scholarly journals Single-Molecule Force Spectroscopy of Mycobacterial Adhesin-Adhesin Interactions

2007 ◽  
Vol 189 (24) ◽  
pp. 8801-8806 ◽  
Author(s):  
Claire Verbelen ◽  
Dominique Raze ◽  
Frédérique Dewitte ◽  
Camille Locht ◽  
Yves F. Dufrêne
Keyword(s):  
Single Molecule ◽  
Coiled Coil ◽  
Bimodal Distribution ◽  
Heparin Binding ◽  
Force Microscopy ◽  
Bacterial Aggregation ◽  
Atomic Force ◽  
Coiled Coil Region ◽  
Force Curves ◽  
Terminal Domains

ABSTRACT The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.

scholarly journals Interaction of the Mycobacterial Heparin-Binding Hemagglutinin with Actin, as Evidenced by Single-Molecule Force Spectroscopy

2008 ◽  
Vol 190 (23) ◽  
pp. 7614-7620 ◽  
Author(s):  
Claire Verbelen ◽  
Vincent Dupres ◽  
Dominique Raze ◽  
Coralie Bompard ◽  
Camille Locht ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Single Molecule ◽  
Coiled Coil ◽  
Force Spectroscopy ◽  
Bimodal Distribution ◽  
Actin Binding ◽  
Heparin Binding ◽  
Single Molecule Force Spectroscopy ◽  
Terminal Domain ◽  
Force Curves

ABSTRACT Although Mycobacterium tuberculosis and related species are considered to be typical endosomal pathogens, recent studies have suggested that mycobacteria can be present in the cytoplasm of infected cells and cause cytoskeleton rearrangements, the mechanisms of which remain unknown. Here, we used single-molecule force spectroscopy to demonstrate that the heparin-binding hemagglutinin (HBHA), a surface adhesin from Mycobacterium tuberculosis displaying sequence similarities with actin-binding proteins, is able to bind to actin. Force curves recorded between actin and the coiled-coil, N-terminal domain of HBHA showed a bimodal distribution of binding forces reflecting the detection of single and double HBHA-actin interactions. Force curves obtained between actin and the lysine-rich C-terminal domain of HBHA showed a broader distribution of binding events, suggesting they originate primarily from intermolecular electrostatic bridges between cationic HBHA domains and anionic actin residues. We also explored the dynamics of the HBHA-actin interaction, showing that the binding force and binding frequency increased with the pulling speed and contact time, respectively. Taken together, our data indicate that HBHA is able to specifically bind actin, via both its N-terminal and C-terminal domains, strongly suggesting a role of the HBHA-actin interaction in the pathogenesis of mycobacterial diseases.

scholarly journals Detection and Localization of Single LysM-Peptidoglycan Interactions

2008 ◽  
Vol 190 (21) ◽  
pp. 7079-7086 ◽  
Author(s):  
Guillaume Andre ◽  
Kees Leenhouts ◽  
Pascal Hols ◽  
Yves F. Dufrêne
Keyword(s):  
Single Molecule ◽  
Bimodal Distribution ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Promising Tool ◽  
Atomic Force ◽  
Bacterial Surfaces ◽  
Force Curves ◽  
Binding Forces ◽  
Detection And Localization

ABSTRACT The lysin motif (LysM) is a ubiquitous protein module that binds peptidoglycan and structurally related molecules. Here, we used single-molecule force spectroscopy (SMFS) to measure and localize individual LysM-peptidoglycan interactions on both model and cellular surfaces. LysM modules of the major autolysin AcmA of Lactococcus lactis were bound to gold-coated atomic force microscopy tips, while peptidoglycan was covalently attached onto model supports. Multiple force curves recorded between the LysM tips and peptidoglycan surfaces yielded a bimodal distribution of binding forces, presumably reflecting the occurrence of one and two LysM-peptidoglycan interactions, respectively. The specificity of the measured interaction was confirmed by performing blocking experiments with free peptidoglycan. Next, the LysM tips were used to map single LysM interactions on the surfaces of L. lactis cells. Strikingly, native cells showed very poor binding, suggesting that peptidoglycan was hindered by other cell wall constituents. Consistent with this notion, treatment of the cells with trichloroacetic acid, which removes peptidoglycan-associated polymers, resulted in substantial and homogeneous binding of the LysM tip. These results provide novel insight into the binding forces of bacterial LysMs and show that SMFS is a promising tool for studying the heterologous display of proteins or peptides on bacterial surfaces.

scholarly journals Single molecule mechanics of the kinesin neck

2009 ◽  
Vol 106 (17) ◽  
pp. 6992-6997 ◽  
Author(s):  
Thomas Bornschlögl ◽  
Günther Woehlke ◽  
Matthias Rief
Keyword(s):  
Single Molecule ◽  
Leucine Zipper ◽  
Structural Integrity ◽  
Mechanical Stability ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Force Microscopy ◽  
Atomic Force ◽  
Kinetics Of ◽  
High Mechanical Stability

Structural integrity as well as mechanical stability of the parts of a molecular motor are crucial for its function. In this study, we used high-resolution force spectroscopy by atomic force microscopy to investigate the force-dependent opening kinetics of the neck coiled coil of Kinesin-1 from Drosophila melanogaster. We find that even though the overall thermodynamic stability of the neck is low, the average opening force of the coiled coil is >11 pN when stretched with pulling velocities >150 nm/s. These high unzipping forces ensure structural integrity during motor motion. The high mechanical stability is achieved through a very narrow N-terminal unfolding barrier if compared with a conventional leucine zipper. The experimentally mapped mechanical unzipping profile allows direct assignment of distinct mechanical stabilities to the different coiled-coil subunits. The coiled-coil sequence seems to be tuned in an optimal way to ensure both mechanical stability as well as motor regulation through charged residues.

Atomic Force Microscopy (AFM) for Topography and Recognition Imaging at Single Molecule Level

2013 ◽  
pp. 102-112
Author(s):  
Memed Duman ◽  
Andreas Ebner ◽  
Christian Rankl ◽  
Jilin Tang ◽  
Lilia A. Chtcheglova ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Recognition Imaging

scholarly journals Atomic Force Microscopy Investigation of the Interactions between the MCM Helicase and DNA

Materials ◽  
2021 ◽  
Vol 14 (3) ◽  
pp. 687
Author(s):  
Amna Abdalla Mohammed Khalid ◽  
Pietro Parisse ◽  
Barbara Medagli ◽  
Silvia Onesti ◽  
Loredana Casalis
Keyword(s):  
Atomic Force Microscopy ◽  
Nucleic Acid ◽  
Single Molecule ◽  
Protein Complex ◽  
The Other ◽  
Contour Length ◽  
Force Microscopy ◽  
Atomic Force ◽  
Mcm Complex ◽  
Dna Double Helix

The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from Methanothermobacter thermautotrophicus by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. We discriminated between two modes of interaction, one in which the protein induces a sharp bend in the DNA, and one where there is no bending. We found that the presence of the MCM complex also affects the DNA contour length. A possible interpretation of the observed behavior is that in one case the hexameric ring encircles the dsDNA, while in the other the nucleic acid wraps on the outside of the ring, undergoing a change of direction. We confirmed this topographical assignment by testing two mutants, one affecting the N-terminal β-hairpins projecting towards the central channel, and thus preventing DNA loading, the other lacking an external subdomain and thus preventing wrapping. The statistical analysis of the distribution of the protein complexes between the two modes, together with the dissection of the changes of DNA contour length and binding angle upon interaction, for the wild type and the two mutants, is consistent with the hypothesis. We discuss the results in view of the various modes of nucleic acid interactions that have been proposed for both archaeal and eukaryotic MCM complexes.

Substrate Specificity of Sugar Transport by Rabbit SGLT1:  Single-Molecule Atomic Force Microscopy versus Transport Studies†

Biochemistry ◽  
2007 ◽  
Vol 46 (10) ◽  
pp. 2797-2804 ◽  
Author(s):  
Theeraporn Puntheeranurak ◽  
Barbara Wimmer ◽  
Francisco Castaneda ◽  
Hermann J. Gruber ◽  
Peter Hinterdorfer ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Substrate Specificity ◽  
Single Molecule ◽  
Sugar Transport ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transport Studies

Breaking Down the Supramolecular Ensemble: Single-Molecule Studies of the Concentration Dependence of Main-Chain Supramolecular Polymer Molecular Weight Distributions on Surfaces

2010 ◽  
Vol 63 (4) ◽  
pp. 624
Author(s):  
Michael J. Serpe ◽  
Jason R. Whitehead ◽  
Stephen L. Craig
Keyword(s):  
Molecular Weight ◽  
Single Molecule ◽  
Monomer Concentration ◽  
Supramolecular Polymer ◽  
Force Microscopy ◽  
Molecular Weight Distributions ◽  
Atomic Force ◽  
Multi Stage ◽  
Single Molecule Studies ◽  
Supramolecular Ensemble

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.

Sign in / Sign up

Export Citation Format

Share Document