Sequence It! Version 1.38 User`s Manual
Contents
1. 14 Sequence It User Manual Modification Options Use 15 0 percent of sample O nMoles of sample 999 0 nMoles remaining Cx Figure 10 Options for Performic Acid Oxidation experiment This is your opportunity to change the default values the program has set up For the tutorial these values are fine Click on the OK button The program now performs a performic acid oxidation on the original protein displaying in a schematic form the process taken in the laboratory After the process is finished the summary window is displayed looking like that in Figure 11 File Edit View etusis Madificatian firsagage Windows Intro Problem Peptide To Be Sequenced y Ferformic Oxidation y Figure 11 Summary Window after Performic Acid Oxidation experiment Sequence It User Manual 15 A new icon is displayed connected to the original protein s icon We term this a descendant It represents the results of the performic oxidation process and the name of the experiment is shown to help you keep track of which icons represent polypeptide derived from which experiments The arrow points to this label which we refer to as the descendant label Open the peptide information window for this newly created polypeptide and enter the length 13 in the same way it was done for the original protein Initial Sequence Determination We are now ready to perform experiments to determine the sequence of the polypeptide For2. BioQUEST A DA WSs t n ie Library VII Allen R Place Thomas Schmidt Sequence It Version 1 38 User s Manual University of Maryland Biotechnology Institute University of Maryland Biotechnology Institute A BioQUEST Library VII Online module published by the BiOQUEST Curriculum Consortium The BioQUEST Curriculum Consortium 1986 actively supports educators interested in the reform of undergraduate biology and engages in the collaborative development of curricula We encourage the use of simulations databases and tools to construct learning environments where students are able to engage in activities like those of practicing scientists Email bioquest beloit edu Website http bioquest org Editorial Staff Editor John R Jungck Managing Editor Ethel D Stanley Associate Editors Sam Donovan Stephen Everse Marion Fass Margaret Waterman Ethel D Stanley Online Editor Amanda Everse Editorial Assistant Sue Risseeuw Beloit College Beloit College BioOQUEST Curriculum Consortium University of Pittsburgh University of Vermont Beloit College Southeast Missouri State University Beloit College BioQUEST Curriculum Consortium Beloit College BioOQUEST Curriculum Consortium Beloit College BioQUEST Curriculum Consortium Editorial Board Ken Brown University of Technology Sydney AU Joyce Cadwallader St Mary of the Woods College Eloise Carter Oxford College Angelo Collins Knowles Science Teaching F
3. Positions from first cleavage OOASCCOLFOACOCODOAOHONOACOO OOAOOCODAGCO DOACCOHOAOCOO F Second cleavage Overlapping area Overlapping area Figure 21 Symbolic representation of overlapping fragments Reconstructing a Protein The tool we provide for the reconstruction of a protein is called the Assemble Window Select the same fragment we ve been using the one from the Performic Acid Oxidation and choose the menu item Assemble Sequence from under the Analysis menu If you ve sequenced all the descendants of this peptide as was explained above each individual subsequence should appear as well as the sequence selected See Figure 22 26 Sequence It User Manual File Edit View etusis Madificatian firsauage Windows Intro Problem Large Font Remove SXIE L Circular Sah TREES EAT EHXEI SGF Pedi fide End Groups SGRE i Peptide 1 by Staphylococcal Protease Figure 22 Initial Assemble Window Reorganize the fragments by clicking on the fragment itself and moving it around inside the window As soon as you click on a fragment it becomes selected the sequence is highlighted its origin is displayed in the bottom portion of the window and the sequence is copied to the scrap You can then paste the sequence into the Assembled Sequence text box at the top of the window at the current insertion point This is a complex window and another perfect opportunity to make the most of the program s on line help
4. between the two cystines it should be easy to determine if a disulfide bond exists Select the original protein s icon and choose the Staphylococcal Protease cleavage again using the defaults the program offers Note the results of this cleavage only three descendants were created To confirm the existence of a disulfide bond you can now perform the modification done on the original on each of the peptides created from the cleavage Here our hypothesis is confirmed the Perform Acid Oxidation of peptide three creates two fragments meaning that the two were joined by a disulfide bond that was destroyed by the modification Figure 24 30 Sequence It User Manual Intro Problem Peptide To Be Sequenced U Staphylococcal Protease Performic Oxidation m VUP Ferformic Oxidation Moved and collapsed descendant label Figure 24 Summary Window after initial experiments for disulfide determination Entering Disulfide Bonds To enter the disulfide bonds so they become part of the overall sequence determined open the Assemble Window again for the original protein Now with two cystines present in the assigned sequence the Setup Disulfides button should be enabled Click on this button to open a window which shows an abstract representation of the protein with the cystine amino acids explicitly shown Your screen should now look like that in Figure 25 Sequence It User Manual 31 File Edit View etusis Madificatia
5. Another way to open the notepad for a peptide is from the Summary Window Select the icon representing the protein and from the Edit menu choose the Open Notepad menu item or take the shortcut and press K We strongly encourage you to make extensive use of the notepads You can make notes to yourself about the importance of a particular experiment or leave hints for others to follow This is your only written record of why or how you did something Also this is the only vehicle currently available for you to get printed output from Sequencelt Saving the Problem It s wise to save the problem now if you haven t done so already No program is perfect and it s possible that unexpected things could occur power outages random acts of God that would cause you to lose all work done to this point To save go under the File menu and select the Save menu item A standard Macintosh dialog will now come up asking you for a name and location for the file which will be created Use any name you like for the manual the name Intro Problem was used so this is what is displayed on all the following screen images Performing a Second Cleavage 24 Sequence It User Manual Go back to the fragment created by the Performic Acid Oxidation select it and choose the Cleavage menu item Clostripain This protease cleaves after arginine amino acids of which we know there is at least one from the Edman experiment performed on peptide 4 f
6. blinking You can also type directly into the sequence edit box using standard text editing commands Lastly you can import a sequence from the clipboard using the Paste command All sequences entered must be in single letter code designations Also you can read in the sequence from another Sequencelt file by using the Read From File button Click there and a standard Macintosh Open File dialog is shown allowing you to choose any file available The sequence from the file is then put straight into the sequence editing box of the window allowing you to change it at will Problem Setup 3 Computer Generated i User Entered ASNIICYS vA O Circular Peptide ASP GLU AL ard Groups N IE afun Pienififpg Amount 200 nMoles Setup Menus Read From File ave To File Sequencelt Figure 27 Problem Setup dialog showing the User Entered options 34 Sequence It User Manual Several Features of this window need some explanation Save To File Button Common to both the User Entered and Computer Generated screens this button allows you to save the protein being generated to a standard Sequencelt file It is useful for instructors setting up a series of problems for students Circular Check Box Selecting this control causes a circular peptide to be generated ignoring all other end group information End Groups Button Opens a window allowing you to set the end groups of the protein to be generated The window looks l
7. in the chain play an important role in the structure of some proteins by tying parts of a single polypeptide chain together or by linking two different polypeptide chains within a protein Disulfide bonds link two cystine amino acids together with a special type of bond distinct from the standard peptide bond is a mixture of amino acids resulting from hydrolysis of a polypeptide chain with 6N HCI at 100 C in vacuo for 24 to 72 hours The visible level of liquid in a tube or amino terminus of a polypeptide is the first amino acid residue in a chain By convention residues are numbered from the N terminus and written left to right in the order they occur in the chain Peptide Bond Polypeptide Proteins Primary Structure R Group Repetitive Yield Sequence Side Group Sequence It User Manual 47 is the link between amino acids An a carboxylate group of one amino acid condenses with the a amino group of another with the loss of a molecule of water This is how polypeptides or proteins are formed with potentially thousands of amino acids linking together The resulting bond is also called an amide bond is generally considered to be a chain of amino acids greater than 50 residues in length Derived from the Greek word proteios meaning primary proteins are linear chains or polymers of amino acids of a protein is the linear sequence of amino acid residues When writing primary structures one
8. now we will use the descendant created by the performic oxidation modification since we know this polypeptide does not contain disulfides Select the small Descendant icon to activate the menu items Now click on the Analysis menu and scroll down to the popup menu item Exopeptidase Degradation Keeping the mouse button depressed move to the right and select the Aminopeptidase M menu item Click on the OK button for the experiment s option dialog The laboratory steps are displayed followed by the experiment s results displayed in a window like Figure 12 Aminopeptidase Mi Peptide 1 by Performic Oxidation Ll Amount Used 10 0 nMoles Legend 16 Sequence It User Manual Figure 12 Results from Aminopeptidase M experiment The exopeptidase procedures both Aminopeptidase and all the Carboxypeptidase experiments are time based meaning that the sequence is inferred by the rate of release of each amino acid with the rate measured at specific time intervals In this experiment for example more than 3 nMoles of the amino acid serine single letter code S were measured at 10 minutes almost 6 nMoles at 20 minutes and so on Unfortunately this was the only amino acid released implying that the next amino acid in the sequence somehow blocks further cleavage by aminopeptidase The sequence can be entered in two ways in this window you can click on the legend item matching the amino acid try it or you can simply type in the s
9. the conditions used to hydrolyze the polypeptide Hydrolysis Options Use i hioles of sample Run for hours 1 196 Cor Figure 6 Hydrolysis Options Window The first window displayed is the Hydrolysis Options Window Figure 6 The default values are displayed here and can be changed if needed All experiments in Sequencelt allow default values to be changed in this manner as you grow more comfortable with the program there will arise many instances where the defaults are unsatisfactory For now just click on the OK button to continue The experiment is performed automatically with the program graphically displaying the steps undergone to perform the acid hydrolysis experiment in a laboratory Sequence It User Manual 9 File Edit View etusis Mindificetian fisagage Windows Acid Hydrolysis Original Peptide 1 00 nM Used T I L SoD AM VMS SKK A E A aa E B a G a ar a a e a nMioles Figure 7 The output of an Acid Hydrolysis experiment After all the steps in the hydrolysis process are completed a window similar to that in Figure 7 will appear This bar graph represents the molar quantities of each amino acid released The quantity of sample used is displayed in the top right corner of the window The three letter code designating each of the standard amino acids is displayed on the left axis of the bar graph The corresponding single letter code is displayed on the far right of the em
10. window or the Assemble window these cursors appear when dragging the link from one cystine amino acid to another Each fragment has associated with it a notepad The empty notepad icon appears when the notepad is empty the full icon when information has been entered Click on either one to open the notepad it represents The microcentrifuge tube or Eppendorf icon represents a peptide The larger icon is used only for the original protein the one to be sequenced Click on one of these to select a peptide for an operation when selected the icon is inverted and double click to open its Peptide Information Window The level of the meniscus gives a rough indication of the amount of sample remaining The amino acid composition tube icon indicates that an acid or base hydrolysis experiment has been performed on that peptide Alter the tree to display these icons by selecting the By Composition item under the View menu The N terminal tube icon indicates that the amino terminus of the represented peptide has been determined Alter the tree to display these icons by selecting the By Amino Terminus item under the View menu 52 Sequence It User Manual UF y HM COOH The C terminal tube icon indicates that the carboxyl terminus of the represented protein has been determined Alter the tree to display these icons by selecting the By Carboxy Terminus item under the View menu The Sequence Tube icon indicates that se
11. 0 Amino Acid ARNOCOQEGHILKHFFPSTHYUX Amino Acid Sequence Entered Amount Used 15 0 nMoles Figure 18 Results of Edman Degradation experiment The output for this experiment is presented as a series of cycles each of which displays the amino acid released at that cycle or step To enter the amino acid either click on the graph at the location of the single letter code i e on the bar in the graph for the cycle or activate the text box labeled Amino Acid by clicking once inside the 22 Sequence It User Manual box and type in the single letter code Do the same for cycle 2 and scroll down to cycle 3 Note that there is nothing shown in the bar graph for this cycle There are a couple of possible reasons for this either the peptide has only two amino acids or the amino acid for that cycle is destroyed by the experimental process which happens to the amino acid cysteine only Refer to the Help message for this experiment to get a detailed explanation of its effects For now assume that there are only two amino acids in this fragment and close the window Open the Peptide Information Window for the fragment on which the Edman Degradation experiment was performed enter into the Length text box the number of amino acids believed to be in the fragment 2 and press lt RETURN gt The Sequence Determined should now show the same two amino acids entered into the Edman Degradation Window Figure 19 Peptide Information
12. Enter the Help mode by typing and click on the item in the window that is unclear A small help window will then come up describing the item selected Sequence It User Manual 27 File Edit View etusis Madificatian firsauage Windows Intro Problem C Grid Large Font Remove L Circular Spdh GRRE Eiiqegi fii S Peptide by Clostripain Figure 23 Fully assembled sequence After aligning all fragments the window should appear like that in Figure 23 Click on the Save button in the lower right hand corner of the window to register the assembled sequence with the Peptide Information Window and close the Assemble Window Go back to the Summary Window select the peptide just assembled and open the Peptide Information Window Now displayed is the entire Sequence Determined Reconstructing the Original Protein Still only a descendant of the original protein has been sequenced To enter the sequence for the original protein select its icon in the Summary Window and then choose the menu item Assemble Sequence to open the Assemble Window for this protein the only fragment displayed will be that just sequenced the descendant created from the Performic Acid Oxidation Select this fragment s sequence by 28 Sequence It User Manual clicking on it and then Paste from the Edit menu the sequence into the Assemble Sequence region Then save this window s configuration by clicking on the Save button Go back
13. Experiments BEE EE UCCELLI Started with 32 5 noles Sample left 17 5 noles pl 7777 units Length 7 2 an Sequence Determined Display Experiment Data Experiment Kolar E 3 of 3 Peptide 1 by Staphylococcal Protease Figure 19 Peptide Information Window after Edman Degradation Continue by performing the same sequence of actions on each of the remaining three fragments created from the Staphylococcal Protease cleavage being sure to enter all amino acids which are displayed in the bar graphs and to enter the number of amino acids into each Peptide Information Window You will come across unknown amino acids labeled X These are created by modifications on the protein remember the Performic Acid Oxidation modification For now enter them as they appear on the graph later on we will determine the actual amino acid Sequence It User Manual 23 Taking Notes Each peptide created by the program has associated with it a notepad These are standard text windows in which you can store pictures pasted in from the clipboard and text typing information in directly or pasting in previously copied text Each experiment window as well as the peptide information window has present a small standard notepad icon L This icon represents the notepad for the subject peptide that one on which the experiment was performed or the one displayed by the peptide information window To open a notepad click on the icon
14. ble you to alter the display of the Summary Window to show or hide elements of particular concern e The first six items in the menu alter the display of the individual peptide icons according to what experiment has been performed on them For example choosing the menu item By Composition changes the icons so the ones on which an Acid or Base Hydrolysis experiment has been performed will contain a small bar graph representative of the display of the experiment s results Check out the Icons section of the manual for a description of the appearance of the icons for each menu item e The Show Fragment Amount item when chosen a check mark will appear next to the item when activated will cause the amount remaining of each selected fragment to be displayed right next to that fragment s icon Also a more immediate visual clue is offered regarding the amount available As the amount of a peptide decreases beyond a certain level the meniscus descends until when there is no sample remaining the icon appears empty e The Full Menus item will display all available experiments at whatever Apprentice level has been setup The menu will then change to Short Menus which reverses the display e The Amino Acid Data item opens a window displaying the name of all amino acids along with their corresponding three and single letter codes like that in Figure 32 40 Sequence It User Manual Amino Acid Data Amino Acid Alanine Arginine Asparagine Aspar
15. can purify a protein determine its amino acid sequence and then clone the gene for it If the protein purified is an enzyme then the kinetic properties can be determined The central idea inherent in all these programs is embodied in the master apprentice relationship Complex biological concepts that are either too costly or too time consuming to duplicate in a teaching laboratory can be experienced through the use of a microcomputer In this Sequence It User Manual 45 simulated research laboratory however one finds an environment that is infinitely patient is inexhaustible in material and is incomparably safe Our philosophy on teaching is best summarized with the ancient Chinese proverb I hear and I forget I see and I remember I do and I understand 46 Sequence It User Manual Glossary of Biochemistry Terms Amino Acid Amino Acid Residues C Terminus Disulfide Bonds Hydrolysate Meniscus N Terminus These are the building blocks of proteins There are 20 different naturally occurring types of amino acids One amino acid differs from another only by its side chain or R group Latin residuus left over are the linked amino acids in a polypeptide During polymerization water is removed to form the peptide bond or carboxyl terminus of a polypeptide is the last amino acid in a chain By convention residues are numbered from the N terminus and written left to right in the order they occur
16. ck on an icon to open the Peptide Information Window for that peptide e Double click on a descendant label to collapse or expand that portion of the summary tree 42 Sequence It User Manual Reference Communicating with Other Programs All communication between Sequencelt and other programs takes place via the Macintosh Clipboard Think of the clipboard as a place you can temporarily store something from one program and then retrieve from another program You proceed by copying material from one program into the clipboard switching to the other program and then pasting the clipboard into the second program This can be quite clumsy if you need to move several different things between the same two programs since the clipboard can only hold one thing at a time In this situation you may want to look into using the Macintosh Scrapbook which can hold more than one thing at a time Copy Window The Copy Window option on the Edit menu is always active as long as there is at least one window open It will put a picture of the current frontmost window on the clipboard Once it is on the clipboard it can be pasted into a notepad or into another Macintosh program The picture of the window looks exactly like the window This is potentially confusing because it is not a window anymore but just a picture of one This means that the Close box and other controls will not work Copy Window Data Several Sequencelt windows are able
17. de groups of glutamic acid increases the hydrogen ion concentration decreasing the pH needed to give the protein a neutral charge The results of an isoelectric point experiment done on the introductory problem are shown in Figure 29 The two symbols plotted and represent the total positive and negative charges on the peptide at each pH increment The solid line represents the net charge number of positive charges the number of negative charges on the peptide The pH at which the solid line crosses the zero charge line at a pH value of about 4 0 estimates the p1 of the peptide This graph shows that the protein is acidic in that it has an overall negative charge This is caused by the large number of glutamic acid residues single letter code of E Isoelectric Point Original Peptide uy Cc mm gm om ch ma Li Isoelectric Point Record pl Figure 29 The output from an Isoelectric Point experiment on the original peptide As you move the mouse across the graph you will notice that the cursor changes to a target site cursor If you click on the point where the net charge line crosses the zero charge line you will notice that the question marks in the Isoelectric Point field will change to the value you clicked on If you click on the Record pI button your estimate will be written to the Peptide Information Window for that protein Sequence It User Manual 37 End Group Analysis End Group Analys
18. e a title displayed at the top For more information see your Macintosh Owner s Guide 50 Sequence It User Manual Hardware and Software Requirements Sequencelt requires a Mac Plus or later Macintosh computer running System 6 0 5 or later with at least 1 megabyte of memory To tell what system you are running choose the About the Finder item from the menu of the Finder program The Finder program is where you start out before you run a program like Sequencelt Cursors and Icons Cursors k raf The arrow is the standard cursor in Sequencelt Use it to click in the title bar and drag a window to move the scroll box to pull down a menu and choose command and to select a peptide item in a window The wristwatch cursor indicates that Sequencelt is performing a lengthy experiment or operation The I bar cursor appears at the insertion point in the editable text area of a window Click to position the insertion point in the window or text The question mark cursor appears when you enter the Help mode Do this by typing a question mark while holding down the command key Use it to select a command or part of screen that you want to learn about The target site cursor appears on some graphs Click in the graph or legend to assign a residue or to obtain a numerical value Icons N p Sequence It User Manual 51 These are disulfide editing cursors When entering disulfides either via the Customize
19. ent s results now You will be able to redisplay this information at a later time Opening the Peptide Information Window A summary of information for each peptide is displayed in the Peptide Information Window Figure 8 Open this window by selecting the icon in the Summary Window then selecting the Peptide Information menu item found under the View menu Sequence It User Manual 11 File Edit View etusis Mindificetian fisagage Windows Intro Problem Peptide Information Experiments MALAL EL CULI ELE Figure 8 Peptide Information Window Redisplaying an Experiment Displayed in a scrollable list in the right portion of the window are all experiments performed on the peptide To open a window with the results of the experiment select the name position the mouse over the text Acid Hydrolysis and click once then click on the button labeled Display Experiment Data The Acid Hydrolysis Window Figure 7 will now open in the same position it was in when the window was closed Note the value in the sum box in the lower right hand corner and close the window again Entering the Estimated Length The sum found in the Acid Hydrolysis Window is a good conservative estimate of the length of the protein being sequenced Enter this value rounded up to 13 in the edit box labeled Length found in the Peptide Information Window just the 12 Sequence It User Manual whole number portion the program will beep if you
20. ft of the cleavage the other representing the fragment to the right You can picture this as a piece of string as shown in Figure 17 The cleavage enzyme is like a pair of scissors cutting the string at specific sights and creating fragments of string Also remember that the help message stated that glutamic acid residues followed by proline amino acids are resistant to cleavage Therefore it is possible that there are even more glutamic acid amino acids present than the three at which the protein was cleaved OAC 2x QACCOHOAO OOHODACOO N Fragment 1 Fragment 2 Fragment 3 Figure 17 Symbolic representation of protein cleavage Sequence It User Manual 21 Performing an Edman Degradation Experiment You now need to determine as a repeat of Step 3 of the common strategy the sequence of each of the fragments produced from the cleavage One of the most frequently used and most useful procedures is Edman Degradation To perform this experiment choose a peptide from the cleavage the leftmost one below the label Staphylococcal Protease and choose the menu item Edman Degradation from the Analysis menu After the default conditions are presented and are used and the program displays the steps used in this experiment you will be presented with a window like that in Figure 18 Edman Degradation Peptide 1 by Staphylococcal Protease Cycle 1of10 Amino Acid 15 00 7 50 0 00 ARHOCOEGHILENFPSTHYUS Amino 4cid 2of1
21. i e an enzyme which cleaves proteins for our first cleavage Determining which cleavage experiment to perform is one of the more difficult phases of protein sequencing not only do you have to know where each cleavage item works but also you need some idea of the sequence of the protein Close the Help Window for now and select the Descendant icon from the Performic Oxidation experiment Click on the Cleavage menu select the menu item Staphylococcal Protease and use the defaults that the program has setup After the computer has shown the steps done during an actual cleavage experiment the Summary Window is shown with a new addition to the tree The program automatically scrolls the tree so the newly created branch is visible your screen should now look like that in Figure 16 20 Sequence It User Manual File Edit View etusis Madificatiagn firsauage Windows Intro Problem Ferformic Oxidation I Lt Staphylococcal Protease ot YUU Figure 16 Summary Window after Staphylococcal Protease There are four descendants from the Staphylococcal Protease experiment This means that there are at least three glutamic acid amino acids in the sequence This deserves a bit of explanation If there are no amino acids that the cleavage mechanism recognizes there will be one descendant the untouched subject of the experiment If there is one such amino acid there will be two descendants one representing the fragment to the le
22. ial cleavage points referred to as overlapping peptides The amino acid sequence of each overlapped peptide orders two or more of the original fragments 6 Reconstruct the Original Protein From the overlapping peptides and information gained from the original protein you should be able to construct a unique sequence for the protein or polypeptide of interest Your overlaps should be at least two amino acids in length 7 Locate the Disulfide Bonds No primary structure analysis of a cyst e ine containing protein can be regarded as complete before the presence and location of disulfide bonds have been established Using a multistep process described in the tutorial the location of these bonds can be assigned Selecting a Polypeptide Use the mouse to position the cursor over the icon in the Summary Window Press once on the mouse button You ll notice the outline of the icon is now white on a black background indicating that it is selected You should also notice that once the polypeptide has been selected many of the menu items become active 8 Sequence It User Manual Performing an Experiment Once you have selected the polypeptide select the Acid Hydrolysis menu item under the Analysis menu This is a good experiment to start with because it gives you a rough estimate of the total number of amino acids in the polypeptide as well as which of the 20 amino acids are present Remember though some amino acids are destroyed by
23. ide Bonds Disulfide cross links complicate the determination of amino acid sequences and usually are cleaved by reduction or oxidation before sequence analysis 3 Perform an Initial N Terminal and C Terminal Sequence Determination Remember that a polypeptide has polarity and therefore a beginning and end However there are proteins that are made up of two or more polypeptides e g insulin Hence you have two beginnings and two ends Also there are polypeptides where the beginning and ends are joined to form a closed structure In this case there are no N terminal and C terminal amino acids Use End group Aminopeptidase and Edman degradation experiments to determine as much of the N terminal sequence as possible and use Carboxypeptidase to determine the C terminal amino acids Sequence It User Manual 7 4 Divide and Conquer Break the polypeptide into fragments by cleaving at specific amino acids Several cleavage methods are available each of which has different specificity i e cleave at different amino acids 5 Repeat Steps 3 and 4 To Determine Subsequences and Create Overlappings The initial cleavage is generally made as specific as possible in order to generate large peptide fragments It is easy to arrange fewer fragments These fragments can be positioned relative to one another after treatment of the original polypeptide by a second cleavage procedure that generates fragments whose sequences extend across the init
24. ike that in Figure 26 select the radio button corresponding to the blocking you re interested in Amino terminal block O Formyl O Acetyl O Pyroglutamyl Unblocked Carboxyl terminal block Amide w Unblocked Figure 28 End Groups dialog Setup Disulfides Button Enabled when at least two cystine amino acids are entered into the sequence box the button will open a window identical to that used to enter disulfide bonds in the introductory problem Amount Entry The default value of 200 nMoles is placed here change it to whatever amount you d like to use to solve the problem Setup Menus Button Use this to filter the menus for different apprentice levels Instructors setting up problems for their students may want to have only the least complex menu items displayed to lessen the chance for confusion and frustration Sequence It User Manual 35 Read From File Button Use this to read in a sequence from another file Click on the button to open a standard Macintosh dialog for opening a file the sequence will be entered into the editing area and can then be modified Analysis Experiments There are several experiments available which were not needed for the determination of the sequence for the tutorial problem These are shown under the Analysis menu and are chosen like any other experiment Base Hydrolysis Base hydrolysis involves treating a polypeptide with a strong base to hydrolyze all pe
25. ingle letter code With the Aminopeptidase experiment we have determined the amino terminal N terminal amino acid Using the Carboxypeptidase experiment we can determine the carboxy terminal C terminal amino acids Close the window with the Aminopeptidase results and click on the Analysis menu again this time choosing the Carboxypeptidase Y menu item After following the same process as with Aminopeptidase digestion a window like Figure 13 will appear Carbouypeptidase Y Peptide 1 by Performic Oxidation Ll Amount Used 10 0 nMoles Sequence It User Manual 17 Figure 13 Results from Carboxypeptidase Y experiment This experiment produces considerably more interesting and less easily interpretable results As you can see at 10 minutes about 1 8 nMoles of threonine is released and only slightly smaller amounts of alanine and glutamic acid After these three amino acids the amounts of each amino acid released at each time point are almost identical making sequence determination beyond the first three amino acids released impossible We now can assign the last three amino acids of the polypeptide Move the cursor over the legend item representing threonine three letter code Thr and click once then move over the item for alanine Ala and select it and do the same for glutamic acid Glu Note that the sequence is being entered from right to left i e from the C terminus toward the N terminus This is normal for carb
26. is Original Peptide T I Oo E I SOC a BE E a e D a a rn a A g A a E a E a G a ar a a B a nMoles Figure 30 Output of End Group Analysis on original peptide Use this experiment to unequivocably determine the N terminal amino acid for the peptide in question The window showing the results Figure 30 closely resembles that obtained from acid and base hydrolysis experiments Each bar represents the quantity of each amino acid reacting with the end group reagent As you move the mouse across the graph you will notice that the cursor changes to a target site cursor If you click on the bar for each amino acid the quantitative value will be entered in the table to the right of the graph The sum or total of each quantity will be tallied for you in the Sum box at the bottom of the window You can also perform a click drag down the graph and fill the table in one operation If you click on the bar representing the quantity of serine the single letter code for serine S will be entered in the small text box representing the N terminus for this peptide You can also type the N terminus assignment directly into this text box A corresponding entry will automatically be made to the Peptide Information Window 38 Sequence It User Manual File Menu Setting Aside a Problem You can only have one problem open at a time in Sequencelt However you can Set Aside a problem to the LabBench Figure 31 for later re examination A
27. ll the work you have currently performed will be saved to a disk when you set aside a problem We implemented this tool to allow you to step away from your current research problem and try other approaches or techniques on other peptides without costing you any of your starting material This strategy of putting aside a complex problem and attempting simpler well behaved problems i e problems in which you know the answer before hand is a useful tactic in science View riaiueis Maodifigcgiign fimsuacge Windows Intro Problem m Figure 31 The LabBench Window containing a previously Set Aside problem Double clicking on the file icon in the LabBench will close the current problem and restore the selected problem to your screen You can have as many problems sitting on the LabBench as you desire but only one open at a time Also choosing the menu item Restore will re open the currently selected problem in the LabBench Sequence It User Manual 39 Printing from Sequencelt The only method of getting printed output from the program is through the notepads To print a notepad make sure that it is the frontmost window then select the Print Notepad menu item under the File menu A standard Macintosh printer dialog will appear allowing you to select the number of copies and the quality of the print View Menu The View menu of the program has several items not described during the tutorial portion of this manual These functions ena
28. lpsssisiiicsnrincneiaiinini aiaiai 12 Removing Disulfide Bod Sresssmcnsicninenrcunanneininiiunnann 13 Initial Sequence Determination e esssssssssssessisieresisestsrssesisiereresisesssrestses 15 Performing a Cle vag eoar ni a E A AAEREN 18 Performing an Edman Degradation Experiment ssesessssereresesesssreseses 21 Taking IN GCS vised incertectlioonanebaaeenciinntemen a ennnaea 23 S ving the Proplemeiiiscsrisoisiinsissn tiaia iiia 23 Performing a Second Cleavage sssesssssessisisisesrrsrereresesisesesenisesrseersnsenes 24 Reconstructing a PROVE II canescjicsicsticeinn sais dancssguinnsiigianeeinanimantecumsssenimustnnbinsins 25 Reconstructing the Original Proteti ss cainaswstsiantinnainsivnadntinasclainensiionieies 27 Moving and Collapsing Branches of the Summary Tree 008 28 Determining the Location of Disulfide Bonds ccccceeseeeeeeeeeees 29 Entering Disulfide Bonds sisi ssisistacivssnsiensoiitaniesinte ons istnasanieunieeaunenmeansins 30 Designing Your Own Problems sssini nnise iite 32 Compu ter Generated siirus iniii ensaia asi iat 32 User EO icici ccotexncasiuatnteleciamoextaadenet a EEE aE REEE E o E Ea 33 Analysis ExperiM ntS yi sxcacncnarssssnncnnaussactvorananisauscunisssiuadeannsdenseimeansanmainanduncncenn 35 Base HydfolysSiS ennn O 35 Isoelectric Point De teria tiOin isisisnassacssctssiedsconaennccarsssdsnanancescdaddeaaunnantaneens 35 End Group Analysis seeno nino a E R 37 File M en ssinieei
29. n firsauage Windows Intro Problem WPeptide 1 by Clostripain Figure 25 Screen appearance for disulfide bond setup To input the disulfide bond click on one of the buttons representing a cystine amino acid drag the mouse to the other then release the mouse button A graphical representation of the bond will then be displayed Click on the OK button to close this window and be sure to save the newly assembled protein using the Save button in the lower right corner of the window The tutorial is now complete you have sequenced the protein SCIENCEISGREAT as the introductory problem This small peptide has been found circulating in the blood stream of every research scientist in the world Hopefully this has given you insight into how the program works as well as made you excited enough to continue allowing the program to generate proteins of increasing difficulty Check out the section of this manual on Designing Your Own Problems to find out how to set the level of difficulty or enter your own protein 32 Sequence It User Manual Designing Your Own Problems Though Sequencelt can be used to study preexisting problems its true power lies in allowing you to conceive define and control all aspects of new problem sets Therefore understanding how to set up a problem from scratch is important in utilizing the program s full potential Computer Generated Start Sequencelt and choose Setup from the Startup Screen dialog Y
30. nches of the tree To move a branch of the tree click on a label e g the text box labeling the Performic Acid Oxidation experiment while holding down the mouse button an outline of the label will move with the cursor move the cursor to the position where you want the label to be displayed then release the mouse button To collapse a branch of the tree select a label by clicking once on the text then move to the View menu and choose the menu item Contract This reduces the branch of the tree so that only the label is displayed Reverse the process by selecting the label again and choosing the menu item Expand from under the View menu Sequence It User Manual 29 Determining the Location of Disulfide Bonds There remains only one step in the full determination of the sequence to find out if disulfide bonds exist and what their locations are Remember that we modified the original protein as one of the first steps and this modification was performed to get rid of the disulfide bonds We then performed several cleavages to divide the modified protein in particular the cleavage using the enzyme Staphylococcal Protease This enzyme cleaves at glutamic acid amino acids single letter code E and now we know that there are three of these in the sequence Since disulfide bonds connect two cysteine amino acids with a particular kind of bridge distinctly different from the standard peptide bond and there is a glutamic acid residue in
31. ndows and throw things away thus controlling the way your working area looks is a window that contains requests for instructions or information For instructions on using dialog boxes see your Macintosh Owner s Guide is the pictorial representation of a task or object Menu Mouse Notepad Scrapbook Window Sequence It User Manual 49 At the very top of your Macintosh computer screen there is a white bar with a number of symbols and words This is called the menu bar each of the symbols and words is the title of a menu For more information about menus and how to use them see your Macintosh Owner s Guide Please see your Macintosh Owner s Guide for information about what a mouse is and how you use it is a window that you can type into or insert pictures or data into using the Copy and Paste procedure It is a place to write notes to yourself or to collect your thoughts for a lab report Each fragment has an associated notepad To open a notepad click on the E symbol in the upper left of the window Empty notepads are denoted by an empty symbol u The scrapbook is a desk accessory that will store more than one thing for you This is an advantage over the clipboard which can only store one thing at a time See your Macintosh Owner s Guide for more information on desk accessories and the scrapbook A window is an area of the computer screen usually rectangular that displays information Most windows hav
32. ng the linear sequence of amino acids composing a protein i e the primary structure is what we view as a biological craft best learned from such masters as Nobel Laureate Fred Sanger This clearly is not possible for most students Moreover protein sequencing has evolved into a highly technical field requiring expensive instrumentation and hazardous chemicals However the tools and logic involved in this craft can be taught to all students whether or not they are science majors Our philosophy requires that you construct meaning as you work by which we mean that beyond discovering a fact or finding you are continuously challenged to evaluate three structures of your knowledge problem statement defense of the real problem to be solved methodology your solution strategy and justification defense of your current knowledge We place peer group learning as an important adjunct to this approach and have accordingly made peer review and criticism of your electronic notebook an integral part of our approach Using this approach the role of your instructor is redefined from one of authority to one of a colleague who earns credibility through demonstrated skill Sequencelt is one of several programs composing The Biotechnologist s Workshop Other programs include protein purification Purifylt enzyme kinetics Ratelt and DNA cloning Clonelt Each program can be used alone or in concert with the others For example you
33. nonoi a Cnet a eae 38 Setting Aside a PPG OU g ancssrt atta satnne ited grolaiattinnas nna 38 View Menu tosenn rides edea nina minced EE aaiae EARE denea iek 39 Windows MenUesssisesisiineniisirasineiin eenaa iaeiaiai 40 Short ttSissin eatea a EE EEEE S R E 41 Summary Window SNOT CUS winintisccassesaniasiscassicaaisiinamaiianeienaaainanta 41 Referente ascsshdivasnsasineiattainsnitgoinedsseassamaeiariaigaimninaeuineiasgianeluna i a ai i 42 Communicating with Other ProgramS s ss sssssssessssesessissesesseseesiseesesn 42 Unkown Cleaya g S ssitniniioncinni skiis asiaa 43 Appendices ine aaa RA ARE E A eid 44 What Is the Educational Philosophy Embodied in Sequencelt 44 Glossary of Biochemistry Terms seicisinctsnaictsissntinminisacnamareanentianensioniiss 46 Glossary oft Computer Terms sisisihin eais a ai 48 Hardware and Software Requirements vicsciscicrassscscnssarsasearcarasaatserieaanaseess 50 Cursors and COME isis isesstasinsitocineimaniontate iasateeinsiaieaseiachuaniineineingalenaanioliumeun 50 Sequence It User Manual 1 What Is Sequencelt Sequencelt is a training tool that allows you to experience the art and logic of protein sequencing through experimentation You are given a polypeptide or protein whose sequence the linear arrangement of amino acids and length are unknown Your objective is to deduce the sequence of this polypeptide using many of the tools available to a practicing protein chemist You specify
34. ou can also setup a problem by holding down the option key when choosing New under the File menu After this you will be presented with the Problem Setup dialog Figure 26 Initially the window displays the options for the Computer Generated setup Here the options are simple just a set of controls called radio buttons As you click in the radio button for each apprentice level you will be provided with a short description describing the general nature of the problem which will be automatically generated after clicking in the Sequencelt button Problem Setup w Computer Generated i User Entered Apprentice Level Beginner 42nd Year O Sth Year O Master The Beginner stage of your apprenticeship generates a protein of up to 10 random amino acids with an amount of not less than 200 nMlioles Save To File Sequencelt Figure 26 Problem Setup dialog showing the Computer Generated options Sequence It User Manual 33 User Entered If you would like to know the sequence before you start or would like to import a sequence of a known protein click on the User Entered radio button at the top of the window The window will change to look like Figure 27 You can now enter a sequence in several ways First if you click on any of the buttons representing the three letter code for each amino acid the single letter code for that amino acid will be entered in the sequence edit box at the current insertion point where the cursor is
35. oundation Terry L Derting Murray State University Roscoe Giles Boston University Louis Gross University of Tennessee Knoxville Yaffa Grossman Beloit College Raquel Holmes Boston University Stacey Kiser Lane Community College Peter Lockhart Massey University NZ Ed Louis The University of Nottingham UK Claudia Neuhauser University of Minnesota Patti Soderberg Conserve School Rama Viswanathan Beloit College Linda Weinland Edison College Anton Weisstein Truman University Richard Wilson Emeritus Rockhurst College William Wimsatt University of Chicago Copyright 1993 2006 by Allen R Place and Thomas Schmidt Copyright Trademark and License Acknowledgments Portions of the BioQUEST Library are copyrighted by Annenberg CPB Apple Computer Inc Beloit College Claris Corporation Microsoft Corporation and the authors of individually titled modules All rights reserved System 6 System 7 System 8 Mac OS 8 Finder and SimpleText are trademarks of Apple Computer Incorporated HyperCard and HyperTalk MultiFinder QuickTime Apple Mac Macintosh Power Macintosh LaserWriter ImageWriter and the Apple logo are registered trademarks of Apple Computer Incorporated Claris and HyperCard Player 2 1 are registered trademarks of Claris Corporation Extend is a trademark of Imagine That Incorporated Adobe Acrobat and PageMaker are trademarks of Adobe Systems Incorporated Microsoft Windows MS DOS and Windows NT are either registe
36. ow Fragment Amount 45 Single Letter Code 15 45 Staphylococcal Protease 24 Summary Tree 11 25 34 47 Summary Window 11 Sequence It User Manual 55 56 Sequence It User Manual Tab Delimited Text 48 Taking Notes 29 Target Site Cursor 15 56 Three Letter Code 15 45 Time Based 22 Unknown Amino Acids 28 User Entered 39 View Menu 15 16 45 57 Windows Menu 46 Wristwatch Cursor 56
37. ox title bar 2 Sequence It User Manual scroll bar and clipboard These terms are all defined in the glossary at the end of the manual It also helps to understand some basic biology and chemistry For example the largest molecules in living things polysaccharides nucleic acids and proteins are polymers built from simpler units of the appropriate type Amino acids are the building blocks of all proteins Figure 1 The amino acids possess both an acidic group carboxyl group and a basic group amino group Both of these chemical groups are attached to the same carbon atom which is call the a carbon The side chains or R groups are what distinguish each amino acid from another It is the properties of the R groups that control the function of polypeptides or proteins Amino Group o 0 N t a Carboxyl Group li e T NH Bme Cam H I lin R Group id Figure 1 The structure of the amino acid glycine Through a process called condensation or dehydration amino acids are linked together into a polymer with the production of a water molecule Figure 2 A linear polymer of amino acids is referred to as a polypeptide A protein is simply a large polypeptide Amino Acid 1 Amino Acid 2 C Terminus pe pe R H R z l l 7 Ho FLOD NH meoo Pg NH EONA ROD H R H R 2 HO 2 N Terminus Peptide Bond Figure 2 The polymerization of two amino acids to form a dipeptide Sequence It User Manual 3 Twen
38. oxypeptidase experiments because the enzyme starts cleavage at the C terminal end of the protein and progresses toward the N terminal end right to left Close the Carboxypeptidase Results Window and open the Peptide Information Window for the descendant icon on which these experiments have been performed It will look like Figure 14 Note that all the experiments are listed that the amount available is reduced by the amount used by the two experiments and most importantly the sequence entered in the exopeptidase experiment windows is reflected in the Sequence Determined box Peptide Information Experiments KEULEN HAA GEGA Aminopeptidase Mi Started with 150 0 nMoles Sample left 130 0 nMoles pl 7777 units Length 7 13 an Sequence Determined Display Experiment Data Ps 1 of 2 Peptide 1 by Performic Oxidation 18 Sequence It User Manual Figure 14 Information after initial sequence determination Performing a Cleavage Now it s time for Step 4 of the common strategy Divide and Conquer Cleavage experiments are used for this step in that they divide a protein into fragments by cleaving at specific sites before or after single amino acids or a group of amino acids Here is where the help system comes in very handy as each menu item under the cleavage menu has a corresponding help which describes the location at which the enzyme or chemical cleaves This time enter the help system by selecting the menu i
39. ptide bonds and return the quantity in nanomoles of each amino acid released from a known quantity of polypeptide The output is displayed in a window identical to that found with acid hydrolysis The major use for this procedure is to obtain accurate estimates for tryptophan which is unstable to acid hydrolysis Many other residues are affected by base hydrolysis among them Cys Arg Thr Ser and Lys Where stable derivatives are produced XAA will appear in the output As with acid hydrolysis Asn and Gln are converted to Asp and Glu respectively Isoelectric Point Determination The isoelectric point of a protein is the pH at which the net charge of the protein is zero i e the point at which the positive and negative charges are equal This experiment is useful in distinguishing peptides with acidic residues from those with their neutral counterparts For example an acid hydrolysis experiment converts all asparagine and glutamine amino acids neutral to aspartic acid and glutamic acid respectively Hence there s no way with this experiment to tell if the subject actually contains the neutral amino acids By determining the isoelectric point or pl of the protein one can make the distinction In the absence of any positively charged side groups a protein with glutamic acid residues will have a lower pI than a protein with glutamine amino acids This is because the presence of the 36 Sequence It User Manual negatively charged si
40. pty table If you need help in remembering this amino acid alphabet you can display a list of them by selecting the Amino Acid Data menu item under the View menu As you move the mouse across the graph you will notice the cursor changes to a target site cursor If you click on the bar for each amino acid the numeric value represented by the bar will be entered in the table to the right of the graph The sum 10 Sequence It User Manual of all quantities will be tallied for you in the box at the bottom right corner of the window You can also perform a click drag dragging while holding down the mouse button over the graph and fill the table in one operation You may be confused by the fact that the quantities of some amino acids are not whole numbers The value graphed represents only a minimum estimate for the true number of each amino acid Welcome to the thorny side of science You will learn with experience that some procedures partially or completely destroy a particular amino acid For example the quantity of the amino acid serine with the single letter code of S is given as 1 47 residues Our experience tells us that there are probably 2 residues of serine in the unknown protein You can edit the value in the table by clicking on the numerical value 1 47 and typing in a new estimate e g 2 00 hitting the return key to enter the value The sum will change for your automatically Close the window displaying the experim
41. quence information exists for that peptide The pI Tube icon indicates that isoelectric point determination has been performed on that peptide These two icons are found in the End Group and Exopeptidase Windows The first represents the N terminal residues of a peptide and the second represents the C terminal residues of a peptide These two icons are found in the Assemble Window and indicate peptides that are neither circular nor have terminal blocking groups These two icons are found in the Assemble Window and indicate that the peptide is circular This icon is found in the Assemble Window and indicates that the C terminus of the peptide is blocked These icons are found in the Assemble Window and indicate that the N terminus of the peptide is blocked represents a formyl blocking group an acetyl blocking group and al pyroglutamyl blocking group Index Acid Hydrolysis 12 14 Amino Acid Composition 12 Amino Acid Data 15 45 Amino Acids 8 Amino Group 8 Aminopeptidase M 21 Amount Entry 40 Analysis Menu 14 21 31 Arrow Cursor 56 Assemble Sequence 31 33 Assemble Window 31 33 58 Assembled Sequence 32 Base Hydrolysis 12 41 By Amino Terminus 57 By Carboxy Terminus 58 By Composition 45 C Terminus 9 Carboxyl Group 8 Carboxypeptidase Y 22 Circular Check Box 40 Cleavage 24 Cleavage Menu 30 Click Drag 16 Clostripain 30 Common Strategy 12 Computer Generated 38 Cursors 56 Descendant 21 De
42. red trademarks or trademarks of Microsoft Corporation Helvetica Times and Palatino are registered trademarks of Linotype Hell The BioQUEST Library and BioQUEST Curriculum Consortium are trademarks of Beloit College Each BioOQUEST module is a trademark of its respective institutions authors All other company and product names are trademarks or registered trademarks of their respective owners Portions of some modules software were created using Extender GrafPak by Invention Software Corporation Some modules software use the BioQUEST Toolkit licensed from Project BioQUEST Table of Contents WV Fie Is SECS LE usensu iinise a akiai a a asoa 1 What Yo Need To RG Wi sicaxitsaccaxassiearaaiociareadanersasacdanaistasmaacateataveaaneaxauceieas 1 A TOUR of SOQ UCI CSIs sasascin ncsescadccnaiaisinavassinsen anc cancrsieatwnasienane damepeoannsaeasalteivariipineanes 4 Starting the PFO STAIN aia caaesssssgicsieasrantiunaigaiveuatuoutianeiaiedniestiuaiessiaedtaowvarasentieiuened 4 A Common Strategy for Protein Sequencing iiss ccisivsiaasianniasiasrsclarncandeats 6 Selecting a Polypeptide nasrin oent E R 7 Performing an ExperiMent sisssssisssiisssessinisisiiiiisisssinisiiriressins 8 Opening the Peptide Information Window ssssssssssesssissesesseseesessesesn 10 Redispl ying an EXxperi ent ssiisrsiesiisisisiisivestioisasininei 11 Entering the Estimated Length sseesssessssseseresesssssisesssrsisesesrsnsennsneneneseses 11 Getting Some He
43. rmation is available on a specified object The icon currently displayed in this window represents a plastic tube containing the polypeptide to be sequenced As we continue the numbers of icons will increase in number and be displayed in an upside down tree format Each of these icons represents a tube containing a protein or a fragment of a protein termed a polypeptide This is a general term we will use throughout the manual 6 Sequence It User Manual A Common Strategy for Protein Sequencing Protein chemists follow a basic strategy when they attempt to determine the sequence of most proteins and polypeptides This strategy is outlined below and described in greater detail as we go through the steps of the tutorial Keep in mind that this strategy is only a guide and should not inhibit your own ingenuity on solving the sequence of a protein 1 Determine the Amino Acid Composition In order to know which amino acids and how many of each amino acid there are in a polypeptide we must break the peptide bonds This can be accomplished with strong acids i e 6N HCl or strong bases or by exhaustive enzymatic digestion By performing an acid hydrolysis or base hydrolysis experiment you obtain a minimum length for the polypeptide This information is useful for determining what cleavages to use in Step 4 However there is a caveat These procedures destroy several amino acids so you must be cautious with your estimates 2 Break All Disulf
44. rom Staphylococcal Protease Sure enough we get a single cleavage and hence two fragments are created After some resizing of the window and scrolling to make everything visible you should be able to get the Summary Window to appear as shown in Figure 20 Intro Problem Peptide To Be Sequenced y Performic Oxidation l j wor Clostripsin Staphylococcal Protease Figure 20 Summary Window after Clostripain cleavage Now you need to sequence the two fragments created Use the Edman Degradation experiment recently described and don t forget to enter the length into the Peptide Information Window to ensure that the program knows how many amino acids to display as the Sequence Determined This is the fifth step of the common strategy repeating Steps 3 and 4 in order to get overlapping peptides Another symbolic figure is supplied here in Figure 21 to help explain this concept The sequence of the fragments from the first cleavage can be determined easily enough and from the Aminopeptidase and Carboxypeptidase experiments we can determine which of the fragments are from the N terminal and C terminal ends of the peptide but how to order the other fragments which goes Sequence It User Manual 25 to the left which to the right By performing another cleavage and sequencing the fragments created it becomes easy to make this determination now the only problem is entering this information into the computer
45. rted with Sequencelt is by taking the Introductory Tour of Sequencelt Feel free to experiment as you go along on your journey 4 Sequence It User Manual A Tour of Sequencelt Starting The Program oth a Sequence It Figure 3 The Sequencelt program icon To start your tour of Sequencelt double click on the Sequencelt program icon Figure 3 File 431 View naiusis Modification fieauvage Windows SOGUENEeT ET An Apprenticeship in Protein Sequencing Sequencelt Lysozyme Introduction _ Figure 4 Sequencelt startup screen dialog The next thing you will see is the Sequencelt startup screen dialog Figure 4 This window gives you the option of beginning the program with an unknown protein Sequence It User Manual 5 the Sequencelt button or with a protein you design the Setup button You can also obtain additional help on using the program the Help button or begin your Sequencelt apprenticeship with the introductory problem the Introduction button Click on the Introduction button now so you can follow along with the tutorial File Edit Diew reissis Mindifinaiian fLissuege Windows Intro Problem Peptide To Be Sequenced y Icon representing original protein Figure 5 Summary Window A Sequencelt problem always starts with the Summary Window Figure 5 With this window you are able to choose a variety of ways to view what you have done and what type of data or info
46. scendant Label 21 47 Disulfide Bonds 12 13 18 19 35 36 Sequence It User Manual 53 54 Sequence It User Manual Disulfide Editing Cursors 57 Divide and Conquer 13 24 Edit Menu 29 Edman Degradation 27 End Group 58 End Group Analysis 43 End Groups Button 40 Eppendorf 57 Exopeptidase 58 Exopeptidase Degradation 21 File 29 File Menu 38 44 45 Full Menus 45 Glossary of Biochemistry Terms 52 Glossary of Computer Terms 54 Hardware and Software Requirements 56 Help Mode 18 Icons 57 Isoelectric Point 41 58 LabBench 44 Length 17 Length 17 Lower Case 18 Meniscus 45 57 Microcentrifuge Tube 57 Modification 28 Modification Menu 18 N Terminal 43 N Terminus 9 New Protein 38 Notepad 29 Notepad Icon 29 57 Open 29 Open Notepad Menu Item 29 Overlappings 13 Peptide Information Window 47 Peptide Information 16 Performic Acid Oxidation 18 19 Polypeptide 8 11 Polypeptide or I Protein 7 Primary Structure 9 Print Notepad 45 Printed 29 Printing 45 Protein 8 Question Mark Cursor 56 R Groups 8 Read From File Button 41 Reconstruct 13 Reconstructing 31 33 Redisplaying an Experiment 17 Reference 48 Remove Info Page 46 Restore 44 Save 29 Save To File Button 40 Saving 29 Selecting a Polypeptide 13 Sequence 7 Sequence Determination 21 Sequence Determined 18 33 Set Aside 44 Setting Aside a Problem 44 Setup Disulfides Button 40 Setup Menus Button 40 Short Menus 45 Sh
47. tem Help with Sequencelt from under the Apple menu with the icon title This opens a larger window with all help topics available in a list Figure 15 What is Sequenceltl Saving Loading Problems When ls Your Peptide Sequenced Notepads Sequencelt Windows The About Box Startup Window Sequencelt Button Setup Button Help Button Lysozyme Picture Figure 15 General Help window Scroll down the list until the enzymatic cleavages are displayed and open the help message for Staphylococcal Protease Note first that this new window with the text for the cleavage help displayed Figure 15a allows you to move quickly to the previous and next help messages as well as back to the topic list Use this to scan through help messages more generally entering the help mode and selecting Sequence It User Manual 19 individual items can get tedious after a while especially if you re just looking around Staphylococcal Protease Staphylococcal protease also referred to as the Vo endopeptidase cleaves on the carboxyl side of glutamyl residues Glu Pro sequences are resistant to hydrolysis k Figure 15a Help for Staphylococcal Protease The message states that this enzyme cleaves on the carboxyl side of glutamyl also called glutamic acid residues We know that there is at least one of these amino acids present remember the results of the Carboxypeptidase experiment so we ll use this protease
48. the operations and the order of their application Success in determining the protein sequence depends on your understanding of the experimental procedures and on the logic you use in executing those procedures Operations available range from simple cleavage reactions enzymatic and chemical which break a polypeptide into smaller fragments to complex sequential analyses e g Edman degradation that provide sequence information Sequencelt replicates laboratory situations with errors and ambiguities and places limits on the amount of material available for study You can set up Sequencelt so as to use only a simple short polypeptide initially and add new complexity as your grasp of protein chemistry grows We have incorporated many organizational tools to help in your hypothesis generation and data retrieval Overall we want you to experience what science is about by doing it What You Need To Know We assume in this manual that you are already familiar with the basic set of standard Macintosh terms and operations If you are not comfortable with these you should work with the Macintosh Owner s Guide or another introduction to the Macintosh before you begin The terms you need to be familiar with are window menu mouse point click shift click drag and double click If you understand these you are ready to get started Before long however you will also need to know several more Macintosh vocabulary words close box zoom box grow b
49. tic Acid Cysteine Glutamine Glutamic Acid Glycine Histidine leqleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Unknown Side Group CH3 CH23 NH CNH NH2 CH2 CONH2 CH2 CO0H CH2 SH CH2 CH2 COMH CH2 CH CO0H H CH2 imidazole CH CHS CH2 CHS CH CHICHZI2 CHZ 4 HH2 CH2 CH2 CH3 CH2 phi N CH2 3 0H CH2 0H CHI CH 0H CH2 indole CH2 phi OH CH CHSI2 Figure 32 Display of Amino Acid Data e The Remove Info Page item active only when the Peptide Information Window is frontmost deletes the displayed page of the information window Windows Menu The Stack Windows command reorders all the open windows by overlapping them The windows are restacked in the standard overlapping pattern that is created when you open several windows in a row This arrangement makes visible as many titles as possible while keeping all windows large To make a stacked window active click its title bar to bring the window to the front The remainder of the menu lists all currently open windows by their titles Select one of these items and that window is brought to the front Sequence It User Manual 41 Shortcuts This section describes ways to speed up use of the program It s useful after you ve become familiar with the more basic functions Summary Window Shortcuts e After selecting an icon use the command key equivalent of I or simply double cli
50. to put a copy of the data they contain on the clipboard in a form that is usable not only displayable by other programs The data are in tab delimited text format that is useable by many word processors spreadsheets data base managers and graphing programs Sequence It User Manual 43 Tab delimited text is text arranged in columns with each column separated by a tab If you paste this into a word processor you will need to set the tab stops to line up the columns nicely Unknown Cleavages To add some variety to the type of problems you can experience in Sequencelt we added three cleavage reactions Protease 1 amp 2 Reaction 1 to the Cleavage menu whose specificities are undefined It is your job to determine what peptide bonds are cleaved by these reactions and the specificity of the cleavages Hint Perform cleavage reactions on short polypeptides of known sequence We have included some test peptides for you to examine They are included in the Test Peptides Folder The file name is the sequence of each test peptide 44 Sequence It User Manual Appendices What Is the Educational Philosophy Embodied in Sequencelt To work under and learn from a master craftsman has been the concept of an apprenticeship since the origin of the word in the Middle Ages A student learns from the experience and method of the teacher and is afforded access to the tools the master originated and developed in perfecting his craft Determini
51. to the Summary Window and open the Peptide Information Window for the original protein Displayed is the entire sequence determined but now we must deal with the unknown amino acids those labeled with the single letter code X One way to figure out which are the actual amino acids in the sequence is by a process of elimination Reopen the window displaying the results of the Acid Hydrolysis experiment and notice that there were two cysteine amino acids counted Since there are none displayed in the Sequence Determined and there are only two unknowns they must be the cystines This one is easy For future proteins it could be much more difficult amino acids could be modified and at the same time destroyed by the Acid Hydrolysis experiment there could be several unknowns of different types etc Go back to the Assemble Window for the original protein and change the unknowns to the single letter code for Cystine C Save these changes and close the Assemble Window Moving and Collapsing Branches of the Summary Tree The number of branches of the tree represented in the Summary Window can quickly become overwhelming Often they represent dead ends in the sequencing process or if one is sequencing a very complex protein the branches could be going all over the place distracting one from the current fragment being sequenced For this reason the program allows you to reorganize the placement of branches and also collapse and then expand bra
52. try to enter the decimal point and press lt RETURN gt Now the Sequence Determined is displayed Since we haven t determined any of the actual sequence only question marks are shown As we continue and begin to enter the sequence this portion of the window will display the single letter codes of all amino acids entered Usually the letters displayed will be upper case If a letter is shown in lower case then there is some conflict in the amino acids assigned for this position in the polypeptide For example you may have assigned one amino acid for the N terminus from the Aminopeptidase experiment and a different one from the Edman degradation experiment The letter displayed is from the first experiment in which an assignment was made Close the Peptide Information Window now by clicking in the Close box Getting Some Help We are now ready for Step 2 of the strategy explained above namely removing disulfide bonds These bonds are a different type of link between amino acids than the standard ones i e peptide bonds which connect them together to form the polypeptide Disulfide bonds are links between two cysteine amino acids while we will have to determine their position later to consider the protein fully sequenced for now their presence complicates the sequencing process by keeping cleaved fragments together We remove disulfide bonds by modifying the R group of cysteine to a less reactive form To determine which modification to
53. ty different amino acids are found in proteins their precise order in a given protein the primary structure or sequence is determined by the order of a different class of building blocks in the corresponding DNA At one end of the polypeptide there is a free amino group the N terminus and at the other end there is a carboxyl group the C terminus All the other amino and carboxyl groups of the amino acids are involved in peptide bonds Thus there is a directionality or polarity to the polypeptide as well as a sequence or linear arrangement of each amino acid The order of amino acids determines what a protein will do how it will look and where it will reside in an organism Proteins occasionally can be crosslinked by a linkage called a disulfide bond which forms between two cysteine amino acids This crosslink helps lock a polypeptide into a particular structure or arrangement The number of different proteins or polypeptides is enormous Since there are 20 different amino acids there are 20 X 20 400 distinct dipeptides and 20 X 20 X 20 8000 different tripeptides Considering that the average protein is approximately 150 250 amino acids in length the number of distinct structures is extremely large Each of the different possible proteins would have its own distinct primary structure It is your job to determine what this arrangement of amino acids is by using the techniques afforded a practicing protein chemist The best way to get sta
54. use we will use the Help system To enter into the Help mode type The cursor will change to a question mark Now go to the Modification menu and select the Performic Acid Oxidation menu item A window will appear and the screen will change to appear like that in Figure 9 Sequence It User Manual 13 File Edit View gaiss Modification fisauage Windows gt Performic Acid Onidation Performic Acid Oxidation removes disulfide bonds by reacting With the SH group of cysteine residues and the S S bond of cystine residues to produce cysteic acid This modified amino acid imparts a negative charge on the peptide pKa 1 5 and is stable to all conditions used in protein sequencing Also methionine residues are converted to methionine sulfone In all subsequent analysis these residues Will be represented as XAA Figure 9 Help for Performic Acid Oxidation menu item This is a detailed description of the Performic Acid Oxidation process and the important part is stated in the first sentence Performic Acid Oxidation removes disulfide bonds Close the Help Window for now but remember that you can get help on any menu item and many window elements by the same process enter help mode and click on the item in question Removing Disulfide Bonds Select the icon for the original protein in the Summary Window and select the Performic Acid Oxidation menu item A new options dialog comes up looking like the one in Figure 10
55. uses either three letter or one letter abbreviations of amino acids The side group of amino acids which distinguishes one from another They range in complexity from a single hydrogen atom to multiple carbon ring structures is the recovery or yield characterizing a sequential reaction or degradation is the linear arrangement of amino acids making up a polypeptide or protein See R Group 48 Sequence It User Manual Glossary of Computer Terms Clipboard Customize Desktop Dialog Box Icon The clipboard is an invisible place where something usually a picture or a few words can be stored for a moment When you Copy text you put it on the clipboard When you Paste you are pulling something off the clipboard to insert it into a document The clipboard is part of the computer not part of the particular program you are using so you can use it to transfer information from one program to another is the ability to change parts of the program to better fit your needs and instructional objectives In Sequencelt you can change the sequence and quantity of the polypeptide the default conditions for a particular procedure available on the menu bar using the option click and difficulty of a problem is analogous to the picture of what lays on your desk surface It is the overall arrangement of menu bars windows icons documents etc that you see on the screen You can move things around open resize and close wi
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