Single-Molecule Force Spectroscopy - Essay Example

Name : xxxxxxxxxxx Institution : xxxxxxxxxxx Course : xxxxxxxxxxx Title : High-throughput single-molecule force spectroscopy and DNA: A Programmable Force Sensor for membrane protein Tutor : xxxxxxxxxxx @ 2009 High-throughput single-molecule force spectroscopy and DNA: A Programmable Force Sensor for membrane proteins Introduction Single-molecule force spectroscopy is majorly useful in solving the low efficiency in information acquisition during the measuring of the forces. This is very significant since the data is obtained comprehensively and thus allowing the precise information to be recorded (Foster 2009). On the other hand, DNA, programmer Force Sensor allows the unbinding forces that are needed in to break intermolecular bonds are measured using a differential format through a comparing with a reference bond that is known. Apart from the method leading to a notable sensitivity increase in addition to force resolution which makes it possible to resolve single base pair mismatches, this method also allows highly specific assays to be performed. This method is most applicable in solving the anti bodies cross reactions in a protein biochip application. Establishing high-throughput single-molecule force spectroscopy for membrane proteins Single-molecule force spectroscopy (SMFS) is a remarkable technique to study the mechanical characterization, unfolding pathways, intermolecular and intermolecular interactions, and energy landscapes of membrane proteins. SMFS procedures' has several steps. First, AFM imaging locates the membranes containing the integral protein of interest. Secondly, when the AFM tip is pushed into membrane proteins, the membrane proteins will retract after a short stopping; this creates curves force distances (F-D) which are recorded. Thirdly, when one termini of protein sticks to the cantilever strongly, it will be unfolded mechanically upon cantilever retraction. In this process, it can obtain data involving data coarse filtering to gain exact curves F-D that present unfolding event, categorization of the latter, the analysis of F-D curves depend on polymer chain models and their alignments. The ability of SMFS on membrane proteins is limited by low accuracy in data acquisition because not every F-D curves result in unfolding event. For this reason, high throughput (HT) procedure for SMFS technique has been developed. Until currently, fully automated SMFS protocols on membrane proteins have not been possible because proteins of interest, that are localized by AFM imaging are covered by lipid membrane and eventually manipulated before SMFS. For instance, in an area it can be revealed that force spectra can be collected by eliminating the upper layer of a collapsed vesicle. Consequently, imaging and sample manipulation are restricted steps in HT-SMFS towards fully automated procedure in membrane proteins. High-throughput single-molecule force spectroscopy for membrane proteins A semi automated high throughput (HT-SMFS) is a powerful protocol that is used to overcome the low efficiency in data acquisition. Data acquisition is not an easy task in HT-SMFS. For instance single curve takes about 1 second to get recorded whereas a complete data set that contains about 200 spectra difficult to get and the unfolding accuracy is less than 1%. Moreover, it can end up losing the relevant force spectra through manual recording associated with online filtering by the operator. Furthermore, several force spectra may be ignored by the operator. Thus, in order to achieve high efficiency in such data, HT-SMFS procedure is applied to extract F-D curves of thoroughly unfolding proteins. Methodology of HT-SMFS In this technique, semi-automated procedure was applied on membrane proteins which record the F-D curves and used off-line course filtering to obtain data. In addition, two dimensional (2D) crystals of the proton pump bacteriaorhodopsin (BR) from Halo bacterium salinarum were employed to act as a control experiment and to proteoliposomes of the L-arginine/agmatine antiporter AdiC from Escherichia coli (E. coli) as a test case. Then, the protein was pulled from N-terminus and from the C-terminus, respectively; it consequently acquired two types of force spectra demonstrated to proteins that unfolded. This was possible through using two different recombinant kinds of the AdiC protein that are different in the length of their termini. After that, it was recorded in data sets in the presence and in the absence of Larginine, D-arginine, and agmatine in order to present molecular interactions between AdiC and its substrates. Subsequently, it recorded approximately 400 000 F–D curves and acquired six data sets that involve the BR control, with roughly 200 force spectra in the presence and in the absence while in the BR, it obtained about 200 curves. Experiment 1- AFM was put on a damp table to reduce noise from the medium. 2- Cantilevers made of silicon nitride were used. 3- The deflection sensitivity (DS) was selected from the slope of D-F curves that recorded in bare mica. 4- Buffer solution from 150 KCl, 20mM Tris-HCl Ph 7, 8 (BR) and150 mM NaCl, and 20 mM citric acid pH 5 was used. 5- By using thermal method, the spring constant of the cantilevers was determined. 6- BR crystals were adsorbed through mica. 7- At room temperature, the mica in the buffer solution adsorbed lipid vesicles that contained AdiC. 8- To eliminate non-adsorbed membrane after adsorption, the same buffer was used to rinse the sample several times. 9- AdiC-substrate interaction was investigated by incubating vesicles that contained densely packed in substrate that contained buffer solution from and150 mM NaCl, 20 mM citric acid pH 5, 10 mM substrate (L-arginine, agmatine or D-arginine). This was performed for 30 minutes at 4°C. 10- At room temperature, mica adsorbed vesicle in the same substrate that contained buffer. 11- Contact mode atomic force microscopy was used before using force spectroscopy to locate proteoliposomes which were densely packed with AdiC. 12- To eliminate the upper layer of collapsed vesicle, the AFM tip was used. 13- A point grid (edge length of 150–300 nm) and a linear point (density .125 nm-1) were used to put on 2D BR crystals. 14- Ten measurements with force 1 nN for 0.1–0.6 s were applied at every point of grid by pushing the cantilever into the membrane proteins which were attached on the AFM tip. 15- The results in the retracted cantilever were 25 s with a velocity of 0.53 μm s−1 (BR) and 0.78 μm s−1 (AdiC). 16- F-D curves were recorded that contained 4096 data point with frequency of 16.4 kHz. Data and filtering analysis In this analysis, only the data of the F-D curve retraction was used. Additionally, the negative force spectrum was attributed to pushing force while a positive force spectrum was attributed to pulling force. Indeed, in order to obtain data set and to extract unfolding event in this procedure, data coarse filtering was used. Subsequently, a manual fine filtering was applied and classification was dependant on fingerprint pattern of AdiC that was obtained repetitively. As a result, it was the removed curves with unfolding that passed through coarse filtering. Finally, data acquisition of Acid unfolding traces until 200 was recorded. Result In this experiment it was found that the N-terminus attaches to the cantilever more than C-terminus because the former is more likely to be longer than latter. In fact, an area densely packed with protein was selected. The area was divided into 625 parts with 10 F-D curves whereby each produced 6250 F-D curves per position. In the filtering process, curves with negative forces were excluded and non-interpretable F-D curves that resulted in a smaller set of force spectra were maintained on good F-D curves. Actually, it allowed the recording of up to 400 00 curves per day and 99% of them were removed whereas useful data was kept. Noticeably, a large amount of data made it possible to select useful force spectra. Moreover, tags that are related to recombinants proteins were very beneficial during establishment of force spectra efficiency. The manual fine filtering was rejected roughly 70 % from the remaining F-D curves. This yielded one data set for BR with 398 spectra (unfolding event) in one day and five data sets for AdiC consisting 200 spectra for each one. As a result, HT-SMFS it was found that reduces the time needed to obtain a complete data due to unbiased data acquisition. In addition, it offers a large data set within a short time which includes information regarding mechanical stability of AdiC and topological information. Lastly, dynamic HT-SMFS can be used in determining the energy landscape for membrane proteins. DNA: A Programmable Force Sensor DNA: A Programmable Force Sensor is a quantitative technique used in computing forces between single molecules. In this procedure, the forces are exerted and measured with microscopic force sensors for instance the AFM cantilevers or beads in optical or magnetic traps. Here, the force resolution is only restricted by thermal changes but these are sensed by the force sensor. In order to get more precise results, a differential force format is used whereby rupture forces of the two molecular complexes are directly compared. The experiment was performed in a buffer solution having 150 mM NaCl at room temperature and this made the thermal off rates to be really low (30) and hence discrimination between mismatch and perfect match sequences is hard to get in convectional equilibrium binding assays. The stable thermal condition ensures that the data is not interfered with by spontaneous strand separation or hybridization efficiencies. The length and the sequence of the reference complex on the sample can be selected on each sample spot accordingly and thus permitting optimum force resolution and background discrimination for each spot. The combination of maximum resolution and local stringency plays a big role in getting exact quantification of interactions (Albrecht 2003). Experiment Here, the cantilever spring is replaced by a polymeric anchor and a known molecular bond which is the reference bond having a fluoresce label. The molecular bond being examined is directly compared to the reference bond which acted as a molecular force standard. During the separation of the two surfaces, the polymeric anchor got elongated and consequently the force acting along the molecular chain having the sample and the labeled reference complex developed suddenly until the time when the bond that was weak ruptured. This was as a result of the difference in stability between the two bonds which broke the symmetry in the experiment. Consequently, the great probability is that the fluorescence label ends up on the stronger side bond and not on the weaker side. Several single molecule force measurements can be done concurrently by use of two identical chip surfaces and different spots having the molecules of interest. Counting the labels on each side, offers a quantitative computation for the differences between the distributions of the bond rupture possibilities of the two molecular complexes. This is the same as measuring fluoresce intensities which are equal to the densities of fluoresce labels. There is a direct comparison of the rupture forces of the DNA strands with different hybridization lengths, a 20-bp duplex and a 25-bp duplex. The two oligonucleotides are joined with a conjugated 65-base oligonucleotide having a terminal fluorescent label. The resultant 20-bp duplex is then joined to an activated glass surface and the 25-bp duplex to a soft polydimetholsiloxane (PDMS) stamp which is between 26-28 using polyethylene glycol (PEG) spacers. Additionally, there are fluorescence images of the glass surface which has the capture oligonucleotide and the labeled sample oligonucleotide before the two surfaces were joined and separated and both the glass which is at the bottom and PDMS which is at the top after the two surfaces separated. Since the PDMS stamp has a grid pattern of trenches to simplify the water flux at the surface when the surfaces are separating, the labels that are transferred form a checkerboard pattern PDMS. There is no transfer that takes place within the trenches so the first label density is maintained on the glass surface whereas within the contact areas which are squared labels are transferred from the glass to the PDMS side (Albrecht 2003). Results In a convectional AFM based single molecule force spectroscopy, a rupture force was used to break a molecule bond and consequently measured using a blue cantilever spring. A differential rapture force follows whereby the rapture force of a red sample bond is measured through comparing it with a known reference bond which is the blue one and it acts as the molecular force standard. The chain of polymer spacers is loaded with sample bond and reference bond whereby the weaker bond possesses a higher probability of rupturing the force than the stronger bond (Albrecht 2003). Therefore, stronger bond ends up with most of the probed fluorescence labels which got a green label after the separation of the two surfaces. On the left, there is the Cy5 fluorescence image of a spot having the molecular chains of polymer spacers, sample and reference duplexes before linking the biotinylated reference duplexes to the second chip surface. At the center, there is Cy5 fluoresce image of both chip surfaces-miscrostructed PDMS which is at the top and a glass at the bottom which are also separated. On the right, there is a PDMS surface at a single molecule resolution and this is after separation of the two surfaces. Finally, the image is acquired through TIRF. Discussion In single molecule force spectroscopy, inter as well as intermolecular forces are exerted and measured using microscopic force sensors for example, like AFM cantilevers or beads in optical or magnetic traps. This therefore creates a room for some limitations which include low accuracy during data acquisition. DNA, programmer Force Sensor is more efficient in computing the forces since it has fewer limitations and has allowed use of single molecule mechanics. This is because the forces are merely limited by thermal fluctuations and these are detected by the force sensor hence almost nullifying almost everything that could interfere with the results. Moreover, the size of the sensor is a bit small and hence improves signal to noise ratio. Both methods made use of a buffer solution that contained 150 mM and in DNA, programmer Force Sensor it was aimed at lowering the thermal rates while in High-throughput single molecule force spectroscopy for membrane proteins the buffer solution was meant for adsorbing the lipid vesicles that contained AdiC. Single molecule force spectroscopy is dependent on the positive and negative forces which facilitate the separation while in DNA, programmer Force Sensor experiment the separation is facilitated by the instability between the bonds whereby the weaker bond gets ruptured. While in the first experiment a cantilever spring is used while in the second experiment this is replaced by a polymeric anchor and a known molecular bond which is the reference bond having a fluoresce label which allows the polymeric anchor elongate during separation and thus can allow several single molecule forces to be measured simultaneously unlike in the first experiment where forces cannot be computed concurrently. From the data provided, it is not possible to judge an approach that is better than the other. This is because both experiments have their own limitations. In Single-molecule force spectroscopy, the major limitation is low accuracy while acquiring data and this can be solved by making use of high throughput method in the technique. In DNA: A Programmable Force Sensor, the obvious limitations are likely to be brought by diverse optical and chemical characteristics of both glass chip and PDMS stamp which affect the quantum yield and the excitation effectiveness of the label. Furthermore, the joining efficiencies to the two chips could vary. Consequently, this should be surmounted through placing both test molecules on the same side of the assay and computing both against a common reference on the other side. Again, the approaches make use of the same scientific approach which is dependant on the forces. Moreover, both experiments are dependant of different forces and so each approach is specific in a way. For instance, the first approach is dependant on the pushing force and pulling force which results into force spectrum which is then used to yield the results. On the other hand, the second approach is also dependant of some rupture forces which eventually yield the results. This depends on the separation that takes place on the two surfaces whereby polymeric anchor gets stretched and thus the force is exerted making the weak bond to be ruptured. The force measurements can then be measured. Finally, both techniques can be used concurrently to come up with comparative results. Conclusion Both approaches allowed the used of automated measurement of large numbers of forces in a molecule. In DNA, programmer Force Sensor experiment, in every force spectrum, sole peaks was detected which led to contour lengths in addition to rupture forces. The approach allowed the observation of various interaction pathways of many molecules and thus giving the required data. Like wise, high-throughput single-molecule force spectroscopy permits the measurement of molecular forces that are responsible of stabilizing single proteins or even antibody – antigen binding. Thus both approaches are in a position to give the required data to compute the forces. Still, both approaches are automated and have different ways of counteracting the limitations and hence it is difficult to point out the approach that is better than the other. Bibliography Albrecht, C., 2003, DNA: A Programmable Force Sensor, Science, and Vol 301 / 367. Foster, S., 2009, Iron-Regulated Surface Determinant Protein a Mediates Adhesion of Staphylococcus aureus to Human Corneocyte Envelope Proteins. Infect. Immune. 77: 2408-2416 Read More