Unraveling Molecular Dynamics
Figure 2: Biotinylated DONs imaged in buffer in the presence of streptavidin. Streptavidin binding/unbinding is seen on top of the DONs as constantly appearing/ disappearing bright dots. The entire 1407-frame high-speed video sequence was recorded with 50 frames/sec. Site-occupation analysis is shown on the bottom. XYZ-scaling in the AFM images is 150, 150, and 2 nm respectively.
showed that the most occupied states featured 2 (39%) and 3 (41%) binding sites. Termodynamic rehybridization of DNA. We have
previously shown that pUC19 plasmids bind to polycationic interfaces in the form of structures that carry a lot of torsional energy, in turn leading to a higher propensity of forming dehy- bridization bubbles [6]. By subjecting that system to high-speed AFM imaging at 40 frames/sec, we could visualize the ther- modynamic state fluctuations of the single DNA strands [15] and ultimately their rehybridization to a double-stranded state (Figure 3). Kinetics of collagen type I fibrillogenesis. Collagen type
I is the most abundant extracellular matrix protein in mam- malian cells. We have previously reported on the application of fast-scanning AFM for studying the epitaxially driven hierarchical self-assembly of collagen type I fibrillar films [16]. Here we have further demonstrated that the kinetics of the fibrillogenesis can be studied with up to 15 frames/sec (Figure 4). Upon closer investigation of the cross-section profile P1,
it becomes apparent that the D-banding periodicity of col- lagen I consists of several sub-D-bands, which have been speculated to be a result of lateral amino acid staggering. Te cross-section profile P2 shows that it is possible to iden- tify several peaks with a lateral dimension of 5–8 nm, which appear to be structural intermediates in the formation of the larger collagen fibrils. Rotational dynamics of mobile annexin V trimmers.
Annexin V (A5) serves as an important regulator of mem- brane repair in eukaryotic cells, where it shows a strong Ca2+
binding affinity to phosphatidylserine [17,18]. We have used high-speed AFM to study the 2D crystal formation in
12
a model system containing supported lipid bilayers and tri- meric A5 molecules, forming a stable honeycomb P6-sym- metry lattice. We demonstrate the lateral dynamics of the mobile A5 trimers and show how the A5 structure orienta- tion can be resolved by acquiring multiple high-speed AFM images (Figure 5). While two-thirds of the trimers occupy the A5 P6-lattice,
the mobile A5 fraction has very high rotational dynamics, in which it intermittently interacts with the honeycomb lattice in preferred orientations at 0° and 60°.
Discussion Under certain experimental conditions, supported lipid
bilayers adopt different states, namely solid (gel) and fluid (liquid-ordered and liquid-disordered) phases. The occur- rence is specific to the chemical nature of the lipids, and transition from solid to f luid phase is related to a positive temperature shift. Previous studies have suggested that increase in temperature can include transition between sev- eral ripple states, namely dominant and metastable phases with different periodicities [13]. Our results indicate that this process can be quantified by means of high-speed AFM, by following the complete 24 to 34 nm periodicity transition while heating the sample from 22.4°C to 23.3°C (Figure 1). The complete loss of periodicity is consistent with the full transition to a fluid state above 26°C. Te bottom-up self-assembly of DONs typically fea-
tures extra molecules that can trigger cell receptor stimu- lation [19] and early signaling events [20]. Te monitored dynamics of biotin-streptavidin binding in such a scenario (Figure 2) indicates that further molecular tailoring can be successfully exploited by featuring enzymes, growth factors, or
www.microscopy-today.com • 2022 May
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