MUTATIONS AND DEVELOPMENTAL PROTEINS FROM DROSOPHILIA
Introduction
You are one of the world’s leading scientists (congratulations!), and you have been doing genetic research with the species Drosophila melanogaster (a.k.a. fruit flies). One day you stumble into the lab and notice that one of your specimens has a pair of legs growing out of its head! You scratch your head and take a second look. Unable to explain, you pull out your handy "Answers to Strange Questions" encyclopedia and learn that this phenomenon is caused by a genetic mutation in a gene that encodes for a protein called the Antennapedia protein. This protein is responsible for suppressing the formation of legs on the segments that give rise to the head during development.
Being the world renowned scientist that you are, you decide to pursue the matter even further. Since you were taught that a protein's amino acid sequence determines its shape and function, a question repeats in your mind "What’s the difference in the structure between the mutated protein and the normal protein?" Unable to find the solution in your book, you decide to use the world-wide web to find an answer. After doing a search on the net you find a nifty tool called the Biology Workbench. The Biology Workbench is a state-of-the-art research tool, which will allow even the non-scientist to search through thousands of protein sequences and perform complex comparisons of these sequences with the greatest of ease.
Primary Author: Nick Exner (
- SETUP
1.1.Open a web browser window and go to the Biology Workbench V.3.2.
1.2.Click on “Set up a free account”.
1.2.1.Fill out the information for the account. Use a user name and password that you can remember in the future (and write this down in a notebook or on a piece of paper), you may want to use this tool again someday.
1.2.2.Click on the “Register” button.
1.3.Click on “Enter the Biology Workbench”, and continue the login process.
DO NOT USE THE BACK BUTTON ON YOUR WEB BROWSER WHEN USING THE BIOLOGY WORKBENCH. ALMOST EVERY PAGE ON THE WORKBENCH HAS A “RETURN” BUTTON, USE IT TO GO BACK TO A PREVIOUS PAGE.
- START A SESSION The Workbench allows you to organize your work by creating sessions.
2.1.Click on “Session Tools”.
2.1.1.In the menu, click “Start a New Session”.
2.1.2.Click “Run”.
2.1.3.In ‘Session Description’ type Antennapedia.
2.1.4.Click on “Start New Session”.
- RUN A MULTIPLE DATABASE SEARCH USING NDJinnThe NDJinn Search is a feature of the Biology Workbench that allows the user to find information on a topic of interest, using specific databases that may be useful. As you use this tool, notice the large number of databases that are available. You will be using a data base called the Protein Data Bank, which contains images of various proteins.
3.1.Click on the "Protein Tools" button.
3.1.1.Highlight the “NDJinn Multiple Database Search” from the scrollbar menu.
3.1.2.Click on the "Run" button.
3.1.3.Scroll down the page, to locate the database named PDBFINDER and click its checkbox. The PDBFINDER (Protein Data Bank) database contains the three dimensional structures of numerous proteins. All of these proteins are in a downloadable ".pdb" format which allows anyone to view this with a pdb protein molecular viewer described later. As you scroll back to the top of the page, check out the descriptions for some of the other databases that are available.
3.1.4.Type the name of the protein sequence that we are interested in, “antennapedia”, in the query space at the top of the page. Do not change any other settings.
3.1.5.Submit your query by clicking on the "Search" button.
3.2.As you scroll down the results page, you will see that the database found nine or more protein structures for your query. These sequences have the word “antennapedia” somewhere in their file. We are interested in the wild type and mutant sequences of the antennapedia protein from Drosophila melanogaster. Select the following records for these sequences by clicking in the checkboxes for:
3.2.1.pdbfinder:2hoa- header: DNA-binding protein
3.2.2.pdbfinder:1hom- header: DNA-binding protein
3.2.3.pdbfinder:1san- header: DNA-binding protein
3.3.Import them to the workbench by clicking the button "Import Sequence(s)".
- ALIGNING SEQUENCES WITH CLUSTALW The next step is to align the sequences you have chosen using CLUSTALW. CLUSTALW is a tool that is used to align a group of protein sequences by their common elements (amino acids) so that they can be compared. You were magically transported back to the Protein Tools window with all of your sequences at the end of the last step.
4.1.Select each of your three sequences by clicking in the small boxes next to the sequences.
4.2.Select “CLUSTALW - Multiple Sequence Alignment” in the scroll down menu.
4.3.Click on the "Run" button.
4.4.The next screen presents an assortment of settings for the CLUSTALW program. Since these are best left at the default, just click the "Submit" button.
4.5.When the results are returned, import them to the workbench (by clicking the "Import Alignments" button).
- COMPARING SEQUENCES WITH BOXSHADE You will be magically transported to the Alignment Tools where your alignment has been saved. You will use BOXSHADE, a program that can be used on your sequence alignment to determine biochemical similarities. This program produces a color-coded output of the protein sequences.
5.1.Select the sequence alignment by clicking in the small box next to them.
5.2.Highlight “BOXSHADE” in the scroll down menu and click on the "Run" button.
5.3.Click the "Submit" button to use the default settings.
5.3.1.In the resulting output, green is for amino acids that are the same (conserved) in all the proteins examined. Yellow is for amino acids that are the same in nearly all proteins and cyan means that the amino acid has the similar structure and charge but is a different amino acid.
5.3.2.Look at your aligned sequences. Notice that a majority of the amino acids are highly conserved. The “1hom” protein sequence is the wild type. The other two proteins contain mutations. If a closer look is taken, it is evident that the “2hoa” and the “1hom” sequences differ by only one amino acid. This single amino acid substitution, all by itself, from cys (cysteine) to ser (serine) at the 39th amino acid is responsible for causing the fruit fly to develop legs on its head. WOW!
5.3.3.Print this screen out, and save it to turn in with your lab report.
5.4.Click on “Return”.
- FINDING THIS PROTEIN IN OTHER ORGANISMS USING BLASTP The above protein sequence is called the homeodomain and is a small part of the whole antennapedia protein. Homeodomain proteins are coded for by a sequence of nucleotides called the homeobox. Homeobox sequences are found in homeotic genes (and some other types of genes). Homeodomain proteins bind to DNA and regulate gene transcription of developmental genes. This particular homeodomain protein binds to DNA associated with a gene or genes that control leg and antenna development. Other organisms use this same type of DNA binding process in the regulation of their development. Now, we will see how the homeodomain of other organisms compares to the antennapedia homeodomain.
6.1.Return to the Protein Tools window.
6.1.1.Select 1HOM by clicking on the small box next to it.
6.1.2.Select the BLASTP program in the scroll down menu.
6.1.3.Click “Run”. The BLAST suite of programs compares molecular sequences of either proteins (amino acid sequences) or DNA (nucleotide sequences) to databases of other sequences and finds the sequences in those databases that are most similar. We will look for similar homeodomain protein sequences in a database called SwissProt. The preliminary screen for BLASTP allows you to set several parameters that determine how similar the sequences are, but we will use the defaults.
6.1.4.Select SwissProt in the scrollbar menu for the databases.
6.1.5.Click on “Submit” at the bottom of the screen.
6.2.Notice that the 2 top “hits” in your BLAST search are the complete antennapedia sequences from two species of Drosophila.
6.2.1.Select these two proteins and the other 7 Hox-B7 (HXB7) proteins.
6.2.2.Click on “Import Sequence(s)”. You will return to the Protein Tools window.
6.3.Now select the 3 structure sequences, and the 9 proteins from the BLAST search (all 12 boxes). Run a CLUSTALW alignment on these proteins and import the alignment (follow instructions from Section 4).
6.4.Run BOXSHADE on the new alignment (follow instructions from Section 5, selecting the box with the 12 sequences). When you get the results, scroll to the end of the alignment where you will see that all 10 proteins are aligned. Notice the similarity among the different species in the homeodomain region. Does any other species have the S mutation related to antennapedia?
- NOTES ON THE STRUCTURE OF A TYPICAL PROTEIN
7.1.In the case of the fruit fly, antennapedia is a single protein that causes legs to grow out of a head instead of a thorax. A single mutation in a single nucleotide base-pair results in the difference between normal development and a fly that develops with legs growing out of its head. To help visualize the effect of this slight mutation, let's to take a look at the structure of the antennapedia protein.
7.2.For proteins, there are 4 levels of structure. The first, primary structure, is the amino acid sequence that makes up the protein. Secondary structures are the -helices that can be clearly seen in the pictures below, and -sheets (which are not seen in this protein). The tertiary structure is the 3-D structure of the protein that allows it to perform its functions. The quaternary structure is the total protein structure that is made when all the subunits of the protein are in place.
7.3.Now that you have seen the mutation in the antennapedia protein’s amino acid sequence that causes the pronounced change in the fruit fly’s phenotype, let's look at what this mutation does to the structure of the protein itself. From the visualization below, we notice that the atoms, marked in red, are arranged slightly differently between the mutant protein and the normal protein. Additionally, a sulfur atom (yellow) has been replaced by on oxygen atom (red). The very subtle difference in this arrangement and content gives rise to the inability of the mutant protein to function as an inhibitor to prevent the formation of legs on the head during early developmental stages.
- VISUALIZING PROTEIN STRUCTURE
8.1.You will now view the structures that were used to create the pictures shown on the previous page.
8.1.1.Type in the Address and hit return. The Protein Data Bank is a database of protein structures.
8.1.2.Type 1HOM in the Search box and click on Search. You will get to the summary page for 1HOM, which tells you how the structure was determined and by whom.
8.1.3.On the right side of this screen there is a picture of your molecule. Under this there is a list of programs for viewing the molecule. Click on WebMol to view your molecule.
8.1.4.A dialog box will pop up asking if you want to view all molecules. Say “No”. If you are curious, come back to this later to see what an NMR structure really looks like.
8.1.5.Open a second web page (you may use ‘File: New: Window from the dropdown list at the top of your webpage), and repeat steps 8.1.1 through 8.1.5. This time, however, type 2HOA into the search box for step 8.1.2.
8.1.6.After completing these steps, you should have two windows open that have images of the proteins – one window with 1HOM and one window with 2HOA.
8.1.7.Arrange the two windows so that you can see as much of both windows as possible.
8.2.Now you will manipulate the structures so that they are in roughly the same orientation.
8.2.1.Change the view of the structure from the default that shows all atoms (AllAt), to just the backbone atoms by selecting Backb in the first drop down menu on the right hand side of the figure for each protein.
8.2.2.If you click and drag on the pictures, it will rotate the molecules. Pick one of the two molecules and rotate it until it is in the same orientation as the other molecule.
8.3.You will now arrange the pictures to clearly show the differences between the sequences. Do the following steps for each structure. Finishing one structure before starting the other is less confusing.
8.3.1.Click on the Select button which is midway down on the right hand menu. The WebMol: Select window will appear.
8.3.1.1.Change Method from Select to SC vs. BB and Color from Current to Yellow.
8.3.1.2.Find the residue that is different between 1HOM and 2HOA (39C or 39S) in the list of residues and highlight the residue name.
8.3.1.3.Click on the apply button. The residue that you selected will turn to a thick yellow line on the structure.
8.3.1.4.Change the Backb button (8.2.1) back to AllAt. This will put the side chain atoms for just the selected residues into the picture.
8.4.Further manipulation of the structures will allow you to explore how the mutation may be affecting the structure. Remember 1HOM is the wild type and 2HOA is the mutant.
8.4.1. Rotating each molecule in the same direction and by the same amount will help you visualize the difference between the two molecules that leads to the development of legs out of the fly’s head. The difference is very subtle.
8.4.2.Click on the Rock button to help with this.
8.4.3.Now print out the two screens showing the molecular structure of the 1HOM and 2HOA proteins. Save these to turn in with your lab report.
8.5.Was it difficult to align the sequences, and then see the structural differences? Here is an easier way to align the structures (but the graphics aren’t as good).
8.5.1.Using internet explorer, go to
8.5.2.There are two boxes marked “PDB ?”. Type 1HOM in one box and 2HOA in the other box.
8.5.3.Click on Calculate Alignment.
8.5.4.When the sequence alignment comes up, click on the button "Press to Start Compare3D".
8.5.5.Select the residues of interest, C and S in the sequence alignment, they will turn into a different color.
8.5.6.Now click on the structures and drag the cursor to rotate them. What part of the protein’s structure seems most affected by the single amino acid substitution?
8.5.7.If you are using a Macintosh computer, you can use the apple-shift-4, then hit the space bar. Center the crosshairs on the window with the aligned structures, and click the mouse button. A .png file will save to your desktop, and you can open it in Preview and print it (IN COLOR). People using PCs running Windows XP . . . I don’t think you can do it except with special software.
LAB REPORT QUESTIONS.
Please answer the following questions in complete sentences. These questions/answers should be turned in, typed, in lab next week along with the three printouts from the lab exercise (the amino acid alignment (1 pt.) and the two structure printouts (2 pts. each)).
- Approximately how many amino acids comprise the antennapedia homeodomain in Drosophila? Clearly describe the difference in the amino acid sequence between the normal, or wild-type form of the antennapedia homeodomain protein and the mutated form. (2 pts.)
- Briefly describe the difference in protein structure between the wild-type and mutated forms of the antennapedia protein (see step 8.5.6). (2 pts.)
- What is the phenotypic result of this difference? (i.e., how do flies carrying the mutated form of the Antennapedia gene develop?) (2 pts.)
- Define the following terms: (1 pt. each)
A)Homeotic gene
B)Homeobox
C)Homeodomain
D)Antennapedia gene