• BLAST questions

Transfer of information

Question 22: We have found a protease in one of our experiments. Its sequence is:

MAFIWLLSCYALLGTTFGCGVNAIHPVLTGLSKIVNGEEAVPGTWPWQVTLQDRSGFHFC
GGSLISEDWVVTAAHCGVRTSEILIAGEFDQGSDEDNIQVLRIAKVFKQPKYSILTVNND
ITLLKLASPARYSQTISAVCLPSVDDDAGSLCATTGWGRTKYNANKSPDKLERAALPLLT
NAECKRSWGRRLTDVMICGAASGVSSCMGDSGGPLVCQKDGAYTLVAIVSWASDTCSASS
GGVYAKVTKIIPWVQKILSSN

Which are its active site residues?
Hint: first figure out which is your sequence (S or Q), and which is the one found in the database?

funny Brasilian rodent

Question 23: In a funny Brasilian rodent we found a strange myoglobin. Its sequence is:

DGQWQQVLNVWGKIEADIAGHGQQVLIRLFTGHPETLERFDKFKHLRTEAE
IKASEDLKKHGTVVLTALGGILKRKGHHEAELRPLAQSHATKHRIPIKYLE
FISDAIIHVIHSKHPGDFGADAEGAMTKALELFRDDIAAKYKEIGFQG

a) Why did I call it a funny rodent?
b) Which residues bind the heme group?
Hint. Spend a few minutes thinking about evolution while you browse through the list of hits that BLAST found. If you search with a rodent sequence, which species' myoglobin do you expect at the top of the hit-list? And what do you actually get from BLAST?

>A protease, or not...
MQSINQSINQSINQSINQSINQSINQSINQSINQSINQSINQIVGFVKTCYKPEEVFHFL
HQHSIPFSSIGGMTNQNVLLNISGVKFVLRIPNAVNLSLINREYEAFNNAQAYRAGLNVE
TPVLDAKSGVKLTRYLENSNTLSQIQLNEQSCLSQVVNNLYRLHNSEFVFRNVFSVFDEF
RQYFSLLENKSAFYQADSRMDKLSAVFWQFEEINKDIILRPCHNDLVPENMLLQDDRLFF
IDWEYSGLNDPLFDIATIIEEAHLSKEAADFLLETYCNQTNKYHKTEFQIAHKRLKIHRF
CQNVLWFLWTKVKEEHGENFGDYALKRLDAAFKLLEELP

function	Mediates phase variation of the LOS 6A2 and 12D9 epitopes.
                Phase variation of H.influenza LOS epitopes expressed by
                 LicA is determined by a translational switch.
domain    	Between 2 and 19 copies of the INQS repeats are to be found
                in different strains; these are thought to serve to generate
                phase-variable expression of this gene.
miscellaneous	There is spontaneous, high frequency switching between three
                discrete levels of expression (++++, +, -) of oligosaccharide
                epitopes.
miscellaneous	Lipooligosaccharide (LOS) is a glycolipid that is a necessary
                component and antigenic determinant of the outer membrane and
                has been shown to be an important factor in the host-parasite
                interaction in a number of Gram-negative species.
miscellaneous	H.influenzae is able to display an extensive repertoire of
                different surface configurations through variability of LOS.
similarity	Belongs to the peptidase S49 family. {ECO:0000305}.

Question 24: Above this question you see two boxes. One with a sequence, and one with a bit of annotation that I got from the corresponding SwissProt file.

I doubt that annotation, especially the bit where it calls it a protease. Look-up where I found this annotation in the SwissProt file. What do you think about this protein? What advice should we send to the SwissProt curators for improving the quality of this SwissProt entry? (Please do not actually send them this advice because they tend to be rather receptive for good advice, and then I have to make a new excercise next year...).

Look at the sequences of LICA1 and LICA2. You will observe that LICA2 is longer and has extra the low complexity motif:

MQSINQSINQSINQSINQSINQSINQSINQSINQSINQSINQ

plus a few more residues; but (and you do not have to check this), the rest of the sequences are identical.

Question 25: Why do I call this a low complexity motif?

The fact that LICA1 does not have this repeat and still seems to function fine tells us that this repeat is likely to have a function that is only important under some experimental conditions that we do not know yet.

And now a really, really difficult question.

Question 26: Can you do the bioinformatics that is needed to design a real in vivo or in vitro experiment that can at least shed a little bit of light on the role of this SINQ repeat?