Lec 8 - BPSL 1958 Flashcards
How was the BPSL 1958 protein discovered?
- 2D PAGE analysis between a pathogenic and non pathogenic strain of Burkholderia
What sigma factor controls the expression of BPSL1958?
RpoS - stress sigma factor
Give the general features of BPSL1958
- around 350 residues - 7 repeated motifs (around 50 residues long) with high sequence similarity arisen from gene duplication events - each motif folds up into 3 B strands (blade) and these 7 then come together to form a propeller
How was BPSL 1958 thought to have acquired this repeated motif?
sequence studies show that this repeat was found in an unrelated organism has it acquired this motif from a horizontal gene transfer?
Draw a rough sketch of the propeller structure and state what residues are likely to be found @ the N/C terminus
N = small residues eg ala
C = larger residues eg Trp
Is it necessary for this protein to have such a high sequence similarity in order to form a propeller structure?
no, this structure is seen in many other organisms w/ v low seq similarity between the blades. we have potentially found this protein early on in its evolution - the non essential residues have not been lost yet
Where are most of the sequence differences between the repeats found?
- at the top and bottom of propeller
- on the outside of the propeller
When BPSL 1958 ran through gel filtration column, a sharp peak was seen at 25kDa (expected 36kDa), what caused this change?
potentially the BPSL 1958 is retarded because it binds to the column matrix - we later found this was the case because it binds glucose (matrix is made up of polyglucose)
What are lectins and how do they relate to determining the function of BPSL 1958?
- lectins are sugar binding proteins, bind to carbohydrates eg glycoproteins on surface of cells
- play a role in immune response and recognitin of self cells
- lectins also adopt a propeller structure - does BPSL 1958 also bind sugars on host cells and therefore play a role in host cell recognition?
State the names of the 3 sugars that were intially discovered to bind to BPSL 1958 and state which of these showed the tightest binding
- glucose
- mannose - tightest binding. Kd = 2mM. identified through Trp fluorescene/NMR studies
- fructose
Because we know that mannose binds to BPSL 1958 tightly what does this suggest about BPSL 1958 function?
because mannose is a surface protein on host cells, as BPSL 1958 binds tightly could it play a role in gaining entry to the host cells
if this is correct then can we design a class of drugs to inhibit BPSL binding to mannose?
How were potential new binding targets of BPSL identified using the knowledge of all the stereoisomers of hexose sugars?
- stereospecificty of the AS of BPSL was looked at and compared to hydroxyl groups and other key features of alll of the stereoisomers to identify potential clashes?
What was seen when comparing a 7-blade B propeller of a lectin (AAL2) to BPSL?
- similar binding pockets
- it was shown that AAL2 can bind chains of sugars - is this true for BPSL?
- as AAL2 has a funciton in host pathogen recognition and in the innate immune system etc is it possible that BPSL invovled in host recognition?
What did the haemagglutination assay involving sheep’s blood and BPSL show?
- haemagglutination was seen at high concns of BPSL showing that BPSL interacts w/ rbc glycan receptors
- at lower BPSL concn haemagglutination was not seen
- the image here shows haemagglutination (yellow particles being BPSL)
What happened to haemagglutination when mannose was added and why?
no haemagglutination seen because mannose competes for binding to BPSL
How did the BPSL 1958 fold evolve? Draw a diagram to aid your explanation
- originally one gene encoding a single subdomain existed. this subdomain could self assemble into a propeller
- due to the strong expression of BPSL shows that it had an important function
- successive gene duplication events led proteins that could still self assemble and create 7 subdomain protein that we see today
