title: 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.
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-IAC, 1060, Wien, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Bonazza
authors.forename: Hanspeter
authors.initials: H
authors.lastname: Rottensteiner
authors.forename: Birgit K
authors.initials: BK
authors.lastname: Seyfried
authors.forename: Gerald
authors.initials: G
authors.lastname: Schrenk
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.forename: Peter L
authors.initials: PL
authors.lastname: Turecek
authors.forename: Gernot
authors.initials: G
authors.lastname: Friedbacher
mesh_terms: D000538:Aluminum Silicates / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry*
chemical_list: D000538:Aluminum Silicates; D055767:Immobilized Proteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D005169:Factor VIII; C011934:mica