Emulating Membrane Protein Environments─How Much Lipid Is Required for a Native Structure: Influenza S31N M2

Abstract

This report investigates the homotetrameric membrane protein structure of the S31N M2 protein from Influenza A virus in the presence of a high molar ratio of lipid. The structured regions of this protein include a single transmembrane helix and an amphipathic helix. Two structures of the S31N M2 conductance domain from Influenza A virus have been deposited in the Protein Data Bank (PDB). These structures present different symmetries about the channel main axis. We present new magic angle spinning and oriented sample solid-state NMR spectroscopic data for S31N M2 in liquid crystalline lipid bilayers using protein tetramer:lipid molar ratios ranging from 1:120 to 1:240. The data is consistent with an essentially 4-fold-symmetric structure very similar to the M2 WT structure that also has a single conformation for the four monomers, except at the His37 and Trp41 functional sites when characterized in samples with a high molar ratio of lipid. While detergent solubilization is well recognized today as a nonideal environment for small membrane proteins, here we discuss the influence of a high lipid to protein ratio for samples of the S31N M2 protein to stabilize an essentially 4-fold-symmetric conformation of the M2 membrane protein. While it is generally accepted that the chemical and physical properties of the native environment of membrane proteins needs to be reproduced judiciously to achieve the native protein structure, here we show that not only the character of the emulated membrane environment is important but also the abundance of the environment is important for achieving the native structure. This is a critical finding as a membrane protein spectroscopist's goal is always to generate a sample with the highest possible protein sensitivity while obtaining spectra of the nativelike structure.

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin

“…Residual dipolar couplings (RDCs) are a sensitive probe of molecular structures in solution . In the case of an α-helical peptide with an expected kink in it, like melittin, NMR-based orientation restraints can tightly define the relative orientation of the two α-helical segments . We utilized the ARTSY technique to measure backbone 1 D NH RDCs in two separate alignment media, liquid crystalline Pf1 filamentous phage (Figure S6) and positively charged stretched polyacrylamide gel, on an 800 MHz spectrometer at 20 °C.…”

“…17 In the case of an α-helical peptide with an expected kink in it, like melittin, NMR-based orientation restraints can tightly define the relative orientation of the two α-helical segments. 18 We utilized the ARTSY 19 technique to measure backbone 1 D NH RDCs in two separate alignment media, liquid crystalline Pf1 filamentous phage (Figure S6) and positively charged stretched polyacrylamide gel, on an 800 MHz spectrometer at 20 °C. Pf1 has a negatively charged surface that has strongly attractive, electrostatic interactions with positively charged melittin.…”

Seeing double: the persistent dimer-of-dimers structure of drug resistant influenza A M2

“…Figure 2B compares the spectrum in DPhPC with a mixture of DOPC and DOPE lipids at an LPRT of 180. These lipids were chosen to match the composition reported by Cross and coworkers, 25,26 and the LPRT of 180 was chosen to exceed the value of 120 reported in their work. 13 C, 15 N S31N M218-60 reconstitued at high and low LPRT.…”

“…Cross and coworkers reported that the difference in reported structures is caused by an insufficient amount of lipids in MAS NMR preparations. 25 The dimer of dimers structure of S31N M218-60 was determined from samples assembled with a lipid to protein tetramer ratio (LPRT) of 24. Two sets of peaks are evident in MAS NMR spectra under these conditions, which is the result of a tetramer structure composed of a dimer-of-dimers.…”

“…Cross and coworkers also pointed out that there are two anomalous isoleucine chemical shifts in the MAS NMR spectra of S31N M2 at an LPRT of 24, which they describe as occurring in a 'non-helical Cα chemical shift range for Ile'. 25 In order to address the above discrepancy in reported CD M2 assemblies in lipid bilayer preparations, here we use MAS NMR as a sensitive probe of protein structure to investigate the influence of lipids, lipid concentration, protein sequence length, and amphipathic helix variants. We additionally investigate the structural underpinnings of the unusual isoleucine chemical shifts.…”