inibacillus sphaericus C3-41, complete genome. Yet, these two subjects have the same maximum score. Briefly explain why this is the case.
Protein Properties Worksheet
In the past 10 years, one of the greatest improvements in biochemistry took place in the field of bioinformatics. Search engines and prediction tools enable us to easily learn a lot about a protein or RNA of interest before we actually start working on a project. The availability of these online tools makes it much easier to tailor a project and assess its feasibility. This problem set introduces you to a few easy-to- use prediction tools. You will learn how to calculate the molecular weight, isoelectric point or amino acid composition of a protein, how to perform sequence alignments to assess sequence conservation, how to predict secondary and tertiary structure.
Follow the step-by-step instructions below and answer all questions
You will use the sequence of the ykkCD multidrug-resistance efflux pump. Efflux pumps are an important part of bacterial defense against antibiotics: they pump antibiotics out of the bacterial cell thereby rendering them ineffective in treating bacterial infections. The RNA sensor (ykkCD riboswitch) you perform mutagenesis on in the biochemistry lab turns on production of the ykkCD efflux pump.
The ykkCD efflux pump is a heterodimer pump meaning that it is made out of two different proteins: ykkC and ykkD. In the first part of the exercise you will perform a series of predictions using these sequences to get an idea about the physico-chemical and structural properties of this pump.
The sequences of the ykkC and ykkD pumps are listed below:
- Generate a sequence alignment between the two pump monomers (C and D) using this link: http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html. Make sure you copy/paste both sequences into the program as it is shown above (names and > included).
- a) Paste the sequence alignment!
- b) How similar are these two sequences (% conservation)?
- c) Is there any trend in the types of amino acids that appear conserved (apolar/polar/charged)?
- d) Based on “c” what prediction would you make about the structural similarity of these two proteins (similar or not and why)?
- Choose the ykkC or the ykkD pump to perform a series of predictions using the link below http://www.expasy.ch/tools/protparam.html; pI (isoelectric point), MW and amino acid composition. You may use the ykkC or the ykkD pump sequence for this excersize!
- a) List pI (isoelectric point) and molecular weight (MW) of this protein.
- b) What are the three most common amino acids in this protein?
- c) What can you tell about the general physical properties (polar/apolar; acidic/basic) of this protein? Would you expect this protein to be water soluble?
- Predict the secondary structure of the ykkC or the ykkD pump by using: http://www.compbio.dundee.ac.uk/www-jpred/
- a) Paste the result of the secondary structure prediction into your report!
- b) What is the major secondary structural motif in this protein?
- c) Based on your secondary structure prediction which amino acids are prone to form a-helix?
- Using the link below predict membrane topology of the pump used in “3”. http://phobius.sbc.su.se/. Answer the questions below
- a) Paste the prediction here.
- b) Based on the secondary structure prediction in “3” and the previous topology prediction sketch how the pump is inserted into the membrane.
- Tertiary structure prediction: Below is the sequence of the MexA multidrug resistance efflux pump from the pathogenic organism Pseudomonas aeruginosa. This organism mostly affects patients with compromised immune system. It is the main culprit behind infections caused by medical implants (like catheter).
Perform tertiary structure prediction on this protein using the link and the sequence below!
Warning! Performing this modeling will take a while (10-20min). Do not use the back button on your browser; just wait patiently until it is done.
MQRTPAMRVLVPALLVAISALSGCGKSEAPPPAQTPEVGIVTLEAQTVTLNTEL PGRTNAFRIAEVRPQVNGIILKRLFKEGSDVKAGQ