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1. A total 0 LM E uc 0 uM Kun uM 1 MT 1 A For this model you need to input the total concentration of A or MT the dissociation constants for the seam Ks lattice K and the binding ratio All inputs must be positive numbers 2 2 3 MAPs Dimerize In this model A can exist either as a monomer or as a dimer and both the monomer and dimer forms can bind to MT 11 Chapter 2 Running MTBindingSim MAPS dimerize A MT AMT A MT AMT A AoA Kaye AIIMTVIAMT K ay AJIMTI TAMT K AAT A A A A total 0 uM uar uM Kaam 0 uM m 0 uM 1 MT 1 A For this model you need to input the total amount of A or MT the dissociation con stant for monomeric A binding MT K the dissociation constant for dimeric A binding MT Ko the dissociation constant for the A dimer KA and the binding ratio All inputs must be positive numbers 2 2 4 Pseudocooperativity This model simulates a situation where the binding of an A to an MT site changes the conformation of another MT site creating an MI site which has a different affinity for A than the MT sites Pseudocooperativity T A MT AMT A MT AMT Kur LAIIMT LAMT Kar ATMT T TAMT MT AMT AMT A total 0 LM m 0 uM Kur o lMT 1 A For this model you need to input the total amount of A or MT the dissociation constant for A binding to MT KAwr the dissociation constant for A binding to MI KAMr and the bindin
2. amp e dingSim Version 0 14 Julia Philip Charles Pence Holly Goodson MTBindingSim is a program to plot binding curves under various conditions partic ularly those encountered in microtubule binding or other polymer binding situations MTBindingSim is licensed under the GNU General Public License and its documen tation is licensed under the Creative Commons CC BY SA 3 0 License University of Notre Dame Department of Chemistry and Biochemistry University of Notre Dame Program in History and Philosophy of Science 1 Getting Started 1 1 Running MTBindingSim for the First Time 0 2 Running MTBindingSim Zl Plotting Options sa es sss RE 4448545658 4440 08 E SR S S Ziel Pxpenmental MeNnod 24 aes ee xk REG Y eie x ees ee ee 24 Piloting Modes s xx these ee hak ba S fee qs uU aS rS 2 Ae weg RO RR S ORUMUROA Rho ee ee ee ee R 214 Na mber or Poms 5 5 oon 8 9e mo EP ES ta ae 4 MEA Modil P C 221 GurSPCROePrDIWEDHEE s otto tee ox so 4 tekita ROS Qo OE S 2 2 2 Seam and Latice Binding s esos i s eee eh US wes SB RR S TE Zee MAF DIME sc kas bes Gh ee RES ee ED eee 224 Pseudocooperativity cee xk RR E 369 RR EROR ROCK eas 22 50 MAPS Bind MT bound NEAPS 424684255 4646625454 UR S 22 6 Two MAPs Bind MT bound MAPs 227 ADDERE NES 44445 9 ov ders oa Se WR wu ROS 928 eS 3 How To Sd Editing and Saving Graphs s s ssw ceros eae ee oe Ts eiras i OOS P aa ose a wla EU C as a
3. First order A total 4 Kaw 2 N71 First order A total 6 K AMT 2 N21 a EC a ga a ao EC a er ua iu pam LL If you d like to start over with a new graph click the clear button This will delete the current graph and subsequent curves will be plotted on a new plot For more detailed information about what you can do with MTBindingSim see the Running MTBindingSim and How To chapters If you have any trouble see the Troubleshooting section Good luck and happy graphing 2 RUNNING MTBINDINGSIM To plot a graph using MTBindingSim select the plotting options and binding model you would like to use enter the parameters you want and click the graph button This section explains what plotting options and binding models are available 2 1 Plotting Options There are serveral sets of plotting options that can be selected These options will apply to all graphed curves 2 1 1 Experimental Method There are three available experimental methods Curves from different experimental methods cannot be plotted on the same graph so changing the experimental method will automatically close an active graph In all methods A is an MT binding protein The concentration of MT is taken as the concentration of polymerized tubulin dimers as is common in MT literature MT binding experiments are generally performed with MT stabilizers so that the con
4. Your graph looking wrong could have several causes First make sure that the x and y axis ranges are appropriate for your graph If you are confident that you are looking at the graph with appropriate axes and it still looks wrong or strange it is possible that the program has calculated your graph incorrectly While we have made every attempt to ensure that MTBindingSim will always calculate the correct graph it is possible that we missed something Please submit a bug report on our website and we will attempt to fix the problem To help us diagnose the issue you are having it would be very helpful if you can run the graph several times tweaking the parameters to determine what parameter or combination of parameters is causing the problem Thank you for helping us keep MTBindingSim working properly 5 3 Saving Your Results 5 3 1 I can t save XLS files MATLAB saves XLS format spreadsheets by communicating with Microsoft Excel in a manner which only works on Windows If you wish to save a spreadsheet on Mac OS X or Linux you can save in CSV format instead CSV files can be opened by any version of Microsoft Excel or your favorite spreadsheet program For more information about the limitations of MATLAB s communication with Mi crosoft Excel see MathWorks Solution 1 2SJUON 5 4 Other 5 4 1 I need help can I contact the developers Or 5 4 2 I think I ve found a bug how do I report it Or 5 4 3 I have an idea for a
5. Al 5 total Kar AllLltota total 1 1 I 1 GIA 1 g 4 This equation is numerically solved for free A and the result is used to calculate bound A and free and total MT 4 4 MAPs Dimerize In this model A can form a dimer with itself and can bind to the MT in both the monomeric and dimeric forms with different disocciation constants 21 Chapter 4 Binding Model Mathematics The binding interaction for this model is A AsA2 A MTSAMT A 2MT A5MTD The disocciation constants for these interactions are Kaa A A Aa Kaur AJ MT AMT Kaamr AMT We can write mass balances for A and MT A total A 2 42 AMT AAR A x AP d c AMI Kur A2 MT A E AP xL A MT xz AP MT MT iig MT AMT n p 2 A MT5 n MT xi AIIMT x A2 MT M 1 2 T Karn A MT Kaur jan Al MT MT 1 kaara Al KzamrKaan Al We can now solve the MT mass balance for free MT to get MT MT total aT KAMT 2 bove AT We can plug this into the mass balance for A u e wm 1 2 2 MT total Alota LA rig lAl Em A KaurKaa Lo gai aa a This equation is numerically solved by the program for free A and the result is used to calculate bound A and free and bound MT 4 5 Pseudocooperativity Cooperative binding of MAPs cannot be modeled using the standard model of coopera tive ligand binding To model cooperative behavior we ha
6. MT SAMT A MT AMT The dissociation constants for this model are Kamm AJ MTi AMT KAur2 AJ MTo AMT The mass balances for this model are Alota A AMT AMT A LAJ MTi K Aum LAJ MT2 KAurra IMT Jtotal MT AMT MT A MT Kamri MT Q A Kamr1 IMT iai MT AMT MT A MTo Kamr2 MT 1 A Kamrz The MT and MT5mass balances can be solved for free MT and MT MT otal pou FAV Rane DM ATI These equations can be substituted into the mass balance for A to get a2 A MT total A MT gt totai Aliota A Kamm 1 14 Kamri Kamro 1Al Kamra This equation is numerically solved by the program to get free A which is then used to calculate bound A and the fraction of A bound Free MT is not calculated because this model cannot be graphed against free MT 25 5 TROUBLESHOOTING 5 1 Downloading and Installation 5 1 1 There are password protected files in your ZIP file or 5 1 2 I can t open your ZIP file If you are running Windows there is a bug in the built in Windows ZIP file extractor that occasionally causes trouble with our releases Try downloading and installing the free and open source 7 Zip ZIP extractor and extracting MTBindingSim using it instead If you are not running Windows or if you have trouble even when using 7 Zip please file a support ticket see our specific information about Filing a Support Ticket
7. a monomer or as a dimer with different affinities e Inputs now default to zero when the experimental mode or model are changed The ratio parameter defaults to one e Various bug fixes A 12 Version 0 5 e Supports the following binding models First order binding of microtubule binding protein to microtubules Classical cooperativity A model where a protein binds the microtubule at the seam and the lattice with different affinities A model where a microtubule binding protein can bind either the micro tubule or a microtubule bound protein allowing two total layers of micro tubule bound proteins A model where a microtubule binding protein can bind either the microtubule or a microtubule bound protein allowing up to three layers of microtubule binding proteins on the microtubule e Three different modes of collecting binding data Holding the protein of interest constant and varying the microtubule concen tration Holding the microtubule concentration constant and varying the concentration of the protein of interest An experiment with two different microtubule binding proteins where one microtubule binding protein and microtubule concentration are held constant and the concentration of the other microtubule binding protein is varied In this mode both microtubule binding proteins are assumed to follow first order binding 32
8. below 5 1 3 MTBindingSim doesn t run On a Mac make sure you have downloaded and installed the MATLAB Compiler Run time MCR available at the MTBindingSim home page On Windows the installer should download and install the MATLAB Compiler Run time MCR if you don t already have it If you get an error that the MCR cannot be found you probably have an old version of the MCR which prevents the installer from installing the new version Uninstall the old version if you don t need it for any other programs and then manually download and install the updated version from the MT BindingSim home page If you have successfully installed all the prerequisites and still cannot get MTBind ingSim to run try Filing a Support Ticket below 5 2 Graphing 5 2 1 My graph doesn t show up If you graph multiple simulations on the same graph the axes will be set to show the full range of all curves If you have curves with very different values you may not be able to see all of the curves on the same graph Try closing the graph and making a new graph paying attention to the range of the X and Y axis Also if you graph two very nearly identical graphs at the same time the line for the last one to be graphed may lie precisely on top of the line for the first to be graphed making the first line invisible Try graphing your curves one at a time and comparing the graphs 26 Chapter 5 Troubleshooting 5 2 2 My graph looks wrong
9. great new feature Head to the next section and learn about Filing a Support Ticket 5 5 Filing a Support Ticket MTBindingSim is hosted on Google Code and we use its Issues system for keeping track of support requests For your best chance at support please file a ticket there rather than e mailing one of the developers When should you file a support ticket In general if you have a question that you just can t get answered in the documentation if you have a bug to report or if you have an idea for a new feature you should send us a message 27 Chapter 5 Troubleshooting Rather than including it in the documentation we keep up to date information about how to file a support ticket on the MTBindingSim website Please visit there for the latest information about how to get MTBindingSim support 28 If you would like to get in touch with the developers by e mail you may do so at the following addresses e Julia Philip e Charles Pence Note that if you are looking for support to file a bug report or to request an issue we would much prefer if you filed an issue in our support system on Google Code See our page about creating support tickets 29 7 ACKNOWLEDGEMENTS Many people have contributed to the process of writing MTBindingSim and we would like to thank them The lead developers are Julia Philip and Charles Pence Julia was supported by the Notre Dame Chemistry Biochemistry and Biology Interf
10. is defined as K A MT AMT AMT We can also write mass balances for total A and total MT A total A AMT A 74 A MT KAMT MT total MT AMT n MT eL lAI MT MT 1 goal We can rearrange the equation for total MT and solve for MT free MT total MN Tega We now can substitute this equation into the equation for total A A A n Ki AIMT tota total OMA AI The program numerically finds the value of A free that solves this equation then uses that to calculate all other necessary parameters 4 3 Seam and Lattice Binding In the seam and lattice binding model it is assumed that there are two different kinds of binding sites on the MT seam sites and lattice sites which have different dissociation constants The seam sites are 1 13 of the total MT and the lattice sites are 12 13 of the total MT 20 Chapter 4 Binding Model Mathematics The binding relationship is A S amp AS A LAL The disocciation constants for these interactions are Kas A S AS Kar A L AL We can write a mass balance for all three species Alota A AS AL A g AJIS g ATIL Slota S AS n S xL AIIS S 1 xEZ A Elota L AL n L g A L L 1 GIA We now can solve for free L and free S S S tota MT A total L lke aA We now can plug these values into the equation for total A A u A n Ky
11. polymer ligand interactions in which one binding event influences other binding events MAPs bind MT bound MAPs A binds to MT and then another A can bind to each MI bound A This model is valid for any protein protein or protein ligand interaction in which one binding event reveals another binding surface Two MAPs bind MT bound MAPs A binds to MT and then two As can bind to each MI bound A This model is valid for any protein protein or protein ligand interaction in which one binding event reveals another binding site This is an extension of the MAPs bind MT bound MAPs model Two binding sites A can bind to two sites on each MT This model is valid for any protein protein or protein ligand interaction with two independent binding sites 2 2 1 First Order Binding First order binding simulates binding under standard first order conditions where one A interacts with one MT dimer and all A MT interactions are identical 10 Chapter 2 Running MTBindingSim First order binding mal A MT AMT K sage AIIMTI TAMT A total 0 LM 1 MT 1 A For first order binding you need to input the total concentration of A or MT the Kp and the binding ratio All of these must be positive numbers 2 2 2 Seam and Lattice Binding Seam and lattice binding simulates a scenario in which protein A binds to the MT seam dimers with a different affinity than the MT lattice dimers MT seam and lattice binding ni Ka AIILI TAL K ASIAS
12. wu RR X uS narea R 3 2 Saving a Graph as a Spreadsheet 64244544655 4646645 RR As Ds Comparnne IWO CUNVES 24484 3x AL padd eri mug aata IECUR RS 4 Binding Model Mathematics 4 1 Polymer Nature of MT s uuo eo eee RAH SERRA GHEY RE ROS LLLI Goncenianon Or zone Geo bh ee S x 9 Eoo p es 412 Dmdie Ralo x93 RE REPE Sp m eh opos de ud 22 TE TOT ee Bi uu mos 9 33 908 8 desig oe wee ees MUR SR RA 43 iem ane LAO RGN 20 saa ados WS ee ae RUE E POR RS do x DH 182i o PP 49 Pscudocooperalivily x 3 422 L443 24Belqc hee TES 4 6 MAPSs Bind MT bound MAPS 25664 6Aes 6s oe eee UR ES EROR DE 47 Two MAPs Bind MI bound MAPS 52 252 99 x om om komme 45 Iwo Binding SIES ss rese bos momo don atorra nEn Bae oS VO O CO CO 10 10 10 11 11 12 12 13 14 15 15 15 15 16 16 Contents 5 Troubleshooting 5 1 Downloading and Installation ed am momo m hes ox RO SER bina da 5 1 1 There are password protected files in your ZIP file oL JL eanmbopen yOUr AIP Ie uou swwek 943 9 9 9954 93 SLs MIBindinp oim doesn TIUN a ak he doe ee 6 wes Gee RESTE DE ON usta ese ons Sees PUER REUS oe eee eee ER RES ES o2 My graph doesn t show up iuo wx eee me RR eae da 9s d Doo JUD graph looks wWIONG 2x39 mx 3 9 E wes E v ee Be s 2o DIVNE Your Resull 6 343 423 2744 9 9 43 Dod Jes peave AUS CS s su ae rais icu ie e Re eae hee CR DO EO UP 5 4 1 Ineed help can I contact the developers sn 5 4 2 IthinkI ve fou
13. ace Program NIH Training Grant T32GM075762 and NSF grant 0951264 We would like to thank Dr Holly Goodson for support and guidance and Eric Bunker for assistance with writing the documentation and troubleshooting the program 30 A MT BINDINGSIM REVISION HISTORY A 1 Version 0 14 e Fix a bug in the CSV file exporter s column headings e Add some more detail to the discussion of the binding model mathematics in the documentation A 2 Version 0 13 e Add an info button which opens a box with information about the currently se lected binding model A 3 Version 0 12 e Fixes to the MTBindingSim installation system A 4 Version 0 11 e Added a new pseudo cooperativity model e Added a new two sites binding model A 5 Version 0 10 e Removed the cooperativity binding model e Added ability to save Scatchard plots A 6 Version 0 9 e Changed calculation method for the binding ratio e New way to save graphs and data A 7 Version 0 8 1 e Fixed an urgent calculation bug A 8 Version 0 8 e Changed terminology for the various dissociation constants e Various bug fixes including a calculation error 31 Appendix A MTBindingSim Revision History A 9 Version 0 7 e Complete documentation is now available for MTBindingSim e Various bug fixes A 10 Version 0 6 1 e Various bug fixes A 11 Version 0 6 e Added anew model where a microtubule binding protein can bind to microtubules either as
14. binding ratio n as an additional parameter that can be used in fitting binding data to accommodate non 1 1 binding ratios We have included the binding ratio n as a parameter that can be set in our binding curve calculations Following Ackmann et al we have defined n such that that binding ratio is n A 1 tubulin dimer However we have differed from Ackmann et al in how we include the binding ratio in our binding equations While they change the number of available binding sites based on the binding ratio we do not change the number of available sites We account for the binding ratio only in the mass balance for microtubules This formulation assumes that the number of available binding sites is the number of free dimers regardless of how many dimers an individual A binds This method will break down in an extreme boundary case where a single A binds so many tubulin dimers that a single MT polymer does not contain enough sites In such a case A would need to encounter two or more individual MT polymers changing 19 Chapter 4 Binding Model Mathematics the binding model However such an extreme case is extraordinarily rare and would most likely occur if two different polymers were interacting with each other directly MTBindingSim does not deal with such cases 4 2 First Order Binding This model is simple first order binding In first order binding the relationship between A and MT is A MTSAMT The dissociation constant
15. centration of polymerized tubulin can be taken as the total concentration of tubulin present Experimental Method Vary MT Vary A Competition Vary MT In this method the concentration of A is held constant and the concen tration of MT is varied The fraction of A bound is graphed on the y axis This is a standard binding experiment and simulates data from many standard experimental methods of measuring binding data including SPR fluorescence anisotropy and trypto phan fluorescence as well as cosedimentation assays Vary A In this method the concentration of MT is held constant and the concen tration of A is varied The concentration of A bound is graphed on the y axis This kind of data cannot be collected using many standard binding assays such as SPR and fluorescence anisotropy but it can be collected using a cosedimentation assay Competition In this method there are two MT binding proteins A and B The con centration of A and the concentration of MT are held constant while the concentration of B is varied The fraction of A bound is plotted on the y axis This kind of data can be collected with many standard methods of measuring binding data such as SPR fluores cence anisotropy tryptophan fluorescence and cosedimentation assays Chapter 2 Running MTBindingSim 2 1 2 Plotting Mode MTBindingSim can plot one curve at a time or it can plot two curves simultaneously and compar
16. e them You may plot as many curves as you wish as long as you don t change the experimental method or X axis settings However only two curves may be directly compared by the program Plotting Mode Q Plot a single curve Compare two curves For more information about comparing two curves see the How To section 2 1 3 X axis In Vary MT mode the X axis can be set to either MT total or MT free X axis 9 MT free MT total MT total is a known quantity in the experiment while MT free must be calculated However the familiar Langmuir Isotherm equation uses MT free as its independent variable and the quick method of determining K p by looking at the MT concentration when the fraction of A bound is 0 5 only works if the x axis is MT free In either X axis mode the Y axis will be the fraction of A bound In Vary A mode there are three possible kinds of plots In the first two the Y axis is the concentration of A bound to MT and the X axis can be set to A free or A total In addition the data can be plotted in a Scatchard plot where the X axis is A bound and the Y axis is is A bound A free This kind of plot is used to make the binding data linear Binding data will be linear for simple interactions and a curved line in a Scatchard plot indicates the presence of cooperativity or other kinds of non simple binding interactions X axis A free J A total J Scatchard Plot Changing th
17. e x axis mode will automatically close the active graph Chapter 2 Running MTBindingSim 2 1 4 Number of Points The number of points plotted may be specified Number af points The default number of points is 100 A larger number of points can be used if a smoother curve is desired A smaller number of points will result in a faster calculation 2 2 Binding Models MTBindingSim can plot curves for several binding models For a detailed description of the math used to generate the curves please see the Binding Model Mathematics chapter Note that all models use the dissociation constant Kp The info button to the right of the model selection box provides a brief description of each model reproduced below First order binding ba First order binding Simple A binds MT interaction This model is valid for any simple protein protein or potein ligand interaction Seam and lattice binding A binds to the MT seam 1 13 of the MT with a different affinity from the MT lattice 12 13 of the MT This model is valid only for binding of proteins or ligands to 13 protofilament MTs with a seam MAPs dimerize A can bind to MT either as a monomer or as a dimer This model is valid for any polymer protein or polymer ligand interaction Pseudocooperativity For every MT site that binds an A another MT site is converted to an MT site which binds to A with a different dissociation constant This model is applicable to polymer protein or
18. ew Insert Tools Desktop Window Help HeWese s Aavgedc a ao Vary MT Binding Assay First order A total 10 Kyr 5 N 1 Seam binding A total 10 K 1 K 100 N 1 ou C 3 m u c c Fract 8 10 12 MT total If the X axis is plotting total MT or total A or in competition mode he program also will calculate the difference between the two curves and display the result Average absolute difference 0 31302 Average percent difference 109 465 Maximum absolute difference is 0 45691 at 20 MT total Maxmum percent difference is 124 827 395 at 0 2 MT total These values are calculated by computing the absolute and percentage difference between the curves at each point and both the average and largest difference are reported for each This feature is designed to help researchers determine whether the predicted differences between two curves are detectable given their expected experimental error This comparison is only done for curves plotted with the X axis as MT total or A total because the x values for both curves are the same in that case so the comparison calculations are unambiguous When the X axis is MT free or A free the x values for the curves will be different and the calculation of a comparison between the curves requires making assumptions about the binding behavior 18 4 BINDING MODEL MATHEMATICS This chapter describes the equations used to simulate the binding curves Though some of
19. for this model are Kamr A MT AMT Kaa A AMT A2MT Kaa A A2MT A3MT Note that the disocciation constant for the first and second MAP binding to the MT bound MAP is the same The mass balances for this model are Alota A AMT 2 A2 MT 3 A3 MT A e A MT g A AMT 4 42M A eH LAJIMT x AP IMT g AP AMT KAMT A xL AI MT grz AP MT Kus Al MT IMT ioiai MT AMT n A2 MT n A3MT n MT xL A IMT gal A MT 3 7 AP MT T KamtTKaan AMTK an MT 1 x4 A pA 1 KAMTKAAn KamrK an 24 Chapter 4 Binding Model Mathematics Notice that as with the MAPs bind MT bound MAPs model all MT A complexes contain a single MT unit The MT mass balance can be solved for free MT MT MT total Lagi AF arias Alt eg AD This can then be substituted into the A mass balance equation yielding Ahoa A eiii KaurKaa AP ante lal KamrK4 A M T total PS z d Au We JA 1 RAMT Alt KAMTSAA a KAMTK a Al This equation is numerically solved by the program to get free A This is then used to calculate bound A and free and bound MT 4 8 Two Binding Sites This model assumes that each tubulin dimer contains two binding sites for protein A sites 1 and 2 with different dissociation constants It is assumed that the two sites do not interact The binding relationships for this model are A
20. free at each point 4 6 MAPs Bind MT bound MAPs In this model A binds MT with a disocciation constant of KAwyr then another A can bind the bound A with a disocciation constant of K AA Okkan The binding relationships are A MTSAMT A AMTSA MT The dissociation constants for these interactions are Kamr A MT AMT Kaa A AMT A2MT We can write the mass balances for this situation 23 Chapter 4 Binding Model Mathematics A total A AMT 2 A2MT A x AIMT giz A AMT A xz AIIMT a 4l MT KAMT MT total ael 1 AMT t MPH MT xi A IMT c r7 AP MT MTJ KAMT A KamrKaan A We can solve the MT mass balance for free MT as follows MT _____ MT rota __ Lt Rare Al KayrKaan Al We can then substitute this equation into the A mass balance to get A total A 1 Al 291 AP a Kur KamrKaa Rann HA i as AP This equation is numerically solved by the program to find the value of free A which is used to calculate bound A and free and bound MT 4 7 Two MAPs Bind MT bound MAPs This model is very similar to the MAPs bind MT bound MAPs model except in this case two layers of bound MAP are possible on top of an MT bound MAP K AA O KAMT LT N pu m amp e a a a as 4q g way oe e a Ra AA The binding relationships for this model are A MTSAMT A AMTSA2MT A AaMTSA3MT The disocciation constants
21. g ratio All inputs must be positive numbers 2 2 5 MAPs Bind MT bound MAPs In this model once one A has bound to an MT dimer another A can bind to it without taking up another MT binding site 12 Chapter 2 Running MTBindingSim MAPs bind MT bound MAPS zl A MT AMT A AMT AMT Kae AIIMT AMT K A AMT A MT A total 0 uM uar 0 uM E 0 pM 1 MT 1 A For this model you need to input the total concentration of A or MT the dissociation constant for A binding to MT Ky the dissociation constant for A binding to MT bound A Ka and the binding ratio All inputs must be positive numbers 2 2 6 Two MAPs Bind MT bound MAPs In this model once one A has bound to the MT two more As can bind to it without taking up additional MT binding sites MAPS bind MT bound MAPS nal A MT AMT A AMT AMT A AMT A MT Kur LEMIMT AMT K AJ AMT A MT K A TA MT A MT A total 0 uM E 0 uM Kas 0 uM l1MT 1 A For this model you need to input the total concentration of A or MT the dissociation constant for A binding to MT Ky the dissociation constant for A binding to MT bound A Ka and the binding ratio All inputs must be positive numbers This model can be used in concert with the MAPs bind MT bound MAPs model described above to begin to understand the behavior of MAPs that use the MT as a nucleation site for MAP polymerization It is impractical to model a case where the MAP can fo
22. n MTBindingSim Saving as an Excel file xls will generate an Excel file containing the x and y values for all curves on the graph Saving as a comma separated value file csv will create a CSV table with the x and y values for all curves on the graph If you are running MTBindingSim in Mac OS X or Linux you cannot save your files as xls You can save them as csv files and then open them in Excel Please see Trou bleshooting for more information 3 3 Comparing Two Curves To compare two curves in MTBindingSim first select compare two curves from the plotting mode box Plotting Made Plot a single curve Compare two curves This will cause a second model selection drop down box and set of model parameter input boxes to appear Enter the parameters for the curves you wish to compare in the two columns The curves are independent of each other An example of two curves to be compared is below 16 Chapter 3 How To Dn E i Vary MT 3 Plot a single curve MT free e Vary A ee Compare two curves e MT total Competition momi o jum gu mere 100 Mmeme 20 uM Seno MT seamana utice wnang A MT AMT A S5 AS A Le AL Ku AIIMT AMT K UAIILI IAL K A ISV AS A total uM A total uM NENNT NUN IMT 1 jA K 10 uM TENE When you click the graph button the curves will be plotted on the active graph or a new graph 17 Chapter 3 How To E Figure 1 File Edit Vi
23. nd a bug how do I report it 5 4 3 Ihave an idea for a great new feature less 595 Filing anupport eke x 4 9 xe pon Rs ER EUR CE RUE ee eee ee Contact Us Acknowledgements MTBindingSim Revision History Bul Version Uae ssiri rerien eee aaoee he eee 9 9 9593992 P VONIS s 20 kk oR REE REGN XR eee ES x A 3 Version 0 12 4 1b 9 R 4cc 39 AX433 S543 3333333w3336 AD Version 0 11 i242 xx aaa L AS Veron U4 sresrrssrss oh URGES eae eee heehee ee oe AO Version US Lu oum e 9 ROMAE RR UEA SEXE UE PU ea ee Per Versionl S Ll 2o sc xx ae ee eee de ooo BERK Oh Eo AO VISION OS sche ake ee eS Hee eee 9 ROOM EH Eee GS A9 Version l0 xm 13 26RD ORE EAE eH eS A 10 Version 0 6 1 lode ea ee eR SRE Eee eee deed Geese ALl Version UO cease eae ebb tae e ebb US ba waa e shee nae dou bad Pol VERSION 2e deus oe eos ea eee hee ee Ree eee IH 1 GETTING STARTED Thanks for checking out MTBindingSim Here s a quick start tutorial designed to get you eraphing binding curves in no time 1 1 Running MTBindingSim for the First Time To begin you will need to download and install MTBindingSim When you run MTBind ingSim for the first time you should see a window that looks like this Vary MT Plot a single curve amp MT free Vary A Compare MT total Em E 3 Competition LE d x Kan AIIMTV IAMT A total uM ure 1MT A Chapter 1 Getting Started MTBindingSim
24. r figure in a LaTeX document you should probably save it either as a PDF file or as an EPS file Finally if you wish to edit the graph changing text or other graph properties it is recommended to save the graph either in Adobe Illustrator format if you have access to Illustrator or in PNG format for editing in any image editing software including Photoshop If you d like to save the curves as x and y values in a spreadsheet see Saving a Graph as a Spreadsheet below 3 1 2 Editing Graphs The graphs generated by MTBindingSim can be edited in several ways From inside the program you can change the location of the legend box by clicking and dragging You also can edit the legend text by double clicking on it If you would like to see a particular part of the graph blown up you can use the zoom in and out tools on the graph window and then the hand tool to drag the graph to the desired region To edit the graph more extensively two options are available If you have MATLAB you can save the graph as a MATLAB Figure FIG file and edit it as you would any other MATLAB graph Otherwise it is recommended to save as an Adobe Illustrator 15 Chapter 3 How To AI file and edit in Adobe Illustrator You may also be able to edit an Illustrator file in the open source Inkscape image editor 3 2 Saving a Graph as a Spreadsheet To save the curves from a graph as x and y values in a spreadsheet click on the save graph button i
25. rm a large polymer but these two models together establish the trend of the binding data in such a case 13 Chapter 2 Running MTBindingSim 2 2 7 Two Binding Sites In this model A can bind to two sites per tubulin dimer site 1 and site 2 with different dissociation constants 2 Sites na A MTI AMTI A MT2 AMT Kam 7 UAIEMTL DAMTI By ZDAJIMT2 TAMTZ A total 0 uM uum 0 uM uz 0 uM In this model you input the total amount of A or MT the dissociation constant for A binding to MTjsites KAyr1and the dissociation constant for A binding to MTosites KamtTz2 All inputs must be positive numbers Note that the total amount of MT is the total amount of MT4and MT This model cannot be graphed with an x axis of MT free 14 This chapter explains how to use some of the advanced features of MTBindingSim 3 1 Editing and Saving Graphs 3 1 1 Saving Graphs To save a graph generated by MTBindingSim for later use or editing click on the save graph button You can save your figure in the following formats e MATLAB figure fig e Adobe Illustrator file ai e EPS file eps e JPEG image jpg e Portable Document Format pdf e Portable Network Graphics file png e TIFF image tif e Excel spreadsheet xls e Comma separated value table csv If you wish to embed your figure in a PowerPoint presentation or a Word document the best choice is probably a JPEG image If you wish to include you
26. s defaults are to plot a graph of an experiment in which the total con centration of microtubules MT is varied along the X axis and the binding model is first order binding See the Running MTBindingSim chapter for more information about these settings and the Binding Model Mathematics chapter for more detailed information about the binding models First enter values for the experimental range of MT Varying between 0 and 10 is a nice start MT total min 0 LM MT total max 10 uM You will also need to enter values for A total Kp and the binding ratio Try 2 for both A total and Kp and leave the binding ratio at its default of 1 A total 2 uMi 1 MT 1 A To calculate the binding curve under these conditions click the graph button Graph Curve You should now see a graph that looks like this Chapter 1 Getting Started E Figure 1 Fille Edit View Insert Tools Desktop Window Hees hA OS94 a E a Ea a ao EC ao er s pam LL Congratulations you ve just plotted your first binding curve using MTBindingSim Try changing the values of A total Kp or the binding ratio to see how the curves change After plotting four curves you ll have a graph that might look something like this E Figure 1 File Edit View Insert Tool Desktop Window Help OGHS s A80dD84 8 0H en Vary MT Binding Assay First order A total 2 KaT 2 N 1 First order A total 1 Kaur 2 N71
27. these equations can be solved analytically the calculations are performed numerically The program breaks either the total A or total MT range into a user defined number of points and then calculates the concentration of free and bound A and free and bound MT at each point 4 1 Polymer Nature of MT Microtubules are polymers composed of tubulin dimers consisting of one alpha and one beta tubulin A microtubule is formed by 13 filaments of tubulin dimers binding head to tail For most of the microtubule the lateral interactions are alpha beta but one set of interactions the MT seam is alpha alpha and beta beta For most binding calculations the polymeric nature of the MT can be ignored however in a few cases it must be taken into account as described below 4 1 1 Concentration of MT By convention the concentration of MT is reported as the concentration of tubulin dimers In most binding assays a microtubule stabilizer is used to make the concentration of free tubulin irrelevant in binding calculations Should this not be the case the contribution of free tubulin dimer both in decreasing the available polymer sites and in possibly com peting with the polymerized tubulin for binding proteins must be taken into account MTBindingSim does not deal with such cases 4 1 2 Binding Ratio Some MT binding proteins appear to bind to MT with ratios other than 1 MT binding protein 1 tubulin dimer Ackmann et al have introduced the
28. ve implemented a pseudoco operativity model where the binding of an A to an MT site with an affinity of KAwr creates an MI site which has an affinity of KAwr for A MT sites are created by the binding of A to both MT and MT sites so the total number of MT sites is equal to the total number of bound A proteins The binding relationships are A MTSAMT A MT AMT x The dissociation constants for these interactions are 22 Chapter 4 Binding Model Mathematics Kamr A MT AMT Kamrx A MTx AMT We can write mass balances for this situation Note that there is an additional mass balance for MT IMT igigy MT AMT AMT AMT This equation can be used to show that MT AMT which we will use later Aliota A AMT AMT A RE LUBE pa 4D ga fain A IMT GC sets IMT totaa MT AMT MT AMT MT AMT AMT AMT MT 2 AMT AMT MT ZAMN 4 AIM X AJMT AJAMT Aner AAMT MI AME t TAI nx KAMT a KAMTKAMT MT 1 KAMT POTRT The MT mass balance can be solved for free MT as follows MT total MT B 1 2 A A KAMT KamTkamtT This equation can then be substituted into the mass balance for A to get MT total Gc e A total A AMT AMT AMT 1 poe o a KAMT E KAMTKAMT This equation is solved numerically by the program to calculate A bound and free and MT bound and

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