Single-Molecule Force Approaches for Experiments - Essay Example

Name : xxxxxxxxxxx Institution : xxxxxxxxxxx Course : xxxxxxxxxxx Title : Assessment single molecule force approaches for two experiments Tutor : xxxxxxxxxxx @2010 Assessment single molecule force approaches for two experiments Introduction Measurement of the molecular forces has been a key research subject for a long time. Several methods have been developed to compute these forces and in this report High-throughput single-molecule force spectroscopy and DNA: A Programmable Force Sensor approaches will be analysed in their application to measuring molecular forces (Bosshart 2008). Atomic force microscope has turned out to be a versatile method of imaging the surface topography of bio molecules at lateral resolution of 5 A and a vertical resolution of 1 under almost local circumstances. Atomic force microscopy based single molecule force spectroscopy is a potent tool assessing the mechanical characteristics, intermolecular as well as intramolecular interactions, unfolding pathways and energy on membrane proteins. High-throughput single-molecule force spectroscopy for membrane proteins has been developed since it has higher efficiency for acquiring data. This approach presents a coarse filter which allows an efficient extraction of protein unfolding events from large information sets. In HT-SMFS approach and the coarse filet were authenticated with the use of the proton pump bacteriorhodopsin (BR) from Halobacterium solinarum and the L-arginine/agmatine antipoter from bacterium Escherichia coli (Bosshart 2008). DNA: A Programmable Force Sensor is a quantitative technique used in measurement of forces between particular molecules. In this process, the forces are applied and measured with microscopic force sensors for example the AFM cantilevers or beads in optical or magnetic traps. In the approach, the force resolution is only restricted by thermal changes but these are sensed by the force sensor. In order to get more exact results, a discrepancy force format is used whereby rupture forces of the two molecular complexes are openly compared (Albrecht 2003). In this approach, it the difference between the two bond that leads to the separation of the bonds and consequently the generation of the rupture forces which are measured to compute the forces between the two bio molecules. These two approaches have their own limitations which limit the approach and some of them include low accuracy while acquiring data in Single-molecule force spectroscopy while in DNA: A Programmable Force Sensor, the limitations are as a result of varied optical and chemical characteristics of both glass chip and PDMS stamp which have an effect on the quantum yield and the excitation efficiency of the labelled molecule (Albrecht 2003). These limitations can be resolved in order to get precise results and allow perfect acquisition of data. High-throughput single-molecule force spectroscopy for membrane proteins There are different steps when using MFS on membrane protein. After putting the tip of AFM above membrane proteins and then retracting the tip, it is possible to record the force- distance (C-D). The data of this process contains some data which are coarse filtering to obtain F-D curves. There is then, alignment and analysis of F-D curves which dependant of polymer chain. The large scale of SMFS on membrane protein is limited because all F-D curves do not contribute to unfolding event. The estimation of unfolding in the bacterial sodium proton antiporter was approximately 3%. It is therefore difficult for the whole automated procedure of SMFS on membrane protein to be feasible. The significant reasons for that is that "lipid membranes containing the protein of interest have first to be located by AFM images and controlled before SMFS. When removing some layer, it can be possible to display the area of force spectra and this area is recorded. The image and the sample which are manipulated by an AFM to force spectroscopy restrict steps towards the procedure of fully automated HT-SMFS for membrane proteins. In the past, automated alignment and also pattern recognition algorithms have been explained but until now the data recording and coarse filtering has not been perfect. There are some bottlenecks of data acquisition towards HT-SMFS for example the record of single F-D curve obtains around 1 second. Moreover, the recording which contains online filtering not only contribute to loss of some relevant force spectra but it can be also decreased the efficiency of the data acquisition. HT-SMFS techniques is used in semi-automated to record F-D curves and it can be used to record the off line coarse filtering. This procedure was introduced by the application of HT-SMFS to two diminution crystals of the proton pump bacteriorhodopsin (BR), which are taken from Halobacterium Salinarum, and also from proteoliposomes of the L-arginine/agmatine antiporter AdiC. In this procedure, BR is used as a control because it is well characterized and also the AdiC protein is employed as a test in HT-SMFS technique. The two major categories of force spectra were taken for AdiC, which is type of the bacterial acid resistance mechanism and an element of the amino acid/polyaminem/organocation transporter superfamily, and ascribed to proteins unfolded by stretching from the N-terminus and also from the C-terminus. The data sets in the absence and also in the presence of L-arginine, D-arginine and agmatine were recorded in order to screen the interaction of molecular between AdiC and the substrates of AdiC. This indicates that approximately 400000 F-D curves which have been recorded to take six data sets. Method AFM was used in HT-SMFS experiments and the AFM was located on an active damping desk in order to eliminate some noise from the surroundings. In addition, the cantilevers in the experiment were made from silicon nitride. It was possible to determine the defection sensitive (DS) from the slope of the F-D curves which were recorded on bare mica in buffer solution. AdiC and BR were adsorbed on mica in buffer solution and at room temperature. After that, the sample was washed many times with the same buffer in order to take out non adsorbed membranes. Contact mode atomic force microscopy located proteoliposomes densely packed with AdiC to force spectroscopy. The tip of AFM was employed to remove any aggregates and also to display appropriate areas of membrane for force spectroscopy. The tip was attached to the membrane proteins by pushing the cantilever above the membrane ten times. After that, the cantilever was pulled for 0.25 s with a speed of 0.53 s-1 RB and 0.78 s-1 AdiC. This made it possible to save all F-D curves and consequently these contain 4096 data. The deflection of cantilever was determined with a position-sensitive photodiode. Data analysis When F-D curve was analyzed, the data of retraction were obtained and used because the positive and negative in forces spectra introduce pulling and pushing forces. The negative forces of F-D curve, which were pushed, would not be important in the analysis. The classification and manual fine filtering is dependant of the frequency pattern of AdiC which was achieved. This stage is capable of removing curves with any non-interpretable unfolding which allowed the coarse filtering. Result After selection of an area that was densely packed with protein In HT-SMFS experiment, AdiC was divided into 625 locations. As a result, the yield of F-D curves per area was 6250 because ten F-D curves were recorded per positions. This method contains the benefit of record all F-D curves and the impartial reduction of this intense data set to a number of force spectra which can be analyzed manually. HT-SMFS result The data set in this procedure contain 398 unfolding event of RB which was recorded within 24 hours only. The procedures of HT-SMFS consist of semi-automated data acquisition and also consist of efficient data filtering which were used to know the mechanical unfolding of the bacterial L-arginine/agmatine antiporter AdiC. If the membrane protein density decreases in lipid bilayers, the probability for unfolding event might decrease also. Semi-automated procedures are the most crucial for SMFS on non crystalline proteoliposomes. The N-terminus attaches the cantilever rather than C-terminus because N-terminus is longer about 2.6 times than C-terminus. The recording of N-terminus during one day was used three different cantilevers. This technique was employed to explain whether in N-His6-AdiC can be altered in the presence of L-arginine, D-arginine and agmatine but there was no major change in the scattering plots. Discussion The data acquisition needed more time in HT-SMFS procedure because it was semi-automation method. The operator in this method obtained 10% of data acquisition time and also this method collected around 40000 curves per day. However, the pure manual data acquisition might have been consuming more time and more labour. HT-SMFS procedure has supplied its ability to introduce amount of data sets within short time on non-crystalline samples. This technique is validated because it has the same result of the yield in early studies. In addition, tags are very useful to collect force spectra. The topological information was not only created by the HT-SMFS but also created and yielded information about the mechanical stability of formal elements in AdiC. The dynamic of HT-SMFS can prove insights into how barriers the energy, stabilize the region in form AdiC in addition to altering their position. The HT-SMFS approach may be used in enormous range of reconstituted member proteins to know their fold and thus useful in studying ligand binding. The semi-automated HT-SMFS method adapts for dynamic SMFS experiments because it reveals the energy of membrane protein. DNA: A Programmable Force Sensor The direct quantification of interaction between bio molecules through single molecule force spectroscopy has turned out to be a very strong tool for materials and life sciences. DNA: A Programmable Force Sensor is an approach whereby there is requirement of unbinding forces to break the intermolecular bonds which are consequently measured in a differential format through which it is compared to a reference bond that is known. Furthermore, there is a marked rise in sensitivity as well as force resolution which permits the approach to resolve single pair mismatches and thus the approach permits for highly specific parallel assays. DNA: A Programmable Force Sensor overcomes cross reactions of antibodies in cross reactions within a protein biochip application. Experiment The cantilever spring was substituted with a polymeric anchor and a known reference bond which had a fluorescence label. The molecular bond which was being examined was directly compared to the known reference bond whose main purpose was to be a molecular force standard. During parting of the two surfaces, the polymeric anchor was elongated and the force that was acting along the molecular chain having the sample and the reference complex that was labelled developed suddenly. It is the variation of the stability between the two bonds that broke the symmetry in this approach. Consequently, the fluorescence label has a higher possibility of going up to the side that has the stronger bond and not on the side with the weaker bond. This methodology is similar to 1-bit analog to digital conversion widened by the thermal changes. Counting the label on every side through use of single molecule optics offered a quantitative measure for the variations between the distributions of the rupture possibilities between both molecular complexes. The actual force was then measured at a single molecule height since every bond sample was probed separately through usage of a single reference bond. To measure the force resolution of the differential force test, the decrease of the unbinding forces which was as a result of single-base pair mismatch within a 20-bp DNA duplex was computed. The experiment was carried out in a buffer solution that had 150 mM NaCl at room temperature. Within these conditions, the thermal off rate were very low and inequity between mismatch and ideal match sequences is hard to get in convectional equilibrium assays (Albrecht 2003). The rupture forces of both DNA strands with dissimilar hybridization lengths (a 20-bp duplex & a 25-bp duplex) are openly compared. Both are then bridged using a 65-base oligonucleotide, having the terminal Cy5 fluorescent label. The resulting 20-bp duplex is joined with the PDMS stamp (26-28) through use of polyethylene glycol (PEG) spacers. Fluorescence images of the glass surface having the capture oligonucleotides and the marked sample oligonucleotide before the surfaces were contacted and then separated and the glass and PDMS after both surfaces were separated (Albrecht 2003). Results The differential force format was enforced by the reference complex which was a restricted boundary state. The sequence as well as the length of the reference complex on a stamp was selected for each sample spot on the chip as a result. This permitted optimum force resolution and background discrimination for each spot. As a result, thermodynamic stringency was global while mechanical stringency was restricted. The amalgamation of maximum resolution and restricted stringency allowed exact quantifications of the molecules interactions. After force dissociation, the molecule complex unzipped the geometry and hence allowing the rupturing to occur. The forces between these rupturing molecules are the ones that were measured and images obtained by using the histogram (Albrecht 2003). Discussion On convectional DNA chips, the single base pair matches where detected through the identification of the variations in the thermal off rate or the equilibrium constant. Reduction of the salt concentration helped in maintaining stringent state and this made the DNA duplexes to be easily assessed at various time scales or bind with differentiable binding ratios (Wang 2005). The decrease in the unbinding forces was measured and this was aimed at examining the force resolution of the differential test. The decrease in the unbinding forces was as a result of single-base pair mismatches within a 20-bp DNA duplex. The discrimination that was there between the strong bond and the weak bond binding modes as well as the concept of mechanical stringency offered key advantages during the application to the field of protein assays. Within this field, it is important to discriminate between specific and non specific interactions and it is hard to characterize a general set of stringent ambient condition for several proteins. Proteins basically interact with each other particularly over well defined binding locations, while non specific interactions with other proteins and with surfaces take place over big surface areas. Discrimination between the two duplexes was established by the use of a low force but high affinity force sensors, for instance a DNA duplex within unzip conformation. In Single-molecule force spectroscopy, the major limitation is low accuracy when getting the necessary data. In DNA: A Programmable Force Sensor, the approach’s limitations take place as a result of varied 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 combination efficiencies to the two chips at times differ. 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. (Bosshart 2008). DNA: A Programmable Force Sensor is the best approach because it has few limitations and has many advantages. The approach has high symmetry and many external disturbances are gotten rid of. Furthermore, most of the applications that involves the approach have an precise measure of the difference which is apparently more important than the two absolute values with their respective error bars or a single –base pair mismatch detection in a DNA sequence (Albrecht 2003). DNA: A Programmable Force Sensor approach is also better because it possesses the art of instrumentation that are automatically sensed by the force sensor and therefore the force resolution is only restricted by thermal fluctuations and these are taken care of by the force sensor which detects the fluctuations. The fact that the sensor is normally small in size improves the signal-to-noise ratio (Albrecht 2003). Moreover, in this approach, the cantilever spring is substituted with a polymeric anchor and a molecular bond that is known and whose purpose is being the molecular force standard and this allows each molecule to be probed independently. This is very important since it allows local application of specific of antibodies and other desired molecules (Wang 2005). Conclusion DNA, programmer Force Sensor is more proficient in the measurement of the forces the forces since it has fewer limitations and has certified use of single molecule mechanics. This is because the forces are only restricted by thermal fluctuations and these are sensed by the force sensor hence almost nullifying all hindrances that could interfere with the results. Moreover, the size of the sensor is a bit small in DNA, programmer Force Sensor as compared to High-throughput single-molecule force spectroscopy for membrane proteins and hence advances signal to noise ratio in the approach. Bibliography Albrecht, C, et al., 2003, DNA: programmer Force Sensor, Science, Vol. 301/367. Bosshart, P., 2008, High-throughput single-molecule force spectroscopy for membrane proteins, Vol. 19/ 15. Wang, J., 2005, Electrochemistry of nucleic acids and proteins: towards electrochemical sensors for genomics and proteomics, Elsevier, Sydney. Read More