I am working on Methicillin resistant Staphylococcus aureus MRSA proteins and would like to know about designing functional studies for hypothetical proteins. Kindly suggest me the softwares and invitro assays for function determination.
I guess the easiest approach here would be a genetic approach - knockout the gene you are interested in and see how the phenotype of the bacteria is changed:
Do they grow faster/slower?
Do they lose the ability to grow on different media?
Are they less resistant to antibiotics?
Are they more susceptible to phagocytosis?
Are they more suscepible to neutrophil NETs?
Do they survive less well inside macrophages/neutrophils?
Alternatively, you could express the gene in a bacteria that lacks it and see how this changes the phenotype (although this approach would require that the protein functioned on its own without a requirement for other proteins).
1) one of the first things should be to go online at expasy or predictprotein or HHpred and look at the amino acid sequence and secondary structure. a true protein should have more than 70% of regular structures like alpha helices or beta sheets and the amino acid distribution should not be excessively rich in a single amino acid say proline etc. also look for presence of leader peptide etc
2) try a simple blast search with the deduced amino acid sequence. if the initial matches are more hypothetical proteins.....don't proceed
3) try to find motifs online at prosite, motifscan or domains at prodom, predictprotein, expasy etc
4) try to get structural homologs by submitting the deduced amino acid sequence at swissmodel, i-TASSER.
5) try to correlate the above information with 1) whether the hypothetical protein is a part of a known operon etc 2) if you know of any expression profile for this protein
I disagree with the first proposition of the precedent answer... What is a true protein for you? And what about Intrinsically disordered proteins or domains? These proteins lack of well defined secondary structure in physiological conditions of pH and salinity and in the absence of their partners, and yet they are biologically active and critical in the proteome. If you find less than 70% of predicted regular structures in your protein sequence, it is nor necessarily abnormal!
If it is the case of some of the proteins that you have identified, this review may interest you http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/22821542
Good luck!
I guess the easiest approach here would be a genetic approach - knockout the gene you are interested in and see how the phenotype of the bacteria is changed:
Do they grow faster/slower?
Do they lose the ability to grow on different media?
Are they less resistant to antibiotics?
Are they more susceptible to phagocytosis?
Are they more suscepible to neutrophil NETs?
Do they survive less well inside macrophages/neutrophils?
Alternatively, you could express the gene in a bacteria that lacks it and see how this changes the phenotype (although this approach would require that the protein functioned on its own without a requirement for other proteins).
@Michael Daws: What about getting a prediction with computational tools and then set experiments accordingly?? Could it be a valid approach??
Yes, certainly. I think that is part of what Anubrata Das was suggesting. You should absolutely look for clues using bioinformatics, then design studies with that information in mind. For example, if predictions indicate the protein binds a lipid, you would examine metabolic and signaling pathways that involve that lipid. I think you would still want to take an initial genetic approach (so easy in bacteria), but you would also measure levels of metabolites specific to the predicted pathway. You could also clone the gene and affinity purify it if there is strong functional homology to a known protein to confirm the function in vitro. Just keep in mind that predictions are just that. They can help you create a rational approach, but stay flexible
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