Purification Methods for Solid-State NMR Analysis of Influenza A M2[18-60]

Sammanfattning: With viral infections becoming an ever-increasing health problem in the world, the study of viral proteins is a cornerstone for understanding and preventing future pandemics. The following work shows the purification and structural characterization of the Influenza A M2 proton channel through solid-state NMR (ssNMR). Despite previously published structures of the M2 proton channel, the method of purification has not been well detailed and requires further optimization in order to produce samples to study at Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP). A truncated version of M2 containing residues 18-60 was studied in this work, which is referred to as M2[18-60]. Purification of labeled and unlabeled M2[18-60] expressed at 22°C and 30°C degrees were performed with varied results. It was found that the expression of M2[18-60] produced the highest yield when expressed in M9 minimal media and that the temperature during expression has no significant impact on the purification results. In addition, there was no difference in purification yield of M2[18-60] using a C3 or C4 column. Both labeled and unlabeled M2[18-60] was able to be reconstituted into liposomes. The acquired 2D 13C-13C correlation spectrum confirmed that presence of M2[18- 60] generated by the previous purification steps. The stability of the M2 protein was confirmed through overlay of previously published assigned spectra of a M2 S31N mutant. Further optimizations of the methods used in this work will be important in order to produce samples of M2[18-60] for future investigations into the design of antiviral drug candidates, ssNMR analysis of binding sites of M2-targeting drugs and assay development.