Tag: biology AFM probes

Real time dynamics of Gating-Related conformational changes in CorA

Magnesium (Mg2+) is a key divalent cation in biology. It regulates and maintains numerous, physiological functions such as nucleic acid stability, muscle contraction, heart rate and vascular tone, neurotransmitter release, and serves as cofactor in a myriad of enzymatic reactions. Most importantly, it coordinates with ATP, and is thus crucial for energy production in mitochondria.*

In order to store Mg2+ in the mitochondrial lumen it is imported via Mrs2 and Alr2 ion channels that are closely related to CorA, the main Mg2+-importer in bacteria. Although these Mg2+-transport proteins do not show much sequence conservation, they all share two trans-membrane domains (TMDs) with the signature motif Glycine-Methionine-Asparagine (GMN) at the extracellular loop.*

CorA, a divalent-selective channel in the metal ion transport superfamily, is the major Mg2+-influx pathway in prokaryotes. CorA structures in closed (Mg2+-bound), and open (Mg2+-free) states, together with functional data showed that Mg2+-influx inhibits further Mg2+-uptake completing a regulatory feedback loop. While the closed state structure is a symmetric pentamer, the open state displayed unexpected asymmetric architectures.*

In the article “Real time dynamics of Gating-Related conformational changes in CorA” Martina Rangl, Nicolaus Schmandt, Eduardo Perozo and Simon Scheuring used high-speed atomic force microscopy (HS-AFM), to explore the Mg2+-dependent gating transition of single CorA channels: HS-AFM movies during Mg2+-depletion experiments revealed the channel’s transition from a stable Mg2+-bound state over a highly mobile and dynamic state with fluctuating subunits to asymmetric structures with varying degree of protrusion heights from the membrane.*

Their data shows that at Mg2+-concentration below Kd, CorA adopts a dynamic (putatively open) state of multiple conformations that imply structural rearrangements through hinge-bending in TM1. They also discuss how these structural dynamics define the functional behavior of this ligand-dependent channel.*

All Atomic Force Microscopy experiments described in the article were performed using NanoWorld Ultra-Short Cantilevers USC-F1.2-k0.15 for high-speed Atomic Force Microscopy ( HS-AFM ). Videos of CorA membranes were recorded with imaging rates of ~1–2 frames s−1 and at a resolution of 0.5 nm pixel−1.

Figure 1 from “Real time dynamics of Gating-Related conformational changes in CorA”:
Sample morphology of CorA reconstitutions for HS-AFM.
 
(a) HS-AFM overview topograph of densely packed CorA in a POPC/POPG (3:1) lipid bilayer exposing the periplasmic side and a loosely packed protein area with diffusing molecules exposing the intracellular face (full color scale: 20 nm). Left: Height histogram of the HS-AFM image with two peaks representative of the mica and the CorA surface (∆Height (peak-peak): 12 nm (20,500 height values)). The dashed line indicates the position of the cross-section analysis shown in (b). (b) Profile of the membrane shown in a), including a cartoon (top) of the membrane in side view. The height profile (~12 nm) corresponds well to the all-image height analysis (a, left) and the CorA structure (Matthies et al., 2016). (c) High-resolution image (top) and cross-section analysis along dashed line (bottom) of the periplasmic face. The height and dimension of the periplasmic face is in good agreement with the structure (left), and the periodicity (~14 nm, n = 40) corresponds well with the diameter of the intracellular face spacing the molecules on the other side of the membrane (full color scale: 2 nm). (d) HS-AFM image of densely packed CorA embedded in a DOPC/DOPE/DOPS (4:5:1) membrane. This reconstitution resulted in two stacked membrane layers, both exposing the CorA intracellular face. The dashed line indicates the position of the cross-section analysis shown in (e). Left: Height histogram of the HS-AFM image with two peaks at ~12 nm and ~17 nm (32,500 height values), corresponding to the proteins in two stacked membranes (full color scale: 20 nm). (e) Section profile of the membrane shown in d), including a cartoon (top) of the membrane in side view. (f) High-resolution view and cross-section analysis along dashed line (bottom) of the CorA intracellular face revealing the individual subunits of the pentamers (full color scale: 3 nm). Inset: 5-fold symmetrized average of CorA. The dimensions of CorA observed with HS-AFM are in good agreement with the structure (left: PDB 3JCF). The structures in (c) and (f) are shown in ribbon (top) and surface (bottom) representations, respectively.

*Martina Rangl, Nicolaus Schmandt, Eduardo Perozo, and Simon Scheuring
Real time dynamics of Gating-Related conformational changes in CorA
eLife. 2019; 8: e47322
DOI: 10.7554/eLife.47322

Please follow this external link to read the full article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927688/

Open Access: The article “Real time dynamics of Gating-Related conformational changes in CorA” by Martina Rangl, Nicolaus Schmandt, Eduardo Perozo 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 http://creativecommons.org/licenses/by/4.0/.

Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability

DNA origami nanostructures are widely employed in various areas of fundamental and applied research. Due to the tremendous success of the DNA origami technique in the academic field, considerable efforts currently aim at the translation of this technology from a laboratory setting to real-world applications, such as nanoelectronics, drug delivery, and biosensing. While many of these real-world applications rely on an intact DNA origami shape, they often also subject the DNA origami nanostructures to rather harsh and potentially damaging environmental and processing conditions.*

In their article “Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability” Charlotte Kielar, Yang Xin, Xiaodan Xu, Siqi Zhu, Nelli Gorin , Guido Grundmeier, Christin Möser, David M. Smith and Adrian Keller investigate the effect of long-term storage of the employed staple strands on DNA origami assembly and stability.*

Atomic force microscopy (AFM) under liquid and dry conditions was employed to characterize the structural integrity of Rothemund triangles assembled from different staple sets that have been stored at −20 °C for up to 43 months.*

NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3 were the AFM probes that were used for the AFM measurements under liquid conditions.*

Figure 1. from “Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability” by Charlotte Kielar et al.
(a) Schematic illustration of the Rothemund triangle DNA origami. AFM images of DNA origami triangles assembled from staple sets aged for (b) 2–7 months, (c) 11–16 months, (d) 22–27 months, and (e) 38–43 months. Measurements were performed either in liquid (left column) or dry conditions after gently dipping the sample into water (central column) or after harsh rinsing (right column). Scale bars represent 250 nm. Height scales are given in the individual images. The insets show zooms of individual DNA origami triangles.

*Charlotte Kielar, Yang Xin, Xiaodan Xu, Siqi Zhu, Nelli Gorin , Guido Grundmeier, Christin Möser, David M. Smith and Adrian Keller
Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability
Molecules 2019, 24(14), 2577
doi: https://doi.org/10.3390/molecules24142577

Please follow this external link to the full article: https://www.mdpi.com/1420-3049/24/14/2577/htm

Open Access: The article « Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability » by Charlotte Kielar, Yang Xin, Xiaodan Xu, Siqi Zhu, Nelli Gorin , Guido Grundmeier, Christin Möser, David M. Smith and Adrian Keller 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/.

IgA tetramerization improves target breadth but not peak potency of functionality of anti-influenza virus broadly neutralizing antibody

Mucosal immunoglobulins comprise mainly secretory IgA antibodies (SIgAs), which are the major contributor to pathogen-specific immune responses in mucosal tissues. SIgAs exist as mainly dimers and tetramers and play critical roles in mucosal immune responses against influenza.*

Detailed characterization of these anti-viral SIgA is important for better understanding of the mechanisms underlying anti-viral immunity.*
In their article “IgA tetramerization improves target breadth but not peak potency of functionality of anti-influenza virus broadly neutralizing antibody” Saito S, Sano K, Suzuki T, Ainai A, Taga Y, Ueno T, et al. (2019) describe a means of generating a recombinant tetrameric monoclonal SIgA to enable exhaustive characterization of tetrameric SIgAs.
The tetrameric monoclonal SIgA possessing variable regions of anti-influenza viruses broadly neutralizing antibody show that tetramerization of SIgA improves target breadth, but not the peak potency, of their anti-viral functions.*
These results broaden the knowledge about the fundamental role of SIgA tetramerization in anti-viral humoral response at the human respiratory mucosa.*

The high speed atomic force microscopy ( HS-AFM ) experiments mentioned in the article were performed using a NanoWorld Ultra-Short Cantilever USC-F1.2-k0.15.

Fig 1. Production of recombinant tetrameric monoclonal SIgAs. (A) Recombinant monoclonal IgA antibodies purified from the culture supernatant of cells co-transfected with A1+L (left upper), A1+L+J (left lower), A1+L+J+SC (right upper), or A2m2+L+J+SC (right lower), were subjected to size exclusion chromatography (SEC) analysis. A chromatogram showing absorbance at 280 nm revealed three major peaks: peak A (retention volume around 10.4 ml), peak B (retention volume around 9.3 ml), and peak C (retention volume around 8.4 ml). Data are representative of three independent experiments. (B) SDS-PAGE and BN-PAGE analysis of IgG and IgA1/IgA2m2 in each peak fraction (peak A, B, and C) purified from cells co-expressing SC (A1+L+J+SC or A2m2+L+J+SC). (C, D, E) High-mass MALDI-TOF MS analysis of the each peak fraction containing recombinant IgA1 purified from the culture supernatant of cells transfected with A1, L, J, and SC. (C) One main peak (arrow) corresponding to monomer (Mo) was detected in the peak A fraction. (D) Two main peaks (arrows) corresponding to a dimer (Di) and a di-cation dimer (Di2+) were detected in the peak B fraction. (E) Three main peaks (arrows) corresponding to a tetramer (Te), trimer (Tr), and di-cation tetramer (Te2+) were detected in the peak C fraction. (F, G) High-mass MALDI-TOF MS analysis of the each peak fraction of recombinant IgA2m2 purified from the culture supernatant from cells transfected with A2m2, L, J, and SC. (F) One main peak (arrow) corresponding to a monomer (Mo) was detected in the peak A fraction. (G) Three main peaks (arrows) corresponding to a tetramer (Te), a trimer (Tr), and a di-cation tetramer (Te2+) were detected in the peak C fraction. (H) Quantification of the amount of each subunit within the peak B or C fraction of recombinant SIgA1 or SIgA2m2 antibodies purified from the culture supernatant of cells transfected with A1/L/J,/SC or A2m2/L/J/SC using LC-MS with stable isotope-labeled standard peptides. The abundance of each subunit to that of J chain is expressed as a ratio. Data are expressed as box-and-whisker plot with minimum, maximum, median, upper and lower quartiles (n = 6–7). (I) HS-AFM image of peak C derived from a recombinant SIgA1 (A1Te) or SIgA2m2 (A2m2Te) antibody purified from the culture supernatant of cells transfected with A1/L/J/SC or A2m2/L/J/SC. Scale bar, 20 nm.

Fig 1. Production of recombinant tetrameric monoclonal SIgAs from ”
IgA tetramerization improves target breadth but not peak potency of functionality of anti-influenza virus broadly neutralizing antibody ” by
Saito S, Sano K, Suzuki T, Ainai A, Taga Y, Ueno T, et al. (2019) :
 
(A) Recombinant monoclonal IgA antibodies purified from the culture supernatant of cells co-transfected with A1+L (left upper), A1+L+J (left lower), A1+L+J+SC (right upper), or A2m2+L+J+SC (right lower), were subjected to size exclusion chromatography (SEC) analysis. A chromatogram showing absorbance at 280 nm revealed three major peaks: peak A (retention volume around 10.4 ml), peak B (retention volume around 9.3 ml), and peak C (retention volume around 8.4 ml). Data are representative of three independent experiments. (B) SDS-PAGE and BN-PAGE analysis of IgG and IgA1/IgA2m2 in each peak fraction (peak A, B, and C) purified from cells co-expressing SC (A1+L+J+SC or A2m2+L+J+SC). (C, D, E) High-mass MALDI-TOF MS analysis of the each peak fraction containing recombinant IgA1 purified from the culture supernatant of cells transfected with A1, L, J, and SC. (C) One main peak (arrow) corresponding to monomer (Mo) was detected in the peak A fraction. (D) Two main peaks (arrows) corresponding to a dimer (Di) and a di-cation dimer (Di2+) were detected in the peak B fraction. (E) Three main peaks (arrows) corresponding to a tetramer (Te), trimer (Tr), and di-cation tetramer (Te2+) were detected in the peak C fraction. (F, G) High-mass MALDI-TOF MS analysis of the each peak fraction of recombinant IgA2m2 purified from the culture supernatant from cells transfected with A2m2, L, J, and SC. (F) One main peak (arrow) corresponding to a monomer (Mo) was detected in the peak A fraction. (G) Three main peaks (arrows) corresponding to a tetramer (Te), a trimer (Tr), and a di-cation tetramer (Te2+) were detected in the peak C fraction. (H) Quantification of the amount of each subunit within the peak B or C fraction of recombinant SIgA1 or SIgA2m2 antibodies purified from the culture supernatant of cells transfected with A1/L/J,/SC or A2m2/L/J/SC using LC-MS with stable isotope-labeled standard peptides. The abundance of each subunit to that of J chain is expressed as a ratio. Data are expressed as box-and-whisker plot with minimum, maximum, median, upper and lower quartiles (n = 6–7). (I) HS-AFM image of peak C derived from a recombinant SIgA1 (A1Te) or SIgA2m2 (A2m2Te) antibody purified from the culture supernatant of cells transfected with A1/L/J/SC or A2m2/L/J/SC. Scale bar, 20 nm.

*Shinji Saito , Kaori Sano , Tadaki Suzuki , Akira Ainai, Yuki Taga,  Tomonori Ueno, Koshiro Tabata, Kumpei Saito, Yuji Wada, Yuki Ohara, Haruko Takeyama, Takato Odagiri, Tsutomu Kageyama, Kiyoko Ogawa-Goto, Pretty Multihartina, Vivi Setiawaty, Krisna Nur Andriana Pangesti, Hideki Hasegawa
IgA tetramerization improves target breadth but not peak potency of functionality of anti-influenza virus broadly neutralizing antibody
PLoS Pathog 15(1): e1007427.
DOI: https://doi.org/10.1371/journal.ppat.1007427

Please follow this external link for the full article: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007427

Open Access: The article « IgA tetramerization improves target breadth but not peak potency of functionality of anti-influenza virus broadly neutralizing antibody » by Saito S, Sano K, Suzuki T, Ainai A, Taga Y, Ueno T, et al. (2019) 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/.