That membrane protein complexes could survive in the gas phase had always seemed impossible. The lack of chargeable residues, high hydrophobicity, poor solubility and the vast excess of detergent contributed to the view that it would not be possible to obtain mass spectra of intact membrane complexes. With the recent success in recording mass spectra of these complexes, first from recombinant sources and later from the cellular environment, many surprising properties of these gas phase membrane complexes have been revealed. The first of these was that the interactions between membrane and soluble subunits could survive in vacuum, without detergent molecules adhering to the complex. The second unexpected feature was that their hydrophobicity, and consequently lower charge state, did not preclude ionization. The final surprising finding was that these gas phase membrane complexes carry with them lipids, bounds specifically in subunit interfaces. This provides us with an opportunity to distinguish annular lipids that surround the membrane complexes, from structural lipids that have a role in maintaining structure and subunit interactions. In this review we track these developments and suggest explanations for the various discoveries made during this research.

Protein allosteric modulation is a pillar of metabolic regulatory mechanisms; this concept has been extended to include ion channel regulation. P2XRs are ligand-gated channels activated by extracellular ATP, sensitive to trace metals and other chemicals. By combining in silico calculations with electrophysiological recordings, we investigated the molecular basis of P2X4R modulation by Zn(II) and ivermectin, an antiparasite drug currently used in veterinary medicine. To this aim, docking studies, molecular dynamics simulations and non-bonded energy calculations for the P2X4R in the apo and holo states or in the presence of ivermectin and/or Zn(II) were accomplished. Based on the crystallized Danio rerio P2X4R, the rat P2X4R, P2X2R, and P2X7R structures were modeled, to determine ivermectin binding localization. Calculations revealed that its allosteric site is restricted to transmembrane domains of the P2X4R; the role of Y42 and W46 plus S341 and non-polar residues were revealed as essential, and are not present in the homologous P2X2R or P2X7R transmembrane domains. This finding was confirmed by preferential binding conformations and electrophysiological data, revealing P2X4R modulator specificity. Zn(II) acts in the P2X4R extracellular domain neighboring the SS3 bridge. Molecular dynamics in the different P2X4R states revealed allosterism-induced stability. Pore and lateral fenestration measurements of the P2X4R showed conformational changes in the presence of both modulators compatible with a larger opening of the extracellular vestibule. Electrophysiological studies demonstrated additive effects in the ATP-gated currents by joint applications of ivermectin plus Zn(II). The C132A P2X4R mutant was insensitive to Zn(II); but IVM caused a 4.9 ± 0.7-fold increase in the ATP-evoked currents. Likewise, the simultaneous application of both modulators elicited a 7.1 ± 1.7-fold increase in the ATP-gated current. Moreover, the C126A P2X4R mutant evoked similar ATP-gated currents comparable to those of wild-type P2X4R. Finally, a P2X4/2R chimera did not respond to IVM but Zn(II) elicited a 2.7 ± 0.6-fold increase in the ATP-gated current. The application of IVM plus Zn(II) evoked a 2.7 ± 0.9-fold increase in the ATP-gated currents. In summary, allosteric modulators caused additive ATP-gated currents; consistent with lateral fenestration enlargement. Energy calculations demonstrated a favorable transition of the holo receptor state following both allosteric modulators binding, as expected for allosteric interactions.

Membrane proteins represent a challenging family of macromolecules, particularly related to the methodology aimed at characterizing their three-dimensional structure. This is mostly due to their amphipathic nature as well as requirements of ligand bindings to stabilize or control their function. Recently, Mass Spectrometry (MS) has become an important tool to identify the overall stoichiometry of native-like membrane proteins complexed to ligand bindings as well as to provide insights into the transport mechanism across the membrane, with complementary information coming from X-ray crystallography. This perspective article emphasizes MS findings coupled with X-ray crystallography in several membrane protein lipid complexes, in particular transporters, ion channels and molecular machines, with an overview of techniques that allows a more thorough structural interpretation of the results, which can help us to unravel hidden mysteries on the membrane protein function.

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single–molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1–aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin. This work was supported by molecular dynamics (MD) simulations to propose an unfolding mechanism of the aptamer–Mg+2 complex. Our results are a step towards the development of new aptamer-based STX sensors that are potentially cheaper and more sensitive than current alternatives.

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