Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy

Abstract

Atomic force microscopy is unmatched in terms of high-resolution imaging under ambient conditions. Over the years, substantial progress has been made using this technique to improve our understanding of biological systems on the nanometer scale, such as visualization of single biomolecules. For monitoring also the interaction between biomolecules, in situ high-speed imaging is making enormous progress. Here, we describe an alternative ex situ imaging method where identical molecules are recorded before and after reaction with a binding partner. Relocation of the identical molecules on the mica surface was thereby achieved by using a nanoscale scratch as marker. The method was successfully applied to study the complex formation between von Willebrand factor (VWF) and factor VIII (FVIII), two essential haemostatic components of human blood. FVIII binding was discernible by an appearance of globular domains appended to the N-terminal large globular domains of VWF. The specificity of the approach could be demonstrated by incubating VWF with FVIII in the presence of a high salt buffer which inhibits the interaction between these two proteins. The results obtained indicate that proteins can maintain their reactivity for subsequent interactions with other molecules when gently immobilized on a solid substrate and subjected to intermittent drying steps. The technique described opens up a new analytical perspective for studying protein-protein interactions as it circumvents some of the obstacles encountered by in situ imaging and other ex situ techniques.

The Effect of Hematocrit on Platelet Adhesion: Experiments and Simulations

“…The end effect is that the rate constants for both bond formation and dissociation in the simulation are two orders-of-magnitude higher than their best physical estimates from the aforementioned articles, but this is needed to make the simulation computationally feasible. In nondimensional units L ¼ 0.1, which translates to~280 nm and is consistent with the high end of Bonazza et al (42). The spring constant is based on an estimate from the prior work of Fitzgibbon et al (30), which used a similar platelet bonding model for a simplified system of a single platelet near a wall and was able to get reasonable agreement to experiments when estimating the translocation distance that a platelet would travel rolling along the wall between the time that it began adhering and the time that it became stationary.…”

The role of ultralarge multimers in recombinant human von Willebrand factor – a review of physico-and biochemical studies and findings in in vivo models and in humans with von Willebrand disease

“…the height seen in the image is significant increased). In contrast, fully stretched rVWF does not bind FVIII, while partially stretched VWF can still bind some but less than its coiled form (17,19). We may therefore speculate that coiled VWF, which is likely the predominant form in which VWF circulates in blood, serves as a molecular transport or delivery system that moves FVIII selectively to anatomic sites where hemostasis is required.…”

“…The latter situation is found at the time of a vascular lesion and persists until coagulation forms the fibrin network which stabilizes finally the hemostatic plug. Almost im-tion of the proteolytic cleavage process of VWF by ADAMTS13 (17). A recently developed method could even be utilized to visualize conformational changes of rVWF and its shear dependent protein-protein interactions on the macro-molecular level (18).…”

A bio-inspired method for direct measurement of local wall shear rates with micrometer localization using the multimeric protein von Willebrand factor as sensor molecule

“…rVWF was recently licensed under the brand name Vonvendi V R as a drug product for the treatment of different forms of von Willebrand's disease. 22,23 All AFM studies were conducted with rVWF lot HN02R00 and were carried out under identical conditions as described by Bonazza et al 24…”

“…Regarding the first option, it should be mentioned that adhesion of sensor molecules to the mica surface is too weak for proper AFM scanning in liquid, even by the use of the tapping mode with the less invasive parameters (displacement of molecules). This problem has been affronted in numerous studies and is usually circumvented by chemical surface modifications 27,28 or imaging in air; 24 hence, the lack of strong attractive forces between untreated mica and protein molecules is well known. Therefore, our model considers stretching of passively transported sensor molecules by the velocity gradient in the carrying liquid followed by a subsequent deposition on the surface.…”