Protein Information Tutorial

Transcription

1 Protein Information Tutorial Relevant websites: SMART (normal mode): SMART (batch mode): HMMER search: InterProScan: CBS Prediction Servers: EMBOSS: Characterizing a protein using protein domain identification and prediction servers on the web. In this tutorial you will use known protein sequence and submit it to a variety of prediction servers to learn how to interpret the output from these servers. Pay attention to the output from the various programs. If you do not understand it, look for help files or links to information explaining the output. The CBS server has a link from the output of most of their programs that describes the output in detail and how to interpret it. There are help files for the EMBOSS programs as well. 1) Use the SMART database in normal mode (single protein submission) Search the SMART database using the human protein NEK2 with the Uniprot accession number P Click on the Normal mode graphic and it will bring up a search window as shown in Figure 1. If you use Uniprot/SwissProt Figure 1: Submission form for the SMART accession numbers, you can simply type in the accession database. number in the text box Sequence ID or ACC. Check the boxes PFAM domains and signal peptides. Then click the Sequence SMART button. The output includes a graphic of any domains found in the protein as shown in Figure 2. The graphic is interactive. If you Figure 2: Domain found in the sequence Nek2_HUMAN (P51955). The vertical lines represent intron/exon boundaries. pause the mouse over one of the intron lines, it will display the position and reading frame. If you pause the mouse over the BCHM Protein Information on the Web Page 1 of 6

2 domain graphic, it will expand the display to show more information about the domain, including the position of the match and the E-value for the match. The domains are represented on the protein sequence in the location that they are found. The bright green horizontal bars represent coiled-coiled regions and the bright pink/magenta color represents regions of low complexity. If you click on the graphic, it will change the window to show the feature details including the alignment. At the top of the new window, there is a link full annotation. Click on that and it will open a new window which shows the sequence of the domain with the catalytic residues highlighted in green (Figure 3). This can be a very useful feature is you are interested in making constitutively active mutants or catalytically inactive mutants. This option of showing catalytic residues is not available for every domain. Figure 3: SMART domain detail page. Catalytic residues are shown in green. At the bottom of the SMART output there are also a number of expandable menus that contain links to additional information about the domain shown. 2) BATCH submission using SMART database 1. Click on the link for the SMART batch submission available on the Exercise 5 homepage. 2. You can submit fasta sequences or Ensemble or Uniprot Protein IDs or accession numbers. 3. Download the Excel file SecretedProteins4ProteinInfoTutorial from the Exercise 5 homepage. This worksheet has 61 proteins that were identified as secretory proteins and most should be recognized by the SMART database. 4. Copy and paste the Uniprot IDs into the Identifiers box on the Batch retrieval page. Click the options for include PFAM Figure 4: SMART Batch output (partial) domains and include signal peptides then click the Submit button. 5. The page the opens will list the IDs that had no matches and then give you a long list of matches with graphical output as shown in Figure 4. BCHM Protein Information on the Web Page 2 of 6

3 If you click on the protein name (i.e. C1QT1_MOUSE) it will bring up a page with the same information as you would get from a single protein submission. This is not the most efficient way of looking at a list of proteins, but it does provide an immediate graphical look at the domains (for proteins available in the SMART database) is one of the few web-based servers that allow batch submission using Protein IDs. You can do batch submission of protein sequences at HMMER, but the results take much longer to come back. 3) HMMER database searches Access to the PFAM database is available via InterproScan or via the HMMER search interface. The difference between them is that InterProtScan combines the searches of several different protein domain databases while the HMMER searches only the PFAM database. Copy the CCR7 protein sequence from the Ex. 5 homepage and paste it into the search box for the HMMER search. Leave the default search against Reference proteomes. You should get a result that shows a match to the 7tm_1 domain. If you click the Show Hit Details, it will provide more detailed information about the hit as well as show a second domain hit. By default, it will show matches for transmembrane domains TM and signal peptides. Figure 5: Output of HMMER search with CCR7 protein Scroll down the window to see the taxonomic distribution of the hits. 4) Sequence submissions using InterProScan InterProScan is well support by EBI and this is well integrated with their databases, particularly Uniprot. BCHM Protein Information on the Web Page 3 of 6

4 Submit the sequence or accession number (P32248) for the CCR7 protein. Once the output comes back you will see that it includes matches to multiple protein domains including Prosite and Prints. If you mouse over the different domain graphics, a pop-up window will provide information on the source and statistical evaluation of the match. The IPR#### represent different families of proteins that have the same domain content and are built using a combination of signatures from the different domain databases. Click on one of those to see what information is available. There is a LOT of information available, particularly for well annotated proteins from human and mouse. If you click the Search tab from the InterProScan homepage, there is an option to search using Domain architecture. So, if there is a particular combination of domains that Figure 6: InterProScan output for CCR7 protein are of interest and you want to see how many proteins contain that particular combination, you can do that also. 5) Using TMHMM program at the CBS prediction server Submit the CCR7 sequence (available from the Ex 5 homepage) to the TMHMM program, using extensive graphics as the output. The output should look like that displayed in Figure 7 The red bars at the top represent the positions of probably transmembrane spanning domains. The pink or blue lines connecting the TM domains provide the topology relative to the cell membrane. If you wanted to design a peptide antibody to this protein, would you choose a region predicted to be on the inside or outside of the membrane? Do these results concur with those obtained from InterProScan? What additional information do you get from this server that you do not from InterProScan? Submit the NEK2 protein sequence to the TMHMM prediction server. Does it predict the presence of any TM domains? 4) Use the prediction servers to look for signal peptides and phosphorylation sites. Scan either the CCR7 or NEK2 protein sequence using the CBS PredictionServers program SignalP to look for signal peptides. Given the results of the TMHMM predictions, Figure 7: TMHMM output for CCR7 protein from the CBS prediction server BCHM Protein Information on the Web Page 4 of 6

5 would you expect this protein to be secreted? This site also predicts signal anchor peptides, which is a more likely result given the number of predicted TM domains. Use the NetPhosK program at the CBS PredictionServers site to predict the potential threonine and tyrosine phosphorylation sites. Keep in mind that these predictions are just that, predictions. While useful for informing possible experiments, they are not necessarily correct. Figure 9: EMBOSS Seqtable output 5) Scan the protein using the antigenic program in EMBOSS. This program is located under the Protein Motif section of EMBOSS. This is a simple program designed to predict potentially antigenic regions of a protein sequence; a good starting point for designing peptide antibodies. Read the manual for it to understand the method behind the program. The output is fairly simple, just a table or list of sequence regions which have a high likelihood of being antigenic. Under the text box where you input the sequence, there is an option for increasing the minimum length of the antigenic region as well as a section for changing the default output. The default output is Emboss_motif that shows the sequence marked by location and the score. You may find the EMBOSS Seq table to be a bit more useful. Try a few different outputs to see what information is provided by them. Figure 8: EMBOSS motif output PATTERN matching A common request I get is to find particular amino acid patterns in a sequence or group of sequences. Often these patterns are somewhat degenerate. There is a program within EMBOSS called fuzzpro which uses PROSITE style patterns to search protein sequences. An example recognition or catalytic site might have the pattern: [FY]-[LIV]-G-[DE]-x(2)-{E} In English that is: F or Y, followed by L, I or V, followed by G, followed by D or E, followed by any 2 amino acids, followed by any amino acid EXCEPT E. An example use is finding potential phosphorylation sites which often have a very short motif, such as SxxS/T or RXS/T. Use fuzzpro to find one of those patterns in the CCR7 protein. Note, that the syntax for either/or is to put the amino acids into square brackets []. Thus, SxxS/T would be written Sxx[ST] in the search box. Do this. How many hits did you get? I got 4 when I did it. BCHM Protein Information on the Web Page 5 of 6

6 Although this example is very simplistic, pattern matching is a very powerful tool both because of the flexibility of the search parameters; something you cannot do using the Find command in Word and also because at the command line you can set it up to search as many sequences as you want. Here is the Prosite signature for GPCR family 1: [GSTALIVMFYWC] - [GSTANCPDE] - {EDPKRH} - x - {PQ} - [LIVMNQGA] - {RK} - {RK} - [LIVMFT] - [GSTANC] - [LIVMFYWSTAC] - [DENH] - R - [FYWCSH] - {PE} - x - [LIVM] Copy and paste that into the search pattern text box for fuzzpro with the CCR7 protein. You should get the match shown in Figure 10. Prosite pattern syntax 1. The standard IUPAC one-letter codes for the amino acids are used in PROSITE. 2. The symbol `x' is used for a position where any amino acid is accepted. Figure 10: FuzzPro output for GPCR signature 3. Ambiguities are indicated by listing the acceptable amino acids for a given position, between square brackets `[ ]'. For example: [ALT] stands for Ala or Leu or Thr. 4. Ambiguities are also indicated by listing between a pair of curly brackets `{ }' the amino acids that are not accepted at a given position. For example: {AM} stands for any amino acid except Ala and Met. 5. Repetition of an element of the pattern can be indicated by following that element with a numerical value or, if it is a gap ('x'), by a numerical range between parentheses. Examples: x(3) corresponds to x-x-x x(2,4) corresponds to x-x or x-x-x or x-x-x-x A(3) corresponds to A-A-A 6. When a pattern is restricted to either the N- or C-terminal of a sequence, that pattern either starts with a `<' symbol or respectively ends with a `>' symbol. In some rare cases (e.g. PS00267 or PS00539), '>' can also occur inside square brackets for the C-terminal element. 'F-[GSTV]-P-R-L-[G>]' means that either 'F-[GSTV]-P-R-L-G' or 'F-[GSTV]-P-R-L>' are considered. Other prediction tools The tools listed here are just a few of the hundreds that are available. If you are interested in a particular feature of a protein, such as if it has a myristylation moiety or is ubiquitinated at certain residues, there are prediction programs for that. Start with a Google search and you can probably find one or more sites that have a web interface to the prediction algorithm. Always keep in mind that a prediction does not mean that the event actually occurs in the cell. If there is more than one prediction algorithm, you might want to test your protein(s) with other prediction algorithms to determine the overlap. BCHM Protein Information on the Web Page 6 of 6