Gelatin is an important natural biological resource with a wide range of applications in the pharmaceutical, industrial and food industries. We investigated the single-chain behaviors of gelatin by atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS), and found that gelatin exists as long chains by fitting with the M-FJC model. By comparing the single-chain elasticity in a nonpolar organic solvent (nonane) and DI water, it was surprising to find that there was almost no difference in the single-chain elasticity of gelatin in nonane and DI water. Considering the specificity of gelatin solubility and the solvent size effect of nonane molecules, when a single gelatin chain is pulled into loose nonane, dehydration does not occur due to strong binding water interactions. Gelatin chains can only interact with water molecules at high temperatures; therefore, no further interaction of single gelatin chains with water molecules occurred at the experimental temperature. This eventually led to almost no difference in the single-chain F-E curves under the two conditions. It is expected that our study will enable the deep exploration of the interaction between water molecules and gelatin and provide a theoretical basis and experimental foundation for the design of gelatin-based materials with more functionalities.

Angiotensin II type 1 receptor (AT1R), a typical G protein-coupled receptor, plays a key role in regulating many cardiovascular functions. Different ligands can bind with AT1R to selectively activate either G protein (Gq) or β-arrestin (β-arr) pathway, or both pathways, but the molecular mechanism is not clear yet. In this work, we used, for the first time, atomic force microscopy-based single molecule force spectroscopy (SMFS) to study the interactions of AT1R with three types of ligands, balanced ligand, Gq-biased ligand, and β-arr-biased ligand, in living cells. The results revealed their difference in binding force and binding stability. The complex of the Gq-biased ligand-AT1R overcame two energy barriers with an intermediate state during dissociation, whereas that of β-arr-biased ligand-AT1R complex overcame one energy barrier. This indicated that AT1R had different ligand-binding conformational substates and underwent different structural changes to activate downstream signaling pathways with variable agonist efficacies. Quantitative analysis of AT1R-ligand binding in living cells at the single-molecule level offers a new tool to study the molecular mechanism of AT1R biased activation. Graphical Abstract Single-molecule force measurement on the living cell expressing AT1R-eGFP with a ligand modified AFM tip (left), the dynamic force spectra of β-arrestin biased ligands-AT1R (middle), and Gq-biased ligands-AT1R (right). The complexes of β-arr-biased ligand-AT1R overcame one energy barrier, with one linear region in the spectra, whereas the Gq-biased ligand-AT1R complexes overcame two energy barriers with two linear regions.

TGF-β/Smad signaling pathway triggers diverse cellular responses among different cell types and environmental conditions. Quantitative analysis of protein-protein interactions involved in TGF-β/Smad signaling is demanded for understanding the molecular mechanism of this signaling pathway. Live-cell single-molecule microcopy with high spatiotemporal resolution is a new tool to monitor key molecular events in a real-time manner. In this review, we mainly presented the recent work on the quantitative characterization of TGF-β/Smad signaling proteins by single-molecule method, and showed how it enabled us to obtain new insights about this canonical signaling process.

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.

Membrane proteins are embedded in lipid bilayers and facilitate the communication between the external environment and the interior of the cell. This communication is often mediated by the binding of ligands to the membrane protein. Understanding the nature of the interaction between a ligand and a membrane protein is required to both understand the mechanism of action of these proteins and for the development of novel pharmacological drugs. The highly hydrophobic nature of membrane proteins and the requirement of a lipid bilayer for native function have hampered the structural and molecular characterizations of these proteins under physiologically relevant conditions. Atomic force microscopy offers a solution to studying membrane proteins and their interactions with ligands under physiologically relevant conditions and can provide novel insights about the nature of these critical molecular interactions that facilitate cellular communication. In this review, we provide an overview of the atomic force microscopy technique and discuss its application in the study of a variety of questions related to the interaction between a membrane protein and a ligand. This article is part of a Special Issue entitled: Structural and biophysical characterization of membrane protein-ligand binding.