Gating Mechanism of the Influenza A M2 Channel Revealed by 1D and 2D IR Spectroscopies

Abstract

Summary The pH-controlled M2 protein from Influenza is a critical component of the virus, serving as a target for aminoadamantane anti-flu agents that block its H+ channel activity. To better understand its H+-gating mechanism, we investigated M2 in lipid bilayers with a new combination of IR spectroscopies and theory. Linear FTIR spectroscopy was utilized to measure the precise orientation of the backbone carbonyl groups, and 2D-IR spectroscopy was utilized to identify channel-lining residues. At low pH (open-state), our results match previously published ss-NMR and X-ray structures remarkably well. However, at neutral pH (closed-state), our measurements point to a large conformational change, that is consistent with the transmembrane α-helices rotating by one amino acid register: a structural rearrangement not previously observed. The combination of isotope-labelled FTIR and 2D-IR spectroscopies, alongside simulations, provides a non-invasive mean of interrogating structures of membrane proteins in general and ion channels in particular.

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

“…For example, a change of ∼50% was observed between a solvated and a hydrophobic site in the M2 tetramer. 32 Despite the relatively small variation, this result suggests that the lifetime of the amide I vibration is sensitive to changes in the local solvent density. 32 As shown (Figures 4(a) and 4(b)), the amide I vibrational lifetimes of Ala30 and Gly34, which were determined by examining the decay of the respective 2D IR peak intensity with waiting time, are identical.…”

“…32,[49][50][51] However, it should be noted that the variation in the amide I vibrational lifetimes of proteins is generally small. For example, a change of ∼50% was observed between a solvated and a hydrophobic site in the M2 tetramer.…”

“…When used in conjunction with isotope labeling, one can obtain residue-by-residue information on hydration dynamics, as has been demonstrated with experiment and theory over the past decade on several membrane protein domains, including CD3zeta, M2, and ovispirin. [29][30][31][32] In this study, we use a double mutant (D44N, R45A) 31,33,34 of the transmembrane domain of the M2 channel, with the pore lining carbonyls of its Ala30 and Gly34 residues isotopically labeled (i.e., 12 C= 16 O to 13 C= 18 O) to serve as site-specific IR probes. [35][36][37][38] The use of this double mutant avoids any potential complexity arising from the 2D IR signals of arginine and aspartic acid, which overlap with those of the 13 C= 18 O groups.…”

Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

“…The 2DIR technique distinguishes β-sheet structures by producing Z shape like spectra [18,19] when this particular structural motif is present. In the past decades a number of 2DIR studies have been reported of peptides and proteins in solution [16,[20][21][22][23][24][25][26][27][28][29][30][31][32][33] or confined in membranes [34][35][36][37][38], revealing structural details and conformational changes from femtosecond (fs) to nanosecond (ns) time scales, and the nature of dynamic environments. For the structure determination of peptides that are in gas phase or micro-solvated (surrounded by few solvent molecules) the mid-infrared spectroscopic technique has become a promising tool [39][40][41][42].…”

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

“…The only difference is the vastly disparate timescales probed by the two methods -picoseconds versus milliseconds -, which refers to the "direct time resolution" inherent to both techniques. In the context of protein dynamics, 2D IR spectroscopy has been applied to investigate the structure of membrane peptides [21,22], fibril formation [23,24], the structural flexibility of enzyme active sites [25], ligand migration in heme proteins [26], ligand binding [27,28], and the dynamics of the protein hydration shell [29,30], to list just a few examples. Recent reviews can be found in Refs.…”