What Are The Benefits Of Single Molecule Biophysics Biology Essay


Single molecule experiments provide a lot of molecular information. However, in conventional ensemble (molecule) experiments, large numbers of molecules are studied simultaneously and average properties are observed. The powerful capabilities of these single molecular experiments helps scientists solve the unanswerable questions in physical, chemical and biological sciences. The first aim of single molecule biophysics is to develop and improve the single molecule technology and supporting methodologies, the second is to use this concept in various scientific fields. There are a variety of reasons which make single molecule methods especially attractive and powerful for the study of various complex and biological processes.

First, in single molecule experiments, molecular properties are studied one molecule at a time. But, in case of conventional experiments, distributions in molecular properties are more directly measured. In modern single molecule experiments rare states are also detected, which would be averaged in conventional methods. Second, dynamics of systems are observed for small molecules under equilibrium conditions. Single molecule experiments easily study the various complex systems like the motion of DNA enzymes along their tracks. The folding and unfolding and kinetic rate constants between various states of a system are easily studied by using single molecule techniques. In addition to these, direct measurements of molecular forces are also molecular structural and various responses to mechanical manipulation and control is easily studied by single molecule manipulation. This review aims to briefly describe the capabilities of single molecule force measurements, which can be used to investigate the relative strengths of bio molecular receptor-ligand interactions.

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High-throughput single molecule force spectroscopy for membrane proteins

The Atomic force microscope has become a versatile tool for imaging the surface topography of biological samples at a very good resolution. Atomic force microscopy-based single molecule force spectroscopy (SMFS) is a powerful tool to study the intermolecular and intramolecular interactions, mechanical properties, unfolding pathways and energy landscapes of membrane proteins. SMFS unfolding curves have a saw-tooth pattern appearance that is characteristic of individual proteins. However, because of lack of covalent catenation between the proteins forming the complex, the complete unfolding pathway of an entire protein cannot be studied. Low efficiency for the data acquisition is the one limiting factor for the large scale applicability of SMFS on membrane proteins. In their method they developed a high-throughput SMFS (HT-SMFS) for efficient data acquisition. In addition to these they use a coarse filter to efficiently extract protein unfolding events from large data sets. They validated their procedure and the filter by using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. The structure for BR has been elucidated and the structure of AdiC is unknown. In their setup they recorded data sets in the absence and in the presence of L-arginine, D-arginine and agmatine to screen the molecular interactions between AdiC and its substrates. In all Bosshart et al. recorded ~400000 force-distance curves and by using their coarse filtering technique to this whole set of data they yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. They took only one to two days to acquire raw data for most of the data and this opens new perspectives for HT-SMFS applications.

The procedure for the SMFS experiment is divided into various steps. First, AFM imaging is done for membranes to localise the integral protein of interest. Then, force-distance (F-D) curves are recorded by pushing the AFM tip into the sample and retracting it after sometime. If one of the termini of the protein adheres strongly with the cantilever, it can be mechanically unfolded upon cantilever retraction. Various steps involved in data processing are (i) data coarse filtering to extract F-D curves that show unfolding events (ii) classification (iii) alignment of the F-D curves, and (iv) their analysis based on polymer chain models.

In their approach they first started with validation of data acquisition and for the filtering process. They checked for the change in deflection sensitivity by manually adjusting the laser to the original position of the photodiode. Cantilever deflection noise and force noise against force spectrum number were also extracted from F-D curves taken on densely packed AdiC membrane. The force noise (σF) obtained showed that real deflection noise of cantilever was between 10 to 18 pN. A comparable drift was observed after repositioning the laser beam, which highlights the reproducible character of the DS change. Long-term drift was exhibited after monitoring the cantilever deflection noise. But the force noise which was calculated for the same F-D curve does not exhibit a drift over time. This shows that forces calculated by their approach are not impaired by long-term drifts. To validate the coarse filter two sets of filtering parameters were used. First, filtering was done for AdiC F-D curves by taking tip-sample separation (tss) as 95nm and minimal pulling force over a certain threshold (FSMFS,thr) as 3.5 σF which results in import of 190 F-D curves out of 22000 curves. During the filtering process they removed the curves where the tip was contaminated. Further filtering was done for the same set of data by using different values for the two parameters and 135 curves was imported. Later by manual filtering 75 force spectra of N-terminally unfolded AdiC were extracted. BR data sets were also filtered by considering different values for the parameters.

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In next step HT-SMFS procedures are used to study the mechanical unfolding of bacterial L-arginine/agmatine antiporter AdiC. Two classes of F-D spectra was obtained from unfolding of N-His6-AdiC, both classes of the F-D spectra had similar length which shows complete unfolding of proteins. The major class of spectra was obtained from N-terminus because it is ~2.6 times longer than the C-terminus. The adhesion of protein to the tip was observed to be pH dependent for both terminals because pH induced protonation of the N- or C- terminal. After obtaining the exemplary force spectrum worm-like-chain (WLC) curves were fitted to all peaks to calculate the contour length, LC , measured in number of amino acids of the unfolded structural elements. Unfolding experiments were performed until ~200 F-D spectra were obtained for AdiC. Scattering plots between force (pN) and tip-sample separation (nm) and contour length histograms between occurrence and contour length (aa) were constructed for BR, N-His6-AdiC and AdiC-His6-C without substrate. Different filtering parameters were used to obtain the scattering plot and length histograms. Later scattering plots and contour length histograms were obtained for N-His6-AdiC and AdiC-His6-C in the presence of L-arginine, agmatine and D-arginine. After that, analysis for the main conserved unfolding events was done to check whether forces in N-His6-AdiC changed in the presence of substrate or not. No major changes were observed in the scattering plots. The analysis of occurrence and average forces stabilising the unfolded structural regions did not reveal major changes either.

Structure prediction is also an important feature of HT-SMFS. After gathering most of the information from the experiment it is easy to design the recombinant structure for N-His6-AdiC and AdiC-His6-C proteins.

Figure 1. Structure predictions and locations of various unfolding barriers in the unfolding of (a) N-His6-AdiC and (b) AdiC-His6-C. Residues highlighted with numbered large circles showing the position of unfolding barriers. Arrows in the termini show the pulling direction. Thick arrows show the change in the position of the unfolding barriers which make up for the thickness of the membrane.

DNA- A programmable force sensor

Single molecule force spectroscopy experiments were carried out in which unbinding forces required to break intermolecular bonds are measured by comparison with a reference bond in a differential format. The reference bond used here is a short DNA duplex, carrying a fluorescent label. In their setup, Albrecht et al. replaced the AFM spring cantilever by a polymeric anchor and the reference bond served as a molecular force standard. On pulling apart the two surfaces, the polymeric anchor was stretched and the force builds up between the molecular chain consisting of the sample and the labelled reference complex, until the weaker bond ruptured. There is always a higher probability for the reference bond to end up as a stronger bond rather than a weaker one. This method is similar to 1-bit analog-to-digital conversion broadened by thermal fluctuations. Many single-molecule force measurements can be carried out simultaneously, using two concurrent chip surfaces and various spots containing different molecules of interest. Single-molecule optics provides the quantitative measure for the differences between the distributions of force rupture probabilities for two molecules. In their setup they compared the rupture force of two DNA strands of different hybridisation lengths one was 20-bp duplex and another 25-bp duplex. 65-base oligonucleotide was used to bridge both oligonucleotides which were labelled using a CY5 fluorescent label. The resulting 25-bp duplex was coupled to a soft polydimethylsiloxane (PDMS) stamp and 20-bp duplex to an activated glass surface, both by means of polyethylene glycol (PEG) spacers. The quantum yield and excitation efficiencies of the two chip surfaces may differ due to different optical properties and chemical properties of glass chip and PDMS stamp. The coupling efficiency of the two surfaces may differ. But, the symmetry of the experiment was maintained by placing the two molecules of interest on the same side of the assay and measuring both against a common reference on the other side. This format is chosen when the single base pair mismatches and different binding modes of DNA were investigated.

They studied the reduction of the unbinding forces caused by single base pair mismatch in a 20-bp DNA duplex to study the force resolution of the differential force test. Histograms were obtained from 20-bp DNA duplex and 20-bp DNA duplex that has a single base pair mismatch (MM). Both duplexes were probed with a 20-bp reference complex which is reverse to the perfectly matching 20-bp duplex. Both duplexes are identical, except for a single base mutation (G→C) which was introduced at position 13 of capture oligonucleotide. They carried this experiment in a buffer solution of 150 mM Nacl at room temperature. Thermal off-rates are extremely low at these conditions and discrimination between two sequences is difficult to obtain in conventional equilibrium binding assays. This high thermal stability in force based assay ensured that the data are not obscured by spontaneous strand-separation events.

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In conventional DNA chips, single base pair mismatches are detected by identifying differences in the equilibrium constant or thermal off-rate. In both these cases, stringent conditions are established by reducing the salt concentrations or by increasing the temperature. By doing these DNA duplexes to be analysed either dissociate at different time scales or bind with distinguishable binding ratios. However, in the differential force method, strictness imposed by the reference complex is a local boundary condition. To allow optimum force resolution and background discrimination for every sample, spot sequence and length of the reference complex chosen accordingly. Thermal stringency is global and mechanical stringency is local. The combination of maximum resolution and local stringency is desirable for the precise quantification of interactions. Another additional and unique feature of force based assays is the discrimination among energetically and kinetically equivalent interactions. In this experiment they studied the two hybrids; one in shear geometry and the one in unzip geometry. Both had the same sequences and therefore had the same binding energies and thermal on-rates and off-rates. But after force dissociation, the histograms showed that the complex in unzip geometry has a 15 times higher probability of rupturing then the complex in shear geometry.

The discrimination between different binding modes and the concept of mechanical stringency offers interesting advantages in the field of protein arrays. It is necessary in this field to discriminate between specific and nonspecific interactions, but at the same time it is difficult to define a common set of stringent ambient conditions for many proteins. They achieved discrimination between specific and nonspecific binding sites for a variety of antibody-antigen interactions by using a low-force but high affinity force sensor, such as a DNA duplex in unzip conformation. In this method, affinity is high enough to provide a stable anchor and antibodies are safely delivered to their respective antigens.


In the HT-SMFS, method data acquisition and pre-processing for BR and AdiC is automated to a large extent. This procedure is validated by using BR, a membrane protein which has been extensively characterised by force spectroscopy and their procedure yields the same results as that of previously published results. In their procedure, they obtained a data set containing ~400000 F-D curves, which were later corrected for variations in cantilever deflection sensitivity and filtered by using various values for two parameters to select ~4000 F-D curves. By manual selection ~1400 interpretable spectra was obtained with high efficiency. In a single day by using semi-automated data acquisition and processing protocol, a relevant set of data can be obtained for a single sample. Therefore, HT-SMFS can be performed speedily and with small amounts of sample. The method of coarse filtering can also be applied to F-D curves recorded with an open-loop piezoscanner. After obtaining the unfolding spectra for AdiC in the absence and presence of substrates, it appears that AdiC and its substrates interact only weakly. The HT-SMFS approach can be applied to a wide range of mutants and various membrane proteins to study their folding and unfolding rates and ligand binding. Interaction of transport proteins with substrates and inhibitors and the protein behaviour under different environmental conditions such as pH, ionic strength and temperature can be easily studied by HT-SMFS. Structure predication can be done for an unknown molecule. Energy landscape of membrane proteins are also explored by using semi-automated HT-SMFS procedure for dynamic SMFS experiments. The drawback of this approach is that it is not fully automated and human interference is required to extract the desired curves. AFM image is taken to select the desired surface which sometimes damages the surface and single-molecule force measurements cannot be performed simultaneously.

In DNA: A programmable force sensor method AFM was used but AFM cantilever was replaced by a polymeric anchor which is helpful in the removal of cantilever deflection and the drift. By using two congruent surfaces and different surfaces containing the molecules of interest, many single-molecule force measurements can be performed simultaneously. In this setup they used a differential measurement format, where rupture forces of two molecules are studied together. This measurement format has various advantages, one of which is that most external disturbances will cancel out due to the high symmetry of the assay and mismatch detection in a DNA sequence. Due to different optical and chemical properties of the sample, direct quantification of the fluorescent label is limited. Coupling efficiency of the two surfaces also differs. Discrimination between two binding sites of a molecule is also possible by the help of this approach. By using this force sensor method, antibodies can be safely delivered to their respective antigens.


Single molecule biophysics is a fast growing field and single-molecule force measurements are widely used to study the receptor-ligand interactions. In this paper two techniques for single-force measurements are discussed. But, out of these DNA: A programmable force sensor technique is found to be more accurate and precise than HT-SMFS. In both the techniques AFM was used but in DNA: A programmable force sensor cantilever was replaced by a polymeric anchor due to which there is no possibility for drift and deflection. AFM imaging of the sample sometimes causes damage to the sample in HT-SMFS. In HT-SMFS technique human interference is required for the extraction of curves which makes it semi-automated and time consuming then another approach. Many single-force measurements can be carried out in DNA: A programmable force sensor approach. Sometimes during the filtering process in HT-SMFS few unfolding curves are also filtered. There are some weak points for DNA: A force sensor like difference in the properties of the two surfaces leads to direct quantification of fluorescent label but these can be overcome easily by using same surfaces. By using DNA: A programmable force sensor antibodies can be safely delivered to their respective antigens and discrimination between two binding sites is possible which has wide applications in protein arrays. Advancements are required in case of HT-SMFS to make it more accurate and less time consuming.