Tidal surge in the M2 proton channel, sensed by 2D IR spectroscopy

Ghosh, Ayanjeet; Qiu, Jade X.; DeGrado, William F.; Hochstrasser, Robin M.

doi:10.1073/pnas.1103027108

Cited by 98 publications

(154 citation statements)

References 49 publications

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“…As discussed above and in ref. 23, the water is more ice-like inside the channel in the Q2 state and exhibits slower orientational dynamics. Because a successful proton transfer event from one water molecule to the next requires collective rearrangement of the surrounding HB network (53,54), the slow dynamics of channel water inhibit PT.…”

Section: Significancementioning

confidence: 98%

“…The high-resolution crystal structure [Protein Data Bank (PDB) code 3LBW] crystallized at pH 6.5 revealed layers of well-ordered water clusters above the His37 tetrad (12). However, the water dynamics in the AM2 protein probed using 2D infrared (2D-IR) spectroscopy revealed that the well-ordered "ice-like" pore water dynamics at pH 8.0 change to more mobile and "liquid-like" dynamics (on the timescale of a few picoseconds) at pH 3.2 (23). This result suggests an interesting pH-dependent behavior of the AM2 protein that is highly relevant for understanding its PT mechanism.…”

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confidence: 99%

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Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

Liang

Swanson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

139

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The influenza A virus M2 channel (AM2) is crucial in the viral life cycle. Despite many previous experimental and computational studies, the mechanism of the activating process in which proton permeation acidifies the virion to release the viral RNA and core proteins is not well understood. Herein the AM2 proton permeation process has been systematically characterized using multiscale computer simulations, including quantum, classical, and reactive molecular dynamics methods. We report, to our knowledge, the first complete free-energy profiles for proton transport through the entire AM2 transmembrane domain at various pH values, including explicit treatment of excess proton charge delocalization and shuttling through the His37 tetrad. The free-energy profiles reveal that the excess proton must overcome a large freeenergy barrier to diffuse to the His37 tetrad, where it is stabilized in a deep minimum reflecting the delocalization of the excess charge among the histidines and the cost of shuttling the proton past them. At lower pH values the His37 tetrad has a larger total charge that increases the channel width, hydration, and solvent dynamics, in agreement with recent 2D-IR spectroscopic studies. The proton transport barrier becomes smaller, despite the increased charge repulsion, due to backbone expansion and the more dynamic pore water molecules. The calculated conductances are in quantitative agreement with recent experimental measurements. In addition, the free-energy profiles and conductances for proton transport in several mutants provide insights for explaining our findings and those of previous experimental mutagenesis studies.T he influenza type A virus is a highly pathogenic RNA virus that causes flu in birds and mammals (1). The influenza A M2 (AM2) protein (2) contains a homotetramer channel that transports protons across the viral membrane and acidifies the virion interior, enabling the dissociation of the viral matrix proteins, which is a crucial step in viral replication (3). The protein has been the target of antiviral drugs amantadine and rimantadine (4, 5). Much effort has been devoted to discovering the structure and proton transport (PT) mechanism of the AM2 channel, resulting in many crystal structures available in the protein data bank (6-14). Based on the crystal structures and electrophysiology experiments, several PT models have been suggested. These mechanisms can be divided into two main categories, delineated by the role of the four histidine residues (a.k.a. the His37 tetrad) that reside in the middle of the AM2 transmembrane domain (AM2/TM) (Fig. 1A), which has been experimentally shown (15) to account for the proton permeation behavior of the full AM2 protein. The "shutter" mechanism (16,17) suggests that the His37 tetrad works as a gate. At low pH the gate opens due to the electrostatic repulsion between the biprotonated, positively charged histidine residues. The excess proton is then transferred through continuous water wire via the Grotthuss mechanism, without changing the pro...

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Section: Significancementioning

confidence: 98%

mentioning

confidence: 99%

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

Liang

Swanson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

139

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“…Moreover, linear infrared spectroscopy has significant limitations in revealing dynamics that are underscored by the vibrational lineshapes; for the M2 channel, these dynamical timescales are characteristic to water structures near the pore lining amides as has been demonstrated before. 31 Thus, the remainder of this work focuses on the 2D spectroscopy of the isotopically labeled amides.…”

Section: A Linear Ir Spectramentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Ghosh

Wang

Moroz

et al. 2014

The Journal of Chemical Physics

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Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs. © 2014 AIP Publishing LLC. [http://dx

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“…For an IR probe that is able to interact with water via H bonding, measurement of its FFCF can provide, sometimes in a site-specific manner, detailed information about the hydration dynamics of the protein molecule of interest. For example, this approach has been used to identify the existence of mobile water molecules inside Aβ40 amyloid fibrils (31,32) and to interrogate the water-assisted drug-binding mechanism of HIV-1 reverse transcriptase (33), among many other applications (34)(35)(36)(37)(38)(39). In the current study, we capitalize on the established sensitivity of the nitrile stretching vibration (C≡N) to local hydration and electrostatic environment (40) and use the unnatural amino acid p-cyano-phenyalanine (Phe CN ) as a local IR reporter.…”

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confidence: 99%

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Pazos

Gai

2014

Proc. Natl. Acad. Sci. U.S.A.

229

237

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Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p-cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect.N ature employs a variety of small organic molecules to cope with osmotic stress. Trimethylamine N-oxide (TMAO) is one such naturally occurring osmolyte that protects intracellular components against disruptive stress conditions (1). In particular, previous studies have shown that TMAO is able to enhance protein stability and to counteract the denaturing effect of urea (2, 3). TMAO (Fig. 1, Inset) adopts a skewed tetrahedral structure with a charged oxygen capable of accepting hydrogen bonds (H bonds) and three hydrophobic (methyl) groups. This amphiphilic structural arrangement makes TMAO a rather special cosolvent, because it can form H bonds with water, self-associate in a manner similar to surfactants, and show preferential interactions with or exclusion (4-12) from certain protein functional groups (13-23). Indeed, these molecular properties of TMAO have been used, either individually or in combination, to rationalize its biological activities. For example, a prevailing view about TMAO is that its osmotic and protective role is caused by the molecule's tendency to be preferentially depleted from protein surfaces, as suggested by physicochemical measurements (24-28). This thermodynamic picture, as described by Bolen and coworkers (29), implies that the protein is preferentially hydrated. Although this notion is consistent with TMAO's being a protecting osmolyte, to the best of our knowledge no experimental studies have been carried out to directly examine the effect of TMAO on protein hydration dynamics, an aspect that is also important to protein function. Herein, we use twodimensional infrared (2D IR) spectroscopy and a site-specific IR probe to explore this critical issue and gain insight toward achieving a microscopic understanding of the molecular mechanism of the protecting action of TMAO.2D IR spectroscopy is capable of assessing the frequencyfrequency correlation function (FFCF) of a given IR probe, which reports on the underlying dynamics of events that lead to fluctuations of its vibrational frequency (30). For an IR probe that is able ...

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Tidal surge in the M2 proton channel, sensed by 2D IR spectroscopy

Cited by 98 publications

References 49 publications

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

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

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

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