Predicting biological properties based on protein sequences

Go to parent Basic bioinformatics concepts, databases and tools#Exercises_during_the_training

Predicting TM helices

Go to the UniProt record of a short fly protein of unknown function.

Obtain the sequence in FASTA format
Go to the Sequences section of the UniProt record Click the FASTA button to download the sequence in FASTA format

We will identify TM regions in the sequence using Phobius.

The tool contains two HMMs, one for TM helices and one for signal peptides. TM regions and signal peptide have very similar amino acid compositions and TM proteins often have a signal peptide to target them to the membrane. Therefore, it is difficult to distinguish between signal peptides and TM helices. This is the nice thing about Phobius: it combines signal peptide and TM predictions. When you use other tools for TM prediction you have to combine them with signal peptide prediction tools like SignalP. If SignalP -and preferably a few other signal peptide prediction tools- agree on predicting a signal peptide you should remove the signal peptide sequence from the full protein sequence since it is not part of the mature protein.

The TM HMM was based on a data set of proteins with experimentally determined topology.

How many TM regions does Phobius predict ?

Go to the Phobius tool. Simply paste the sequence in FASTA format in the text area Click Submit The tool compares the query sequence to the HMM and output the result of the prediction: You see the location of the predicted TM region, together with the regions that are expected to be located NON CYTOPLASMIC (outside of the cell) and CYTOPLASMIC (inside the cell). So if the whole sequence is labeled as NON CYTOPLASMIC or CYTOPLASMIC, the prediction is that it contains no membrane helices. It is important to realize that the location is not completely accurate. The prediction gives the most probable location and orientation of transmembrane helices in the sequence.

Phobius is making a binary prediction on the existence of a TM region in the fly sequence. Remember that it is advised to use multiple prediction tools and compare their results. Therefore, we will repeat the prediction with MEMSAT, the TM prediction tool of PSIPRED as an alternative TM prediction tool.

Psipred will run both MEMSAT and MEMSAT-SVM, the former a neural network based and the latter an SVM based TM helix prediction tool.

Does MEMSAT agree with the Phobius prediction ?

Go to the PSIPRED website. In the Prediction methods section select MEMSAT3 and MEMSAT-SVM Paste the sequence in FASTA format in the text area in the Input sequence section Provide an ID for the sequence in the Submission details section Click Predict Psipred analyses can take a while. The results page show the results of both tools. Go to the MEMSAT-SVM tab. Scroll down to the MEMSAT-SVM cartoon: you see that a TM region is predicted from position 57 to position 73. Scroll further down to the MEMSAT cartoon: you see that a TM region is predicted from position 55 to position 74.

So you see that the predictions of the different tools agree. In this case you can be more or less sure of the prediction.