameter k on by AFM, as also noted by previous reports on other systems [34][35][36][37] . On the other hand, a direct comparison is not appropriate because the K d values are estimated under very different experimental conditions in AFM and in homogenous solution.…”

“…In the AFM configuration, the Os-terpy complex is immobilized on an Au(111) surface and its dissociation is induced by external mechanic forces (that is, through retraction of the AFM cantilever). Direct comparison for other systems has posed similar challenges [34][35][36][37][38] . The relative values of the parameters estimated by AFM force spectroscopy within the same system under different experimental conditions are thus more meaningful.…”

“…The method has been used for a wide range of chemical and biological systems [35][36][37][38] , although in some cases the parameters estimated could differ by an order of magnitude even for the same systems. Our AFM measurements were focused on the LR-dependent force spectra at three different oxidation states of the Os-terpy complex.…”

“…5). A recently reported approach was used to estimate the binding kinetic rate constant (k on ) 37 . The apparent dissociation constant (K d ) or affinity constant (K a ) can then be obtained by Equation 2: ARTICLE states is thus obtained using the following relations 38 .…”

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…AFM cantilevers (PFQNM‐LC from Bruker) had nominal spring constants of ≈0.08 N/m, had resonance frequencies of ≈60 kHz in buffer, and carried a SiO 2 stylus with a nominal apex radius of ≈65 nm. The cantilevers were functionalized with NH 2 groups, then coated with heterobifunctionalized 27‐unit long PEG linkers (NHS‐PEG 27 ‐maleimide), and quenched with β‐mercaptoethanol to lower the adhesion to imaged surfaces (Wildling et al , 2012). Images were taken using force‐distance curve‐based AFM (Dufrêne et al , 2013; PeakForce mode, Bruker) in buffer solution (20 mM Hepes, 50 mM NaCl, pH 7.4) at room temperature.…”