The oligomerization of Influenza A Virus matrix 1 (M1) protein was analyzed usin

Question

The oligomerization of Influenza A Virus matrix 1 (M1) protein was analyzed using affinity chromatography followed by gel filtration.(A) Purified recombinant M1 (with a C-terminal His6-tag) from nickel affinity chromatography eluted in peak 4.The fraction containing peak 4 (from panel A) was collected.SDS-PAGE analysis determined that the peak 4 fraction was 100% pure.(B) Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 7.4 (physiological pH).(C) Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 5.0.

Figure adapted from: Zhang K, Wang Z, Liu X, Yin C, et al. (2012). PLoS ONE 7(5): e37786. doi:10.1371/journal.pone.0037786

Which peak in panel B contains protein(s) with the highest molecular weight?

What experimental condition causes multiple peaks in panel B to elute as a single peak in panel C

Propose a reasonable scientific hypothesis about the biochemical basis for multiple peaks in panel B, but a single peak in panel C.  

Which peak in panel A contains protein(s) with the highest affinity for the Ni+2 Agarose column?       

Explanation / Answer

1. The 10.1 ml fraction contained protein with highest molecular weight of 580KDa in 0.5mg/ml concentration beacuse of oligomerisation.

2. In the panel B, the proteins were eluted as various states of oligomers, but in the panel C when the pH is changed to 5.0 the interactions between the oligomers were destroyed and protein was eluted as a single oligomer unit of dimers.

3. The influenza M1 protein at pH 7.4 forms different states of oligomers due to which they are eluted at different peaks indicating their molecular weights and among them the smallest oligomer state is dimer of molecular weight 52KDa, in the panel C the elution of protein as a single peak is due to the loss of oligomerisation state at low pH, hence all the proteins form dimers and eluted as single peak.

4. The protein in peak 3 has the highest affinity for Ni+2 agarose because of its lesser elution, which suggests that most of the protein is still bound to the column showing its high affinity.

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