Correlation of membrane protein conformational and functional dynamics

Membrane proteins (MPs) reside in the plasma membrane and perform various biological processes including ion transport, substrate transport, and signal transduction.*

Function-related conformational changes in membrane proteins occur in times scales ranging from nanoseconds to seconds.*

Obtaining time-resolved dynamic information of MPs in their membrane environment is still a major challenge.*

Although High Speed Atomic Force Microscopy (HS-AFM) images label-free samples such as DNA, soluble proteins, MPs, and intrinsically disordered proteins at ~1n~m lateral, ~0.1 nm vertical and ~100 ms temporal solution in aqueous environment and at ambient temperature and pressure, its temporal resolution is too slow to characterize many dynamic biological processes.*

In order to overcome this limitation Raghavendar Reddy Sanganna Gari, Joel José Montalvo-Acosta, George R. Heath, Yining Jiang, Xiaolong Gao, Crina M. Nimigean, Christophe Chipot and Simon Scheuring in their article Correlation of membrane protein conformational and functional dynamics use High Speed Atomic Force Microscopy Height Spectroscopy ( HS-AFM-HS) to characterize the microsecond timescale conformational changes of an integral-MP model system, i.e., the outer membrane protein G (OmpG) in a membrane environment.*

The positioning of the AFM tip is guided by HS-AFM imaging immediately before HS-AFM-HS-operation.*

NanoWorld Ultra-Short Cantilevers (USC) of the USC-F1.2-k0.15 type were used for the HS-AFM and HS-AFM-HS presented in the article.*

Figure 1 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al:
HS-AFM imaging of OmpG in lipid bilayers at pH 7.6 and pH 5.0.
a OmpG at pH 7.6 (Supplementary movie 1, left; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. Arrowheads in t = 11.6 s: Loop-6 fluctuating over the lumen. Arrowhead in t = 12.0 s: Fully open state. b Correlation average (n = 2752) of the HS-AFM movie frames (344 frames recorded over 68.8 s, full color scale: 0.0 nm < height < 1.25 nm, where the membrane level was set to 0.0 nm). c Correlation average of OmpG dimers. The topography outline (based on the molecular structure in 1e), serves as a visual guide to locate loop-6 and loop-2 in the topography and is highlighted by the dashed outline (the position of loop-6 is indicated by the asterisk based on its location in the structure (e)). Inner dashed outline show barrel lumen. d Standard deviation (std) map (n = 2752) from the averaging process in (b) (full color scale from blue to red: 0.05 nm < std < 0.19 nm) and topography outlines as in (c). e X-ray structure (PDB 2iwv) of the open OmpG conformation. Loop-6 (arrowhead L6) stands out of the image plane towards the viewer. Loop-2 (L2) forms a beta strand pointing away from the β-barrel, well detected by HS-AFM in the open state (b). f OmpG at pH 5.0 (Supplementary movie 1, right; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. g Correlation average (n = 2472) of the HS-AFM movie frames (309 frames recorded over 61.8 s, full color scale: 0.0 nm < height < 0.7 nm, where the membrane level was set to 0.0 nm). h Correlation average of OmpG dimers. For comparison, the topography outline of the open state (e) is shown (the position of loop-6 is indicated by the asterisk). i Standard deviation (std) map (n = 2472) from the averaging process in (g) (full color scale from blue to red: 0.04 nm < std < 0.07 nm) and topography outlines as in (h). j X-ray structure (PDB 2iww) of the closed OmpG conformation shown in the same orientation as in (e). Loop-6 (L6) folds over the β-barrel lumen in a lid-like manner. Loop-2 (L2) does not form a β-strand in the closed state, in agreement with absence of topography in this region in (h). Black dashed line: outline based on (e) for comparison.
NanoWorld USC-F1.2-k0.15 AFM probes were used for the HS-AFM.
Figure 1 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al:
HS-AFM imaging of OmpG in lipid bilayers at pH 7.6 and pH 5.0.
a OmpG at pH 7.6 (Supplementary movie 1, left; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. Arrowheads in t = 11.6 s: Loop-6 fluctuating over the lumen. Arrowhead in t = 12.0 s: Fully open state. b Correlation average (n = 2752) of the HS-AFM movie frames (344 frames recorded over 68.8 s, full color scale: 0.0 nm < height < 1.25 nm, where the membrane level was set to 0.0 nm). c Correlation average of OmpG dimers. The topography outline (based on the molecular structure in 1e), serves as a visual guide to locate loop-6 and loop-2 in the topography and is highlighted by the dashed outline (the position of loop-6 is indicated by the asterisk based on its location in the structure (e)). Inner dashed outline show barrel lumen. d Standard deviation (std) map (n = 2752) from the averaging process in (b) (full color scale from blue to red: 0.05 nm < std < 0.19 nm) and topography outlines as in (c). e X-ray structure (PDB 2iwv) of the open OmpG conformation. Loop-6 (arrowhead L6) stands out of the image plane towards the viewer. Loop-2 (L2) forms a beta strand pointing away from the β-barrel, well detected by HS-AFM in the open state (b). f OmpG at pH 5.0 (Supplementary movie 1, right; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. g Correlation average (n = 2472) of the HS-AFM movie frames (309 frames recorded over 61.8 s, full color scale: 0.0 nm < height < 0.7 nm, where the membrane level was set to 0.0 nm). h Correlation average of OmpG dimers. For comparison, the topography outline of the open state (e) is shown (the position of loop-6 is indicated by the asterisk). i Standard deviation (std) map (n = 2472) from the averaging process in (g) (full color scale from blue to red: 0.04 nm < std < 0.07 nm) and topography outlines as in (h). j X-ray structure (PDB 2iww) of the closed OmpG conformation shown in the same orientation as in (e). Loop-6 (L6) folds over the β-barrel lumen in a lid-like manner. Loop-2 (L2) does not form a β-strand in the closed state, in agreement with absence of topography in this region in (h). Black dashed line: outline based on (e) for comparison.
Figure 2 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al:
Single channel electrophysiology and HS-AFM height spectroscopy recordings of OmpG in lipid bilayers.
Representative 60-ms segments of OmpG single channel recordings at pH 7.6 (a) and pH 5.0 (b) at +40 mV membrane potential (longer traces in Supplementary Figs. 2 and 3). Cartoon representation of single channel recording experimental setup is shown in inset of (a). OmpG (yellow) in open state (PDB:2IWV) is placed in a lipid bilayer (green) surrounded by buffer (light blue shade) and potassium and chloride ions are shown as red and blue spheres. Red arrow indicates ion flow through OmpG in response to voltage application. Dwell time histograms of open and closed states at pH 7.6 (c) and pH 5.0 (d) from single-channel recordings (see Supplementary Table 1). Representative 60-ms segments of OmpG HS-AFM-HS recordings at pH 7.6 (e) and pH 5.0 (f) (longer traces in Supplementary Fig. 4). Cartoon representation of HS-AFM height spectroscopy experimental setup is shown in inset of (e). An oscillating AFM tip (orange) detects conformational changes of loop motion. Dwell time histograms of open and closed states at pH 7.6 (g) and pH 5.0 (h) from HS-AFM-HS recordings (Supplementary Table 2). In HS-AFM-HS the low state represents the open state, where the HS-AFM tip can descend into the β-barrel, and the high state represents the closed state, where loop-6 covers the beta barrel barring access of the HS-AFM tip to the cavity. All current-time and height-time traces were filtered at 20 kHz during analysis. The state dwell-time histograms are shown using log binning for better visualization of the components49. Red traces in (a) and (b) represent idealized current-time traces using clampfit software. Red traces in (e) and (f) represent idealized height-time traces using the STaSI algorithm (see Methods).
NanoWorld USC-F1.2-k0.15 AFM probes were used for the HS-AFM-HS.
Figure 2 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al:
Single channel electrophysiology and HS-AFM height spectroscopy recordings of OmpG in lipid bilayers.
Representative 60-ms segments of OmpG single channel recordings at pH 7.6 (a) and pH 5.0 (b) at +40 mV membrane potential (longer traces in Supplementary Figs. 2 and 3). Cartoon representation of single channel recording experimental setup is shown in inset of (a). OmpG (yellow) in open state (PDB:2IWV) is placed in a lipid bilayer (green) surrounded by buffer (light blue shade) and potassium and chloride ions are shown as red and blue spheres. Red arrow indicates ion flow through OmpG in response to voltage application. Dwell time histograms of open and closed states at pH 7.6 (c) and pH 5.0 (d) from single-channel recordings (see Supplementary Table 1). Representative 60-ms segments of OmpG HS-AFM-HS recordings at pH 7.6 (e) and pH 5.0 (f) (longer traces in Supplementary Fig. 4). Cartoon representation of HS-AFM height spectroscopy experimental setup is shown in inset of (e). An oscillating AFM tip (orange) detects conformational changes of loop motion. Dwell time histograms of open and closed states at pH 7.6 (g) and pH 5.0 (h) from HS-AFM-HS recordings (Supplementary Table 2). In HS-AFM-HS the low state represents the open state, where the HS-AFM tip can descend into the β-barrel, and the high state represents the closed state, where loop-6 covers the beta barrel barring access of the HS-AFM tip to the cavity. All current-time and height-time traces were filtered at 20 kHz during analysis. The state dwell-time histograms are shown using log binning for better visualization of the components49. Red traces in (a) and (b) represent idealized current-time traces using clampfit software. Red traces in (e) and (f) represent idealized height-time traces using the STaSI algorithm (see Methods).

*Raghavendar Reddy Sanganna Gari, Joel José Montalvo-Acosta, George R. Heath, Yining Jiang, Xiaolong Gao, Crina M. Nimigean, Christophe Chipot and Simon Scheuring
Correlation of membrane protein conformational and functional dynamics
Nature Communications volume 12, Article number: 4363 (2021)
DOI: https://doi.org/10.1038/s41467-021-24660-1

Please follow this external link to read the full article: https://rdcu.be/cAE2S

Open Access : The article “Correlation of membrane protein conformational and functional dynamics” by Raghavendar Reddy Sanganna Gari, Joel José Montalvo-Acosta, George R. Heath, Yining Jiang, Xiaolong Gao, Crina M. Nimigean, Christophe Chipot 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/.

HS-AFM video of Covid’s Docking Method

Johannes Kepler University in Linz Austria has published a High-Speed Atomic Force Microscopy video of human lectin CLEC4G binding to glycans on a SARS-CoV-2 spike. This video was recorded by Daniel Canena and Peter Hinterdorfer and is, according to the two researchers, the first short film of the active structure the virus uses to attach to cell

Congratulations!

NanoWorld Ultra-Short AFM Cantilevers of the USC-F1.2-k0.15 type were used for the HS-AFM video.

Please follow this external link to the Johannes Keppler University webpage to watch the video: https://www.jku.at/en/news-events/news/detail/news/film-of-covids-docking-method/ or have a look on Youtube

NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)
NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)

High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action

The current pandemic is not the only health threat worldwide. Another worry is the increasing antibiotic resistance which increases the fear to run out of effective antibiotics.

This is one of the reasons why antimicrobial peptides (AMPs) are gaining more and more interest.

The lipopeptide Daptomycin ( DAP ) has been therapeutically used as a last resort antibiotic against multidrug-resistant enterococci and staphylococci in the past. Unfortunately, some strains have become resistant to Dap in recent years. There still is a knowledge-gap on the details of Dap activity. It is therefore important to understand the structure-activity relationships of AMPs on membranes in order to develop more antibiotics of this type as a countermeasure to the spread of resistance.*

High Speed Atomic Force Microscopy ( HS-AFM ) makes it possible to observe dynamic biological processes on a molecular level.

In the article “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” Francesca Zuttion, Adai Colom, Stefan Matile, Denes Farago, Frédérique Pompeo, Janos Kokavecz, Anne Galinier, James Sturgis and Ignacio Casuso describe how, by using the possibilities offered by high speed atomic force microscopy, they were able to confirm some up until now hypothetical models and additionally detected some previously unknown molecular mechanisms. *

The HS-AFM imaging made it possible for the authors to observe the development of the dynamics of interaction at the molecular-level over several hours. *

They investigated the lipopeptide Daptomycin under infection-like conditions and could confirm Dap oligomerization and the existence of half pores. *

They also mimicked bacterial resistance conditions by increasing the CL-content in the membrane. *

By correlating the results of other research techniques such as FRET, SANS, NMR, CD or electrophysiology techniques with the results they achieved with high speed atomic force microscopy F. Zuttion et al. were able to confirm several, previously, hypothetical models, and detect several unknown molecular mechanisms. *

It is to be hoped that the possibilities offered by HS-AFM imaging will stimulate new models and insight on the structure-activity relationship of membrane-interacting molecules and also open up the possiblity to increase the throughput of screening of molecular candidates considerably. *

NanoWorld USC ( Ultra-Short AFM Cantilevers) of the USC-F1.2-k0.15 type, which are specially designed for the use in high speed atomic force microscopy, were used for the HS-AFM imaging described in the article cited below. These AFM probes have a typical resonance frequency of 1200 kHz and have a wear resistant AFM tip made from high density carbon.

Figure 4 Sub-MIC Dap on POPG at 37 °C. Tens of minutes from “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” by Francesca Zuttion et al. NanoWorld Ultra-Short AFM Cantilevers USC-F1.2-k0.15 AFM probes for HS-AFM imaging were used.
Figure 4 Sub-MIC Dap on POPG at 37 °C. Tens of minutes from “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” by Francesca Zuttion et al.
Intermediate stages a A new structure appeared: dimples, zones of thinner membrane thickness, whose diameter was in the range 7 ± 2 nm. Most dimples diffuse, but some remained static (colour scale: 3 nm). Movie details: frame rate 97 ms; zoom of a full image of 150 nm × 90 nm and 256 × 160 pixels. b The dimple diffusion consisted of swinging trajectories, implying membrane-mediated dimple-dimple attraction (colour scale: 3 nm). b, right, Energy profile of the interaction of the dimples obtained derived from 120 centre-to-centre distance measurements that contains as the oligomers two energy minima. Movie details: frame rate 83 ms; full image of 150 nm × 150 nm and 256 × 256 pixels. c In some membrane zones, clusters of dimples, reminiscent of cubic phases, developed (colour scale: 4 nm). Movie details: frame rate 74 ms; full image of 90 nm × 60 nm and 256 × 160 pixels. d The clusters of dimples were moderately dynamical in time, with moderate internal rearrangements (colour scale: 4 nm). Movie details: frame rate 74 ms; full image of 25 nm × 16 nm and 256 × 160 pixels. e The other deformation found was elongated-humps on top of the POPG membrane. e, left, An elongated-hump in the proximity of a cluster of dimples (colour scale: 4 nm). e, right, A close-up and a profile of an elongated-hump. Additional images of elongated-humps on Supplementary Fig. 1. Movie details: frame rate 479 ms; zoom of full image of 250 nm × 200 nm and 300 × 256 pixels. f It was observed that the dimples and the elongated-humps fused and gave yield to pores of toroidal structure where a protruding ring surrounds the pore (colour scale: 4 nm). Movie details: frame rate 74 ms; full image of 40 nm × 40 nm and 256 × 160 pixels.

*Francesca Zuttion, Adai Colom, Stefan Matile, Denes Farago, Frédérique Pompeo, Janos Kokavecz, Anne Galinier, James Sturgis and Ignacio Casuso
High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action
Nature Communications volume 11, Article number: 6312 (2020)
DOI: https://doi.org/10.1038/s41467-020-19710-z

Please follow this external link to read the full article: https://rdcu.be/ciaW2

Open Access : The article “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” by Francesca Zuttion, Adai Colom, Stefan Matile, Denes Farago, Frédérique Pompeo, Janos Kokavecz, Anne Galinier, James Sturgis and Ignacio Casuso 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/.