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.

Explanation / Answer

The M 1 protein always play an important role in the throughout the virus lifecycle .the oligomerization of the M protein is helpful for the formation of the viral matrix layer during assembly and budding process.

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.

the M 1 exists a pure form only at the neutral pH. it can display different forms of dimers and oligomers is the smallest oligomerization state . thge pure M 1 protein is a typical alpha helical protein .it will show a single band in SDS page with single band

b.Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 7.4 (physiological pH).

At ph 7.4, N terminal domain of the M1 alone exists as a multiple order oligomers but c terminal domain exists as exclusivly stable dimer . the purified M1 from nickel chromatography was eluted into 4 fractions , the apparent molecular mass is determined it was about 52kda and most of the M1 existed as oligomers and small farction as a dimer. it was further analysed using crosslinking assay ,showed two bands one at molecular mass of 28kda and other is at the 56 kda with M1 existing as dimer . when this is again ultrafilteration is done using the different concentrations we get differnt types of oligomers with differnt molecular weight . these concentration are again diluted with 0.05 mg/ml ,the elution volumes didnt change no other oligomerisation state is formed on gel filteration couln at ph 7.4 . this shows that it form s the stable oligomers at pH7.4.

Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 5.0.

the effect of the acidic pH is determined based on the oligomerisation state at PH 5.0, gel filteration column ,15.8 ml was collected as elution ,no dimerisation is seen at this pH ,M1dimer is only seen . the samples which were collected at 7.4 again loaded at pH5.0 to see he oligomers ,but the oligomers were not seen . this suggests that at acidic pH the M1 protein exists as a monomer , when these samples are analysed at neutral pH and pH7.4 it forms the dimers .this shows the stablity of the compound

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