Bin Wang

Transporting individual molecules across cell membranes is a fundamental process in cellular metabolism. Although the crystal diffraction technique has greatly contributed to our understanding of the structures of the involved transporters, a description of the dynamic transport mechanism at the single-molecule level has been extremely elusive. In this study, we applied atomic force microscopy (AFM)-based force tracing to directly monitor the transport of a single molecule, D-glucose, across living cell membranes. Our results show that the force to transport a single molecule of D-glucose across cell membranes is 37 ± 9 pN, and the corresponding transport interval is approximately 20 ms, while the average speed is approximately 0.3 μm s⁻¹. Furthermore, our calculated force profile from molecular dynamics simulations showed quantitatively good agreement with the force tracing observation and revealed detailed information regarding the glucose transport path, indicating that two salt bridges, K38/E299 and K300/E426, play critical roles during glucose transport across glucose transporter 1 (GLUT1). This role was further verified using biological experiments that disrupted these two bridges and measured the uptake of glucose into the cells. Our approaches led to the first unambiguous description of the glucose transport process across cell membranes at the single-molecule level and demonstrated the biological importance of the two salt bridges for transporting glucose across GLUT1.

The emerging nanotechnologies have greatly facilitated the development of label-free biosensors. The atomic force microscopy (AFM) has been used to study the molecular mechanism of the reactions for protein and aptamers. The surface plasmon resonance (SPR) have been used in fast detections of various pathogens such as bacteria. This study used both AFM and SPR to investigate the complex reactions between aptamers and outer membrane proteins (OMPs) on the surface of S. typhimurium. Two DNA aptamers were used for the label-free detections of S. typhimurium by AFM and SPR. The aptamers have specific binding affinities to the OMPs of S. typhimurium. At single-molecule level, the high resolution AFM topography and recognition images distinguished the OMPs on the bacteria surface, which is the first time the location of individual outer membrane protein have been determined on Salmonella surface. E. coli in the control experiments didn’t generate recognition signals, which proved the specificity of these two aptamers to S. typhimurium. The off-rate values for the interactions of these two aptamers to the OMPs were estimated as 5.2 × 10−3 and 7.4 × 10−3 s−1, respectively, by the AFM dynamic force microscopy (DFS). The force and extension values form DFS measurements were used to distinguish the two aptamers. The surface membrane model was proposed to explain the complex correlations among force and extension values. Next, these two aptamers were used in the bulk solution detections of S. typhimurium. The gold chips in SPR experiments were modified with carboxymethylated-dextran (CD), followed by aptamers immobilization, to reduce the non-specific binding signals. The limit of detection (LOD) was determined as 3 × 104 CFU mL−1. The AFM single-molecule study revealed detailed information about the unbinding force and extension of the aptamer in complex biological reactions. The careful analysis of the experimental results provide better understanding of the molecular mechanism of OMPs reactions. The single-molecule measurements are helpful in evaluating the specificity of binding reagents, such as aptamers, in bulk solution detections. The protocols used in the SPR detections can be expanded into the label-free detections of other bacterial pathogens.

The aggregates of the full-length human recombinant prion protein (PrP) (23–231) on model membranes were investigated by combining the atomic force microscopy (AFM) measurements and theoretical calculations at pH 5.0, showing the great effect of PrP concentration on its supramolecular assemblies on the lipid bilayer.

Understanding the correlation between structure and charge transport properties of a DNA-based single molecule device is crucial to the creation of nanoscale functional devices. Now, a DNA-based molecular rectifier with a high rectification ratio has been constructed by site-specific intercalation of small molecules (coralyne) into native double-stranded DNA.

The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

Aggregations of human prion protein (23–231) were monitored by atomic force microscopy in real-time under pH 4. Prion dimers and trimers were determined as the basic units by AFM images and simulated structures. Aggregates aligned with the herringbone structures of an Au(111) reconstructed surface via Au–S bonds as the first layer, while the second layer was formed by non-covalent interactions.

We used atomic force microscopy (AFM) and surface plasmon resonance (SPR) to study the surface conformations of an anti-ricin aptamer and its specific binding affinity for ricin molecules. The effect of surface modification of the Au(111) substrate on the aptamer affinity was also estimated. The AFM topography images had a resolution high enough to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The aptamer on a Au(111) surface modified with carboxymethylated-dextran (CD) showed both similarities to and differences from the one without CD modification. The influence of CD modification was evaluated using AFM images of various aptamer conformations on the Au(111) surface. The affinity between ricin and the anti-ricin aptamer was estimated using the off-rate values measured using AFM and SPR. The SPR measurements of the ricin sample were conducted in the range from 83.3 pM to 8.33 nM, and the limit of detection was estimated as 25 pM (1.5 ng mL⁻¹). The off-rate values of the ricin–aptamer interactions were estimated using both single-molecule dynamic force spectroscopy (DFS) and SPR as (7.3 ± 0.4) × 10⁻⁴ s⁻¹ and (1.82 ± 0.067) × 10⁻² s⁻¹, respectively. The results show that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single-molecule measurements, the various conformations of the aptamer immobilized on the gold surface determined the availability of each specific binding site to the ricin molecules. The SPR bulk solution measurements averaged the signals from specific and non-specific interactions. AFM images and DFS measurements provide more specific information on the interactions of individual aptamer and ricin molecules.

We studied the binding kinetics of family 3 carbohydrate-binding module (CBM3a) molecules to crystalline cellulose fibrils extracted from the poplar cell wall by atomic force microscopy (AFM) recognition imaging. The free CBM3a molecules of different concentrations were added to the buffer solution to bind to the crystalline cellulose sample immobilized on the AFM substrate. During in-situ AFM imaging, the CBM molecules were observed to bind to cellulose efficiently and regularly, especially in the first 60–120 min. A 1:1 single-molecule binding model was used to study the kinetics of the CBM3a–cellulose interaction. The saturation time when the concentration of occupied binding sites is 99% of the maximum bound CBM3a concentration at the end of reaction, t_(0.99), was determined by fitting different concentrations of CBM3a against reaction time using the high resolution AFM images and the single-molecule kinetics equations. Based on the experimental data and kinetics calculations, the minimal effective initial CBM3a concentration was estimated to be 5.1 × 10^–7 M at 287 min reaction time. This study provides an in-depth understanding of the binding mechanism of CBM with crystalline cellulose at single molecule level.