Millisecond dynamics of an unlabeled amino acid transporter

Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft.*

In the article “Millisecond dynamics of an unlabeled amino acid transporter “ Tina R. Matin, George R. Heath, Gerard H. M. Huysmans, Olga Boudker and Simon Scheuring develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution.*

Among the bulk and single-molecule techniques, high-speed atomic force microscopy ( HS-AFM ) stands out with its ability to provide real-time structural and dynamical information of single molecules. HS-AFM images label-free molecules under close-to-physiological conditions with ~0.1 nm vertical and ~1 nm lateral imaging resolution. Furthermore, HS-AFM has typically ~100 ms temporal resolution, giving access to structure–dynamics relationship of proteins, though the achievable imaging speed depends on sample characteristics like scan size and surface corrugation.

Recently in a quest to achieve higher temporal resolutions, the authors of the cited article used HS-AFM line scanning (HS-AFM-LS) for the analysis of single-protein dynamics. *

Line scanning, using a conventional AFM, has been used to study protein–protein interactions earlier. In HS-AFM-LS, the slow-scan axis (y-direction) is disabled. Therefore, instead of imaging an x/y-area, the scientists scan over one horizontal x-line several hundreds to thousands of times per second, thus reaching millisecond temporal resolution. The topographical readouts of this line are stacked one after another, resulting in kymographs of the dynamical behavior of the molecules. Therefore, HS-AFM-LS has between 2 and 3 orders of magnitude higher temporal resolution than HS-AFM imaging and should allow the detection of fast transporter dynamics and possible intermediate states that have so far escaped kinetic characterization. *

All AFM images presented in this study were taken using a HS-AFM operated in amplitude modulation mode (with typical free and setpoint amplitudes, Afree = 1.0 nm and Aset = 0.9 nm, respectively using optimized scan and feedback parameters. NanoWorld Ultra-Short Cantilevers ( NanoWorld’s AFM probe series especially dedicated for High Speed Scanning) of the USC-F1.2-k0.15 type were used. In the presented experiments, four different buffer conditions were used. *

As the authors state in their article they find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.*

Figure 2 from “Millisecond dynamics of an unlabeled amino acid transporter” by Tina R. Matin et al.
HS-AFM line scanning (HS-AFM-LS): millisecond temporal resolution of unlabeled transporter dynamics.:
a HS-AFM image of a membrane packed with GltPh exposing the extracellular face before HS-AFM-LS (apo condition: 20 mM Tris-HCl, pH7.5, 150 mM KCl). Dashed lines indicate the position of the central scan line where subsequent HS-AFM-LS is performed. b Six seconds of a HS-AFM-LS kymograph with 3.3 ms line acquisition speed. Each transporter domain appears as a vertical line. c Projection (top) and height profile (bottom) of b. d HS-AFM image after HS-AFM-LS. The lateral position of recognizable features in a–d are indicated by arrowheads. e One second high-magnification views of dashed regions 1, 2, and 3 in b. Transport domain excursions to the inward-facing state appear as dark dwells along the vertical time axis. f Projection (top) and height profile (bottom) of e. Arrowheads indicate the position of the seven protomers in the kymograph (red: active protomer #5). g Height/time traces (gray) and state fits (red) of the active domain (protomer #5) in e. This figure is representative of the experimental sequence for the >50 replicates analyzed in this work.
NanoWorld Ultra-Short Cantilevers ( NanoWorld's AFM probe series especially dedicated for High Speed Scanning) of the USC-F1.2-k0.15 type (8 μm length, nominal spring constant of 0.15 N/m, nominal resonance frequency of ∼650 kHz and quality factor of ∼1.5 in buffer) were used.
Figure 2 from “Millisecond dynamics of an unlabeled amino acid transporter” by Tina R. Matin et al.
HS-AFM line scanning (HS-AFM-LS): millisecond temporal resolution of unlabeled transporter dynamics.:
a HS-AFM image of a membrane packed with GltPh exposing the extracellular face before HS-AFM-LS (apo condition: 20 mM Tris-HCl, pH7.5, 150 mM KCl). Dashed lines indicate the position of the central scan line where subsequent HS-AFM-LS is performed. b Six seconds of a HS-AFM-LS kymograph with 3.3 ms line acquisition speed. Each transporter domain appears as a vertical line. c Projection (top) and height profile (bottom) of b. d HS-AFM image after HS-AFM-LS. The lateral position of recognizable features in a–d are indicated by arrowheads. e One second high-magnification views of dashed regions 1, 2, and 3 in b. Transport domain excursions to the inward-facing state appear as dark dwells along the vertical time axis. f Projection (top) and height profile (bottom) of e. Arrowheads indicate the position of the seven protomers in the kymograph (red: active protomer #5). g Height/time traces (gray) and state fits (red) of the active domain (protomer #5) in e. This figure is representative of the experimental sequence for the >50 replicates analyzed in this work.

*Tina R. Matin, George R. Heath, Gerard H. M. Huysmans, Olga Boudker and Simon Scheuring
Millisecond dynamics of an unlabeled amino acid transporter
Nature Communications volume 11, Article number: 5016 (2020)
DOI: https://doi.org/10.1038/s41467-020-18811-z

Please follow this external link to read the full article: https://rdcu.be/cbuOU

Open Access : The article “Millisecond dynamics of an unlabeled amino acid transporter” by Tina R. Matin, George R. Heath, Gerard H. M. Huysmans, Olga Boudker and Simon Scheuring is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

The free energy landscape of retroviral integration

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved.*

In the article “The free energy landscape of retroviral integration” published in Nature Communications Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser and Jan Lipfert use biochemical assays, atomic force microscopy (AFM), and multiplexed single-molecule magnetic tweezers (MT) to study tetrameric prototype foamy virus (PFV) strand-transfer dynamics.*

Their finding that PFV intasomes employ auxiliary-binding sites for modulating the barriers to integration raises the question how the topology of higher-order intasomes governs integration of pathogenic retroviruses, most notably HIV. The single-molecule assays developed in this work are expected to be particularly useful to further unravel the complexity of this important class of molecular machines.*

The AFM images were recorded in amplitude modulation mode under ambient conditions and by using NanoWorld high resolution SuperSharpSiliconSSS-NCH cantilevers ( resonance frequency ≈300 kHz; typical end-radius 2 nm; half-cone angle <10 deg). Typical scans were recorded at 1–3 Hz line frequency, with optimized feedback parameters and at 512 × 512 pixels.*

Figure 2 e, f and g from “The free energy landscape of retroviral integration” by Willem Vanderlinden et al. 
(please refer to the full article for the complete figure 2  https://rdcu.be/b0R63 ) :
  e Atomic Force Microscopy image of intasomes incubated briefly (2 min) with supercoiled plasmid DNA, depicting a branched complex as found in ~50% of early complexes.
  f  Atomic Force Microscopy image of a bridging complex that dominates (~80%) the population of complexes at longer (>45 min) incubation. 
 g  Atomic Force Microscopy image of a gel-purified STC
Figure 2 e, f and g from “The free energy landscape of retroviral integration” by Willem Vanderlinden et al.
(please refer to the full article for the complete figure 2 https://rdcu.be/b0R63 ) :
 e AFM image of intasomes incubated briefly (2 min) with supercoiled plasmid DNA, depicting a branched complex as found in ~50% of early complexes.
 f AFM image of a bridging complex that dominates (~80%) the population of complexes at longer (>45 min) incubation.
g AFM image of a gel-purified STC

*Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser, Jan Lipfert
The free energy landscape of retroviral integration
Nature Communications volume 10, Article number: 4738 (2019)
DOI: https://doi.org/10.1038/s41467-019-12649-w

Please follow this external link to read the full article: https://rdcu.be/b0R63

Open Access The article “The free energy landscape of retroviral integration“ by Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser and Jan Lipfert is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

High-speed AFM height spectroscopy reveals microsecond-dynamics of unlabeled biomolecules

In their recent publication “High-speed AFM height spectroscopy reveals μs-dynamics of unlabeled biomolecules” in Nature Communications George R. Heath and Simon Sheuring develop and apply HS-AFM height spectroscopy (HS-AFM-HS, a technique inspired by fluorescence spectroscopy), a technique whereby the AFM tip is held at a fixed x–y position and  the height fluctuations under the tip in z-direction with Angstrom spatial and 10µs temporal resolution are monitored.

They demonstrate “how this technique can be used to simultaneously measure surface concentrations, diffusion rates and oligomer sizes of highly mobile annexin-V molecules during membrane-binding and self-assembly at model membranes and derive its kinetic and energetic terms. Additionally, HS-AFM-HS at specific positions in the annexin lattice where the freedom of movement is restricted to rotation allowed determination of the interaction free energies of protein-protein contacts.”* The applicability of this technique is wide and is discussed at the end of the publication.

NanoWorld Ultra-Short Cantilevers (USC) for Fast-/High-Speed AFM  ( USC-F1.2-k0.15 ) were used.

Congratulations to the authors to this publication which pushes the speed limits of AFM even further!

Increasing the temporal resolution of HS-AFM by reducing the dimensionality of data acquisition. a HS-AFM image of a DOPC/DOPS (8:2) membrane in the presence of annexin-V and NP-EGTA-caged Ca2+. Blue arrows illustrate the slow- (vertical) and the fast-scan axis (horizontal). Images can be captured at up to 10–20 frames s−1. b HS-AFM movie frames of A5 membrane-binding, self-assembly and formation of p6 2D-crystals upon UV-illumination induced Ca2+-release. c Average height/time trace of the membrane area in b. d Averaged HS-AFM image of an A5 p6-lattice overlaid with the subsequent line scanning kymograph, obtained by scanning repeatedly the central x-direction line as illustrated by the blue arrow with a maximum rate of 1000–2000 lines s−1. e Line scanning kymograph across one protomer of the non-p6 trimer, marked by * in d and e at a rate of 417 lines s−1 (2.4 ms per line). f Histogram of state dwell-times of the molecule in e. g HS-AFM image of an A5 p6-lattice partially covering a DOPC/DOPS (8:2) SLB surface during self-assembly. HS-AFM height spectroscopy (HS-AFM-HS) is performed following halting the x- and y-piezos to capture height information at a fixed position at the center of the image (illustrated by the target). h Schematic showing the principle of HS-AFM-HS. The AFM tip is oscillated in z at a fixed x,y-position, detecting single molecule dynamics such as diffusion under the tip. i Height/time trace obtained by HS-AFM-HS with the tip positioned at the center of image (g). The height/time trace allows determination of the local A5 concentration analyzing the time fraction of the occurrence of height peaks. j Dwell-time analysis of each height peak of diffusing A5 from 60 s height/time data and subsequent fitting of the distribution to multiple Gaussians (possible molecular aggregates corresponding to the fits with distinct dwell-times (τD) are shown above the graph). All scale bars: 20 nm, NanoWorld Ultra-Short Cantilevers (USC) for Fast-/High-Speed AFM ( USC-F1.2-k0.15 ) were used.
Figure 1 from “High-speed AFM height spectroscopy reveals μs-dynamics of unlabeled biomolecules”: Increasing the temporal resolution of HS-AFM by reducing the dimensionality of data acquisition. a HS-AFM image of a DOPC/DOPS (8:2) membrane in the presence of annexin-V and NP-EGTA-caged Ca2+. Blue arrows illustrate the slow- (vertical) and the fast-scan axis (horizontal). Images can be captured at up to 10–20 frames s−1. b HS-AFM movie frames of A5 membrane-binding, self-assembly and formation of p6 2D-crystals upon UV-illumination induced Ca2+-release. c Average height/time trace of the membrane area in b. d Averaged HS-AFM image of an A5 p6-lattice overlaid with the subsequent line scanning kymograph, obtained by scanning repeatedly the central x-direction line as illustrated by the blue arrow with a maximum rate of 1000–2000 lines s−1. e Line scanning kymograph across one protomer of the non-p6 trimer, marked by * in d and e at a rate of 417 lines s−1 (2.4 ms per line). f Histogram of state dwell-times of the molecule in e. g HS-AFM image of an A5 p6-lattice partially covering a DOPC/DOPS (8:2) SLB surface during self-assembly. HS-AFM height spectroscopy (HS-AFM-HS) is performed following halting the x- and y-piezos to capture height information at a fixed position at the center of the image (illustrated by the target). h Schematic showing the principle of HS-AFM-HS. The AFM tip is oscillated in z at a fixed x,y-position, detecting single molecule dynamics such as diffusion under the tip. i Height/time trace obtained by HS-AFM-HS with the tip positioned at the center of image (g). The height/time trace allows determination of the local A5 concentration analyzing the time fraction of the occurrence of height peaks. j Dwell-time analysis of each height peak of diffusing A5 from 60 s height/time data and subsequent fitting of the distribution to multiple Gaussians (possible molecular aggregates corresponding to the fits with distinct dwell-times (τD) are shown above the graph). All scale bars: 20 nm

*George R. Heath & Simon Scheuring
High-speed AFM height spectroscopy reveals μs-dynamics of unlabeled biomolecules
Nature Communicationsvolume 9, Article number: 4983 (2018)
DOI: https://doi.org/10.1038/s41467-018-07512-3

Please follow this external link for the full article: https://rdcu.be/bdaKU

Open Access The article “High-speed AFM height spectroscopy reveals μ s-dynamics of unlabeled biomolecules” by George R. Heath & Simon Scheuring is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.