Unraveling Molecular Dynamics
in TE-1x buffer (40 mM Tris, 2 mM EDTA, 12.5 mM Mg2+
,
pH 8) were incubated on clean substrates for 3 min. Te fluid cell was topped with 1.5 mL TE-1x buffer, followed by adding 10 μL of 1 μM streptavidin (STV) stock (Sigma-Aldrich). Plasmid DNA. Freshly cleaved mica substrates were coated
with 10 μl of 0.01% 30-70 kD poly-L-ornithine stock solution (Sigma-Aldrich) for 10 min. Aſter rinsing with ultra-pure water, 10 μL of pUC19 vector solution (MoBiTec) was deposited on the surface, to reach a final concentration of 1 nM DNA in the chamber volume. Aſter another rinsing step with ultra-pure water, the substrates were subjected to imaging in fluid. Collagen type I nanomatrices. Aſter being freshly cleaved,
the mica substrates were incubated with PBS-1x buffer (200 mM KCl, pH 7.3) for 30 min. Aſter adding 15 μL of type I bovine atelocollagen stock solution (Advanced Biomatrix) to a final volume collagen concentration of 30 μg/ml, the samples were immediately subjected to imaging in PBS-1x buffer. Annexin V lattices. Coagulation solutions were prepared
from Coag Reagent II, containing DOPC:DOPS (7:3) (Avanti Lipids). Briefly, 10 μL of Coag Reagent II were deposited in clean Eppendorf tubes and vacuum-dried for 3 hr, before being diluted in 1 mL coagulation buffer (20 mM HEPES, 150 mM NaCl, 2 mM CaCl2
, pH 7.4), and ultra-sonicated for
30 min. 10 μM A5 aliquots were prepared from 33 kD human placental A5 (Sigma-Aldrich) in A5 buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.6). Aſter freshly cleaving the mica sub- strates, 10 μL of Coag solution was deposited for 2 min and rinsed with Coag buffer. Te fluid cell was topped with 1 ml of Coag buffer, and the A5 concentration was adjusted to 100 mM. High-speed AFM imaging. Samples were analyzed using High-Speed AFM (Bruker), which features a
a NanoRacer®
customized fluid cell with optional temperature control. Te instrument was placed in an acoustic isolation housing on an active antivibration table. Unless stated otherwise, all sam- ples were measured in liquid under ambient temperature in amplitude-modulation AC mode. We used fast-scanning high- resonant ultra-short cantilevers (USC-F1.2-k0.15, NanoWorld, Switzerland) with a nominal resonance frequency of 1.2 MHz in air, spring constant of 0.15 N/m, reflective chromium/gold- coated silicon chip, and high-density carbon tips with a radius of curvature smaller than 10 nm.
Results Temperature-induced lipid phase transition. The
temperature-driven increase in mobility between solid and fluid phases in supported lipid bilayers oſten results in intermediate ripple-like structures, where both states coexist with a constant periodicity [13]. To study the dynamics of that process in detail we have imaged supported DMPC lipid bilayers in the temperature range from 22°C to 24°C (Figure 1). Te results indicate that there are
two concurring structural periodicities existing in the studied temperature range, namely 24 and 34 nm. Te gradual tem- perature ramping from 22.4°C to 23.3°C leads to a complete 34 nm ripple phase transition. Further increase in tempera- ture above 25–26°C led to a complete loss in periodicity. For representative pur- poses, the periodic transition has been fit with a Poisson-Boltzmann theory, which is typically applied for model membranes with mobile charged lipids [14]. DNA origami streptavidin-biotin
binding kinetics. DNA origami nano- structures (DONs) are emerging as molecular pegboards for the immobili- zation of ligands on surfaces, oſten fea- turing high-affinity biotin-streptavidin bridges. We have studied DONs that carry five equidistantly spaced covalent biotin tags (Figure 2). By supplementing the biotinylated
Figure 1: Sequence of 424 AFM phase images recorded at 1 frame/sec, indicating the thermodynamic transition between two distinct DMPC ripple phase periodicities. The ripple phase transition kinetics has been fit with a Poisson-Boltzmann equation. XYZ-scales in the AFM images are 800 nm, 800 nm, and 3 deg respectively.
2022 May •
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DONs with streptavidin, it is possible to visualize the dynamics of binding/ unbinding of
individual streptavidin
molecules. Quantification of the binding properties by occupation site analysis
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