Force‐induced globule–coil transition in laminin binding protein and its role for viral–cell membrane fusion

The specific interactions of the pairs laminin binding protein (LBP)–purified tick‐borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single‐molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves‐based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus–cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus–cell membrane fusion, SMDFS data reveal the existence of a force‐induced (effective already for forces as small as 30–70 pN) sharp globule–coil transition for LBP and LBP–fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first‐order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus–cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF₁₆₅ was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF₁₆₅ was dependent on the concentration of VEGF₁₆₅. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF₁₆₅ complexes was revealed, which was similar to that of the intact aptamer/VEGF₁₆₅. Besides, the dissociation rate constant (k_off) of split aptamer/VEGF₁₆₅ was close to that of intact aptamer/VEGF₁₆₅, and the interaction force of split aptamer/VEGF₁₆₅ was higher than the force of intact aptamer/VEGF₁₆₅. It indicated that split aptamer also possessed high affinity with VEGF₁₆₅. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level.     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Elucidation of the effect of aptamer immobilization strategies on the interaction between cell and its aptamer using atomic force spectroscopy

The immobilization strategy of cell‐specific aptamers is of great importance for studying the interaction between a cell and its aptamer. However, because of the difficulty of studying living cell, there have not been any systematic reports about the effect of immobilization strategies on the binding ability of an immobilized aptamer to its target cell. Because atomic force spectroscopy (AFM) could not only be suitable for the investigation of living cell under physiological conditions but also obtains information reflecting the intrinsic properties of individuals, the effect of immobilization strategies on the interaction of aptamer/human hepatocarcinoma cell Bel‐7404 was successively evaluated using AFM here. Two different immobilization methods, including polyethylene glycol immobilization method and glutaraldehyde immobilization method were used, and the factors, such as aptamer orientation, oligodeoxythymidine spacers and dodecyl spacers, were investigated. Binding events measured by AFM showed that a similar unbinding force was obtained regardless of the change of the aptamer orientation, the immobilization method, and spacers, implying that the biophysical characteristics of the aptamer at the molecular level remain undisturbed. However, it showed that the immobilization orientation, immobilization method, and spacers could alter the binding probability of aptamer/Bel‐7404 cell. Presumably, these factors may affect the accessibility of the aptamer toward its target cell. These results may provide valuable information for aptamer sensor platforms including ultrasensitive biosensor design. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigation of the interactions between aptamer and misfolded proteins: From monomer and oligomer to fibril by single‐molecule force spectroscopy

Increasing knowledge on the understanding interactions of aptamer with misfolded proteins (including monomer, oligomer, and amyloid fibril) is crucial for development of aggregation inhibitors and diagnosis of amyloid diseases. Herein, the interactions of lysozyme monomer–, oligomer‐, and amyloid fibril–aptamer were investigated using single‐molecule force spectroscopy. The results revealed that the aptamer screened against lysozyme monomer could also bind to oligomer and amyloid fibril, in spite of the recognition at a lower binding probability. It may be attributed to the inherent structural differences of misfolded proteins and the flexible conformation of aptamer. In addition, dynamic force spectra showed that there were similar dissociation paths in the dissociation process of lysozyme monomer–, oligomer‐, and amyloid fibril–aptamer complexes. It showed that the dissociation only passed 1 energy barrier from the binding state to the detachment. However, the dynamic parameters suggested that the oligomer‐ and amyloid fibril–aptamer were more stable than lysozyme monomer–aptamer. The phenomena may result from the exposure of aptamer‐recognized sequences on the surface and the electrostatic interactions. This work demonstrated that single‐molecule force spectroscopy could be a powerful tool to study the binding behavior of the aptamer with misfolded proteins at single‐molecule level, providing abundant information for researches and comprehensive applications of aptamer probes in diagnosis of amyloid diseases.     

Modulation of protein–ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3‐kinase SH3 domain as model system

To photomodulate the interaction of the phosphatidylinositol 3‐kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic‐1) with a photolabile side chain‐to‐side chain linker was synthesized. The conformation of cyclic‐1 differs from that of the parent linear peptide, but becomes identical by UV‐irradiation. Accordingly, the binding affinity of cyclic‐1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K_d: 3.4 ± 1.7 and 0.9 ± 0.3 mM , respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide–protein interactions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Moderate disturbances accelerate forest transition dynamics under climate change in the temperate–boreal ecotone of eastern North America

Several temperate tree species are expected to migrate northward and colonize boreal forests in response to climate change. Tree migrations could lead to transitions in forest types, but these could be influenced by several non‐climatic factors, such as disturbances and soil conditions. We analysed over 10,000 forest inventory plots, sampled from 1970 to 2018 in meridional Québec, Canada, to identify what environmental conditions promote or prevent regional‐scale forest transitions. We used a continuous‐time multi‐state Markov model to quantify the probabilities of transitions between forest states (temperate, boreal, mixed, pioneer) as a function of climate (mean temperature and climate moisture index during the growing season), soil conditions (pH and drainage) and disturbances (severity levels of natural disturbances and logging). We further investigate how different disturbance types and severities impact forests' short‐term transient dynamics and long‐term equilibrium using properties of Markov transition matrices. The most common transitions observed during the study period were from mixed to temperate states, as well as from pioneer to boreal forests. In our study, transitions were mainly driven by natural and anthropogenic disturbances and secondarily by climate, whereas soil characteristics exerted relatively minor constraints. While major disturbances only promoted transitions to the pioneer state, moderate disturbances increased the probability of transition from mixed to temperate states. Long‐term projections of our model under the current environmental conditions indicate that moderate disturbances would promote a northward shift of the temperate forest. Moreover, disturbances reduced turnover and convergence time for all transitions, thereby accelerating forest dynamics. Contrary to our expectation, mixed to temperate transitions were not driven by temperate tree recruitment but by mortality and growth. Overall, our results suggest that moderate disturbances could catalyse rapid forest transitions and accelerate broad‐scale biome shifts.