title: Proton association constants of His 37 in the Influenza-A M218-60 dimer-of-dimers.
abstract: The membrane protein M2 from influenza-A forms a single-pass transmembrane helix that assembles in lipid membrane as homotetramers whose primary function is to act as a proton transporter for viral acidification. A single residue, histidine 37 (His 37), is known to be responsible for selectivity and plays an integral role in the protein's function. We report pH-dependent (15)N MAS NMR spectra of His 37 within the influenza-A proton conduction domain of M2, M218-60, which has been previously shown to be a fully functional construct and was recently determined to adopt a dimer-of-dimers structure in lipids. By extracting the ratio of [His]/[HisH(+)] as a function of pH, we obtained two doubly degenerate proton disassociation constants, 7.63 ± 0.15 and 4.52 ± 0.15, despite a possible maximum of four. We also report the (1)HNε chemical shifts at pH 6.5 recorded at 60 kHz MAS in a CP-based (1)H-(15)N spectrum. We were unable to detect resonances indicative of direct proton sharing among His 37 side chains when the tetramer is in the +2 state. In the neutral state, His 37 is exclusively in the τ tautomer, indicating that the δ nitrogen is protonated solely as a function of pH. We also found that the plot of [HisH(+)]/[His] as a function of pH is qualitatively similar to previously reported proton conduction rates, indicating that proton conduction rate is proportional to the level of histidine protonation within the channel. Two-dimensional (13)C-(13)C and (13)C-(15)N correlations suggest that at low pH multiple conformations are populated as the spectra broaden and eventually disappear as the acidity is increased. A second highly resolved state at low pH was not observed.
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Colvin
authors.forename: Loren B
authors.initials: LB
authors.lastname: Andreas
authors.forename: James J
authors.initials: JJ
authors.lastname: Chou
authors.forename: Robert G
authors.initials: RG
authors.lastname: Griffin
mesh_terms: D000595:Amino Acid Sequence; D006639:Histidine / Q000737:chemistry*; D009980:Influenza A virus / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D011522:Protons; D014763:Viral Matrix Proteins / Q000737:chemistry*; D000086082:Viroporin Proteins
chemical_list: D006639:Histidine; D011522:Protons; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins