[Frontiers in Bioscience 8, d1288-1297, September 1, 2003]
1288
RELAY AND BLOCKAGE OF PROTONS IN WATER CHAINS
Régis Pomès and Ching-Hsing Yu
Structural Biology & Biochemistry Programme, Hospital for Sick Children, and Department of Biochemistry, University of
Toronto, Toronto, Ontario, Canada
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Molecular Determinants of Proton Conduction
3.1. Wire Nucleation
3.2. Hydrogen Bonding
3.3. Hydrogen-bond polarization
3.4. Proton Solvation
3.5. Stabilization of Bonding Defects
4. Proton Relay in Gramicidin
5. Blockage by Methanol
5.1. Method
5.2. Results
5.2.1. Unprotonated Methanol
5.2.2. Protonated Methanol
5.3. Discussion
6. Conclusions
7. Acknowledgments
8. References
1. ABSTRACT
The movement of H+ is one of the most important
and ubiquitous reactions to take place in biological
systems. The gramicidin A (gA) dimer, which forms a
water-filled channel selective to small monovalent cations
in biological membranes, is used as a model system to
study the molecular determinants of biological proton
transport with computer simulations. The single-file chain
of water molecules, or water wire, embedded in the channel
interior mediates the translocation of H+ via a hop-and-turn
Grotthuss relay mechanism. Earlier work showing how the
mobility of the excess proton in gA is essentially
determined by the fine structure and the dynamic
fluctuations (structural diffusion) of the hydrogen-bonded
network is summarized. The structure and fluctuations of a
methanol-containing water chain in the channel lumen
suggest a molecular mechanism for the experimentally-
measured attenuation of proton conductance by methanol.
2. INTRODUCTION
The transfer and long-range transport of H+ are
among the most important phenomena in chemistry and
biology. These reactions are central to acid-base equilibria
in aqueous solutions and to energy transduction in living
systems. The translocation of protons across biological
membranes is a fundamental requirement of bioenergetics.
The production of ATP is driven by an electrochemical
gradient resulting from the pumping of H+ across the
bioenergetic membrane, a process known as chemiosmotic
coupling (1). For that reason, the control of proton
translocation across protein assemblies spanning energy-
transducing membranes is essential to life.
The transport of an excess proton is thought to
involve a Grotthuss relay mechanism consisting of
successive transfers of hydrogen nuclei between water
molecules forming hydrogen-bonded networks. In such
relay processes, the molecular mechanism governing the
long-range transport of H+ is determined both by the nature
of the hydrated proton and by structural fluctuations of the
hydrogen-bonded network (2,3). In bulk water, the
elementary step for the transfer of H+ between two water
molecules involves the interconversion between the Eigen
and Zundel forms of the hydrated proton, respectively
OH3+(OH2)3 and H+(OH2)2. In the first of these two limiting
forms, the excess proton is hosted by one water molecule
and in the second, it is shared by two water molecules in a
strong hydrogen bond. Theoretical studies have shown that
this elementary exchange is driven by the rearrangement of
hydrogen bonds in the second hydration shell of the excess
proton, a process that has been called structural diffusion
(2,4).
Likewise, the translocation of H+ across channels
and proteins embedded in biological membranes can also
occur by way of a Grotthuss mechanism. The long-range
relay of H+ involves water molecules and titratable amino-
acid side chains forming hydrogen-bonded networks of low
dimensionality, or proton wires (5,6). In linear hydrogen-
bonded arrays, this process may be described as a hop-and-

Relay and Blockage of Protons
1289
turn mechanism, in which the hop steps consist of
successive H+ transfers along a polarized hydrogen-bonded
chain. Because hops invert the donor-acceptor pattern of
the chain, the subsequent rearrangement (turn) of proton
relay groups is required to restore the original polarization
of the wire. Structural evidence for the involvement of
water molecules in the relay of protons through the interior
of energy-transducing proteins includes bacteriorhodopsin,
a light-activated proton pump from the purple membrane of
Halobacterium salinarum (7). The complete elucidation of
proton translocation mechanisms in complex energy-
transducing enzymes is a formidable challenge due to the
coupling of long-range proton transfer to photochemical or
redox reactions and to protein conformational changes (8).
Because of its comparative simplicity, gramicidin A (gA), a
peptide that forms ion channels in various bilayers,
constitutes a good model for the study of biological proton
translocation. gA is a pentadecapeptide whose alternating
sequence of D- and L- amino acids adopts a right-handed
beta6.3 helix fold in lipid bilayers (9-11). Head-to-head
association of two gA monomers results in the formation of
a pore permeable to water and to small monovalent cations
(12,13). The functional form of the dimer defines a
cylindrical cavity lined with the peptide backbone,
exposing its predominantly hydrophobic amino-acid side
chains to the core of the bilayer. The lumen, which is
approximately 25Å in length and 4Å in diameter,
accomodates a single-file chain of water molecules that
mediates passive proton translocation.
In the remainder of this paper, we first
summarize the molecular determinants of proton transport
that have emerged from previous studies of gA,
emphasizing the concepts and properties likely to be of
general relevance to other biological water wires. Among
other factors, the balance of molecular interactions leading
simultaneously to the solvation and the mobility of protons
is highlighted. As an extension and an illustration of these
concepts, we then examine the molecular mechanism of
attenuation of proton transport by methanol in a dioxolane-
linked gA dimer.
3. MOLECULAR DETERMINANTS OF PROTON
CONDUCTION
For the effective permeation of any molecular
species to occur, a channel must satisfy a dual requirement:
(i) it must provide a local environment suitable to the
proper solvation of the permeating species, and (ii) this
stabilization must not be so strong as to hinder permeant
mobility. In narrow pores, the permeant is at least partly
stripped of surrounding water molecules, and the channel
itself must provide the stabilizing interactions resulting in
partition from bulk solvent into the channel. In turn,
interactions with specific groups on the inner walls of
narrow channels, which have been called surrogate waters,
offer the possibility of controlling the selectivity of the
channel. In a membrane channel, this may be achieved
locally, by a selectivity filter, or throughout a more
extended region in which the permeant and water
molecules proceed as a single file. Permeant mobility is
achieved as long as the permeant does not bind too strongly
relative to the fully-hydrated state, which could result in
channel blockage. In the case of a single-file environment,
the repetition of the channel-solvation motif can help attain
this balance both in a thermodynamic sense and kinetically
(14).
The molecular forces leading to the selectivity
and transport of monoatomic ions are emerging from
studies of the gramicidin channel (15,16), of the potassium
channel KcsA (17), and of the chloride channel ClC (18).
In addition, the molecular determinants of water
permeation through aquaporins have recently been
investigated by spectroscopic (19,20) and computational
(21,22) studies. Both in gA and in KcsA, the surrogate
solvation of alkali metal ions such as potassium is provided
by carbonyl groups of the channel backbone in narrow
single-file regions. Gramicidin mediates the permeation of
small monovalent cations but hardly discriminates between
various alkali ions (11,13). In contrast, the coordination of
K+ by KcsA is snug enough to confer specificity and to
allow the close proximity of several ions despite the strong
coulombic repulsion between like charges (23-25). In
channels from the aquaporin family, permeating water
molecules also proceed in single-file and form hydrogen
bonds both with backbone carbonyl groups and with amide
groups, but the molecular origin for the blockage of ions,
including H+, is still a matter of debate.
Protons constitute a very special case of
permeating cations because they can form covalent
interactions with relay groups or molecules such as water.
The molecular basis of the hop-and-turn or Grotthuss relay
mechanism for the conduction of an excess proton by a
wire is shown schematically in figure 1. In this scheme, the
successive transfer or hop of hydrogen nuclei between
adjacent relay groups forming a polarized hydrogen-bonded
chain is complemented by the reorganization (turn) of the
hydrgogen-bonded network so as to restore the polarity of
the wire prior to the passage of another proton in the same
direction (5). The propagation of hop and turn steps may be
described respectively in terms of the mobility of an ionic
defect and of a bonding defect. In water wires, the ionic
defect corresponds to the hydrated proton, whereas a
bonding defect is defined by the interruption of the
polarized chain due to the reorientation of a water
molecule. Before illustrating the conduction and blockage
of protons in gA channels, we first summarize some of the
governing principles and mechanistic aspects implied by
the hop-and-turn mechanism in proton wires. The interplay
of equilibrium properties and non-equilibrium effects is
emphasized. Dynamic fluctuations are paramount to any
transport mechanism; in hop-and-turn processes, each of
the requirements listed below is modulated by thermal
motions giving rise to the transport of ionic and bonding
defects.
3.1. Wire Nucleation
A preliminary requirement to the conduction of
protons by a relay mechanism is the presence of relay
groups in a channel or channel-like cavity such as those
found in the interior of proton pumps bacteriorhodopsin
and cytochrome c oxidase (26). In the case of water wires,

Relay and Blockage of Protons
1290
Figure 1. Schematic depiction of the hop-and-turn Grotthuss relay mechanism for the transport of H+ by a hydrogen-bonded
chain (proton wire). Dashed lines represent hydrogen bonds in the pseudo-unidimensional proton wire whereas dotted lines
indicate stabilizing hydrogen bonds between the wire and the channel lumen. Cases in which all the relay groups are water
molecules (R = H) and where one of these water molecules has been replaced by a molecule of methanol (R = CH3) are
considered in the text. In the latter case, there is no hydrogen bonding between R and the channel.
the relay groups may not be present at all times but could
instead nucleate transiently, as has been proposed for the
leakage of H+ across pure lipid bilayers by transient
hydrogen-bonded water chains (see Ref. 27 and references
therein) or the protonation of the Schiff base in
bacteriorhodopsin (7).
3.2. Hydrogen Bonding
For proton hopping to occur, two consecutive
relay groups must be hydrogen-bonded to each other. This
is because the transfer of H+ cannot happen through
distances longer than that of a hydrogen bond. Not only are
protons too reactive to be found in free form in biological

Relay and Blockage of Protons
1291
systems, but also, contrary to that of electrons, their de
Broglie wavelength (approximately 0.05nm) is too short to
enable their quantum tunneling through extended regions of
space.
Hydrogen transfer between two water molecules
strongly depends on the length of the hydrogen bond. The
potential energy profile for the transfer of a H atom in a
(moderately strong) water-water hydrogen bond is a
bistable well with a substantial activation energy barrier.
The height of that activation energy barrier increases with
the length of the hydrogen bond. In the presence of an
excess proton, the hydrogen bond is much shorter (and
stronger); the potential energy barrier opposing H+ transfer
is either absent or considerably reduced, thereby facilitating
the transfer (28,29). Thus, the interchange between
hydronium and Zundel cations, the two predominant forms
of the hydrated proton in a water wire, is characterized by
changes in hydrogen bond length.
Inversely, the reorganization of the hydrogen-bonded chain
required for the translocation of a bonding defect implies
that each hydrogen bond be broken in turn. A good proton
duct is one that assists the proton-relay chain in tackling the
dual requirement of a proton wire: to enable strong
hydrogen bonds between relaying groups for the efficient
transfer and relay of H+; and to help weaken these
hydrogen bonds so as to facilitate the reorientation of
proton-relaying groups (30).
3.3. Hydrogen-Bond Polarization
Since the translocation of both ionic and bonding
defects inverts the polarity of each hydrogen bond in the
chain, thermally-accessible conformations of the wire must
include alternate polarization of each hydrogen bond
(whereby the donor-acceptor pair is arranged alternately
left and right in the process depicted in Fig.1) suitable to
directional proton transport. It has been proposed that the
adverse polarization of the water chain induced by two
alpha helices lining the single-file region of aquaporins is
what prevents the intrusion of protons in these channels
(22).
3.4. Proton Solvation
As emphasized above, the channel must lead to
the stabilization of the hydrated proton (ionic defect). In
general, this may be achieved by long-range electrostatic
forces or locally, by hydrogen-bonding interactions. In a
single-file environment, the stable states of the hydrated
proton are dominated by hydronium and Zundel forms, and
proton hopping occurs via successive exchanges between
these two forms (3,28,29,31). Because water molecules
hosting the excess proton are coordinated to exactly three
hydrogen-bond acceptors (2,4), an environment favoring
such a coordination is suitable to the solvation of the
hydrated H+, as depicted schematically in figure 1.
3.5. Stabilization of Bonding Defects
Likewise, molecular interactions favoring the
presence of a bonding defect in the chain are important for
the reorganization of the hydrogen-bonded network. Thus,
the permeation of an excess proton along a chain of water
molecules requires adequate solvation both of a hydrated
proton and of unprotonated water molecules. In model non-
polar pores devoid of hydrogen-bonding partners to the
water chain, the turn step of the Grotthuss mechanism was
shown to be a thermally-activated process opposed by a
significant free energy barrier (27,32).
The above considerations provide guidelines to
gauge the possibility of proton conduction by a relay
mechanism and the performance of a hydrogen-bonded
network in that process. In the next section, we discuss how
these concepts are met for rapid relay of H+ in a good
proton duct, gramicidin; inversely, in section 5 we show
how their partial violation may lead to the attenuation of
proton currents by methanol.
4. PROTON RELAY IN GRAMICIDIN
Evidence for the relay of protons by the single-
file water chain embedded in the lumen of gA has been
provided by experimental measurements (12,33). The
molecular basis of the hop-and-turn mechanism in gA has
been the object of detailed computational studies (3,30),
which have opened the way to kinetic models of H+
permeation linking atomistic simulations to the large body
of conductance data (34-37). The simulations revealed how
the equilibrium structure and the dynamic fluctuations of
the entire hydrogen-bonded network involving the water
chain give rise to H+ conduction in a narrow biological
pore. In particular, the effect of hydrogen bonding
interactions between channel and water molecules on the
hop-and-turn conduction mechanism was analyzed by
comparison to the results obtained in studies of model non-
polar channels, in which such interactions were missing
(3,27-32).
In preformed and pre-polarized hydrogen-bonded
water chains found in gA and in non-polar pores,
spontaneous, small-amplitude oscillations in the length of
water-water hydrogen bonds are sufficient to drive the
exchange between hydronium and Zundel cations within
picosecond time scales (28,29,31). As depicted
schematically in figure 1, hydrogen bond donation from
water molecules to the gA channel backbone carbonyl
groups completes water-water hydrogen bonds to provide a
coordination well suited for proton solvation and proton
mobility in the proton wire by stabilizing both elementary
forms of the hydrated proton, hydronium and Zundel ions.
The translocation of the ionic defect is thus facilitated by
the fact that water molecules in the chain are primed for
hosting the excess proton by adequate tri-coordination; in
other words, the single-file region of the channel provides
presolvation of the hydrated proton throughout most of the
single-file region. The repetition of hops along the
polarized section of the wire results in the long-range
displacement of H+ without necessitating disruption of the
hydrogen-bonded network. In addition, because the
periodicity of the gA channel backbone provides
approximately the same local environment to each single-
file water molecule, the relay groups have comparable
proton affinities, which favors high mobility of the ionic
defect (30).

Relay and Blockage of Protons
1292
By virtue of the symmetry inherent to the head-
to-head dimer organization, gA possesses no net dipole
moment on average. Thus, the channel does not impose a
preferential polarization to the water molecules in the
single-file region, which, in the absence of an excess proton
and of an electric field, are equally likely to point towards
one mouth or the other. Accordingly, in the channel lumen,
as in model non-polar pores, the water chain adopts either
one of two polarized conformations due to strong water-
water dipole interactions. Likewise, the inversion of the
chain’s polarity is an activated process involving the
sequential reorientation of water molecules. However,
hydrogen-bond donation to the channel wall stabilizes
intermediate conformations of water molecules in the
process of reorienting between their two prefered polarized
states (see the right-hand side of figure 1), reducing the
magnitude of the activation free energy that opposes
reorganization of the chain in non-polar pores. Thus the
“solvation” of bonding defects by the channel results in
catalysis of the turn step of the Grotthuss mechanism (30).
In summary, molecular simulation studies of
proton transport in gA have shown how structural diffusion
in biological pores arises from subtle local fluctuations of
the hydrogen-bonded network and how these properties are
themselves determined by the hydrogen-bonding
coordination, arrangement, and topology of proton-relay
groups. Water chains form highly modulable hydrogen-
bonded networks, whose hydrogen-bonding properties are
harnessed by carbonyl groups lining the lumen of gA to
assist both hop and turn steps of the Grotthuss mechanism.
In that sense, the single-file region of the gA channel
provides a blueprint for the rapid (~ns), passive
translocation of protons across biomembranes.
5. BLOCKAGE BY METHANOL
Together with native gA, linked gramicidin
channels offer an avenue to refine the emerging
understanding of biological proton relay mechanisms.
Covalent linkage of the N-termini of two gA monomers by
a dioxolane group results in two diasteroisomeric forms, SS
and RR, of the channel (38). Experimental measurements
of single channel conductance in planar bilayers showed
that methanol attenuates proton currents significantly in the
SS dioxolane-linked gA channel, leading to the proposal
that methanol partitions inside the pore and delays the
passage of protons through the single file (39). How does
methanol affect H+ transport? When inside the channel, a
molecule of MeOH could either block proton relay
completely or slow it down relative to all-water wires. The
unambiguous discrimination between these two
mechanisms depends, among other things, on the fraction
of time that the channel is occupied by methanol. Two
limiting cases can be considered: 1) if methanol resides in
the lumen for a fraction of the time, complete blockage
would result in the attenuation of proton currents by the
same fraction, whereas 2) if methanol occupies the channel
permanently, proton permeation would require relay
between water and methanol molecules, as shown in figure
1. Since the fractional occupancy of gA by methanol is not
known, it is necessary to determine whether or not protons
are transferred between water and methanol molecules in
order to distinguish between blockage and slow-down of
proton transport in gA channels. Here we investigate the
structural basis for a relay mechanism using molecular
simulations.
Support for a relay mechanism is found in the
anomalously fast transport of proton in pure alcohol
solutions. Structural diffusion of H+ in liquid methanol was
recently investigated with computer simulations, revealing
a chain-like arrangement of methanol hydroxyl groups
which is reminiscent of the single-file chain of water
molecules found in the interior of gA (40). In the interior of
proteins, the direct participation of hydroxyl groups in
proton relay is supported by the role of serine residues in
enzymes such as green fluorescent proteins (41) and
photosynthetic reaction centers (42). In addition, a
structural model of a continuous and polarized proton wire
with the hydroxyl group of a Tyr side chain joining two
chains of water molecules was proposed to explain
functional uptake of protons in mutant forms of bacterial
cytochrome c oxidase (43). For relay mechanisms to be
competent, the properties of the hydrogen-bonded network
must be compatible with both hop and turn steps of the
Grotthuss mechanism (see above), suggesting that dynamic
studies of hydrogen-bonded networks can provide clues as
to their ability to function as proton wires. Thus, the role of
hydrogen-bonded network fluctuations in shuttling of
protons by the carboxylic moiety of a glutamic acid residue
was investigated in the proton uptake D-pathway of bovine
cytochrome c oxidase (44).
In order to explore the physical basis for the
attenuation of proton currents in gramicidin channels, we
present molecular simulations in which the lumen contains
water molecules as well as one molecule of methanol. Both
protonated and unprotonated states of methanol are
considered in turn. Analysis of the structure of the
hydrogen-bonded network and its fluctuations at 300K
indicates that the hydrogen-bonded chain is interrupted by
intercalation of the methyl moiety of methanol, suggesting
that a hop-and-turn mechanism is precluded when methanol
is present in the single-file region of the channel.
5.1. Methods
The model system consisted of the SS dioxolane-
linked channel embedded in a hydrated glyceryl-
monooleate (GMO) bilayer (figure 2). The CHARMM
program (45), version 27, was used in all molecular
dynamics (MD) simulations. Water molecules were
described by the TIP3P model (46). The CHARMM22
force field (47,48) was used to model MeOH, the 18-
carbon-chain GMO molecules and the peptidic moiety of
the gA channel. The force field for the dioxolane linker was
taken from a previous study (49). The internal geometry
and non-bonded parameters for MeOH2+ were obtained
from ab initio calculations; non-bonded interaction
parameters were optimized to reproduce the structure and
energy of the two hydrogen bonds in a single-file
MeOH2+(OH2)2 cluster. Further details of the calculations
will be given elsewhere (Yu and Pomès, in preparation).

Relay and Blockage of Protons
1293
Figure 2. Overview of the system. GMO molecules are
shown in dark blue, the gA channel is shown in yellow. A
molecule of methanol is visible in the middle of the single-
file region of the channel.
In both simulations reported here, the Langevin
piston algorithm (50) was used to maintain a pressure of
1.0 atm and zero surface tension (51). A friction coefficient
of 5 ps-1 was imposed on all heavy atoms. The SHAKE
algorithm (52) was employed to fix the length of all
covalent bonds involving hydrogen. The Hoover thermostat
(53) was used to maintain the temperature at 300K.
Periodic boundary conditions were applied together with a
Particle Mesh Ewald treatment of long-range electrostatic
interactions. Lennard-Jones interactions were cut off at
12Å. The time step for the integration of the equations of
motion was 2fs.
The system comprised 122 GMO and 3210 water
molecules, yielding a water:GMO ratio of 26:1. The initial
surface area of a GMO molecule was set to the
experimental value of 37.9Å2 (54,55). Following the
prepartion, equilibration, and a 5.8ns simulation of the
water-filled SS channel, a molecule of methanol was
inserted into the lumen. Two insertion modes differing by
the orientation of the CO axis were considered
successively. In conformation 1, the methyl group was
placed in the way of the water chain, with the CO bond
approximately parallel to the channel axis. In conformation
2, the CO bond was oriented perpendicularly to the channel
axis (see figure 1). Both conformations were subjected to
500 steps of steepest-descent energy minimization, first
with channel atoms fixed, then again with all atoms
moving; both systems ended up in conformation 1 (figure
3). The system was then equilibrated for 200ps followed by
a 7ns production MD run. The starting structure for
simulations of protonated methanol was taken from the
methanol run at 3.8ns. The protonated methanol was moved
to the center of the channel and the system was equilibrated
for 200ps before sampling for 2ns.
5.2. Results
In this subsection, we describe the hydrogen-
bond network and the dynamics of a methanol-containing
water column in the lumen of SS dioxolane-linked gA. In
light of the analysis of relay mechanisms presented above,
these results offer clues as to the molecular origin for the
attenuation of proton conduction by methanol.
5.2.1. Unprotonated methanol
Although MeOH fits and remains in the lumen, it
undergoes significant displacements along the channel axis
during the course of the simulation. In general, the contents
of the lumen fluctuate between seven and eight water
molecules. The dynamic behavior of the chain alternates
between small-amplitude oscillations around a mean
position and rapid burst-like events displacing the column
by up to half of the length of the lumen. The time evolution
of the interatomic separation between the O atom of
methanol and those of its two water neighbors in the single-
file region is shown in figure 4. Only one of these two
hydrogen bonds is present at any given time. MeOH
accepts a hydrogen from a neighboring water and donates
its hydrogen to a carbonyl O atom of the channel. As a
result, the only stable state observed in the relatively long
(7ns) simulation of the MeOH/H2O column involves
interruption of the hydrogen-bonded chain by methanol.
Significant gaps of up to 12Å arise occasionally in the
column due to this interruption and to the non-polar nature
of the methyl group of MeOH, which repels water in the
confined channel lumen (figures 2,3). Rapid tumbling of
methanol, which occurs eight times during the course of the
simulation, exchanges the direction of the C-O bond with
respect to the channel axis and the connectivity to water
molecules located above and below (figure 3). These
transitions are completed within 1ps. Conformations in
which MeOH bridges the water chain may exist at best as a
transient species in the reorientation process. Because both
bridging and interrupting conformations involve MeOH
forming two hydrogen bonds, the hydrogen-bond energy of
the bridging conformation is comparable to that of the
interrupted conformer. Thus, the strong preference for the
latter is essentially due to steric repulsion between the
methyl group of MeOH and the channel wall.
5.2.2. Protonated methanol
Contrary to the results reported above for
methanol, MeOH2+ is expelled from the channel; this
happens relatively quickly (within 0.8ns) despite its initial
equilibration near the dimer junction. The ion then spends
the rest of a 2ns simulation hovering at the channel/water
interface (near z = 12Å). While inside the lumen, MeOH2+
remains in an orientation that interrupts the hydrogen-
bonded chain (figure 5), similarly to the results obtained for
MeOH. No tumbling is observed during that time; instead,
the ion proceeds to exit the lumen with its polar OH2+

Relay and Blockage of Protons
1294
Figure 3. Representative conformations of methanol and
water molecules in the lumen of the gA channel. With its
CO bond approximately colinear with the channel axis,
methanol donates its H atom to a backbone carbonyl O
atom of the channel and accepts one from an adjacent water
molecule; the methyl group interrupts the hydrogen-bonded
chain. Tumbling of methanol exchanges the two
conformations, exposing the hydroxyl group alternatively
to polarized water chains above and below.
Figure 4. Structural fluctuations of the hydrogen-bonded
network around unprotonated methanol, MeOH. The time
evolution of the two OO distances separating the O atom of
methanol from those of the two closest water molecules
located respectively above and below it are shown
respectively in green and red. Insets depict hydrogen
bonding by methanol.
group first while maintaining its hydrogen bond with the
water molecule above and its molecular orientation relative
to the channel axis. The absence of bridging conformations
suggests that proton relay cannot take place while methanol
is present in the channel. In stark contrast, MeOH2+ is able
to form two hydrogen bonds with water as soon as its
methyl group pops out of the singe file.
5.3. Discussion
The above results underline properties of
hydrogen-bonded networks that are important for structural
diffusion. From the point of view of a relay mechanism,
methanol is analogous to a water molecule with one of its
(H) arms in a bulky (Me) cast. Thus, the perturbation
induced in the water wire by the introduction of one
molecule of methanol is the disappearance of a hydrogen
donor. While most water molecules form exactly three
hydrogen bonds in the single-file region of gramicidin (30),
both MeOH and MeOH2+ form two hydrogen bonds while
in the channel interior, one with water and one with the
channel backbone. Thus, the coordination of the protonated
form of methanol, which donates its two H atoms, is
consistent with the hydrogen-bonded structure in liquid
methanol (40). In that sense, MeOH2+, like OH3+ and
O2H5+, is adequately solvated in the lumen of the gA
channel. However, unlike protonated water, protonated
methanol does not permit the formation of a continuous
hydrogen-bonded chain with two water neighbors unless it
is located at the channel mouth, with the methyl group
outside the channel.
While the relay of H+ by MeOH could in
principle still be achieved by the rotation or reorientation of
MeOH2+, such events, which are not observed in the
simulation, are relatively unlikely. The tumbling or
reorientation of a charged protonated species, which
necessitates breaking hydrogen bonds, is hampered by the
strength of the hydrogen bonds that it forms with its
neighbors (56). Such a property is consistent with structural
diffusion in bulk water, where reorientation takes place in
the second hydration shell of H+ but not closer (2). In pure
water wires such as that found in the gA channel, proton
transport can occur over a primed chain of as many as six
or seven water molecules without any bond making or
breaking (3), consistently with the idealized picture of the
hop-and-turn mechanism (figure 1). The lesser strength of
the MeO(H)—HOH and H2O—HOH hydrogen bonds
makes the unprotonated forms of water and methanol better
suited to turns than their protonated counterparts.
Accordingly, the present results suggest that MeOH is
indeed well suited as a bonding defect, since it alternatively
connects with two polarized water chains (figures 3, 4).
6. CONCLUSIONS
In the above overview, we have outlined some of
the general aspects of structural diffusion of relevance to
the study of biological channels, membrane proteins, and
enzymes. In recent years, molecular simulations of proton
transport have begun to provide meaningful insight into the
balance of forces underlying the Grotthuss relay
mechanism in biological systems. Studies of single-file
water chains embedded in narrow pore environments have
helped to clarify the role of quantum effects and have
shown how the long-range movement of H+ via a hop-and-
turn mechanism is controlled by low-amplitude fluctuations

Relay and Blockage of Protons
1295
Figure 5. Structural fluctuations of the hydrogen-bonded
network around protonated methanol, MeOH2+. The time
evolution of the two OO distances separating the O atom of
methanol from those of the two closest water molecules
located respectively above and below it are shown
respectively in green and red. The methyl group of
methanol proceeds to exit the single file at t = 753ps and
dwells at the channel/water interface for the remainder of
the simulation. Insets depict hydrogen bonding before (left)
and after (right) extrusion of the methyl group from the
single-file region.
in the fine structure and the topology of hydrogen-bonded
networks (3,27-31,56,57). Furthermore, comparative
studies of non-polar pores and of gramicidin have
highlighted the interplay between the solvation of ionic and
bonding defects and their mobility in a proteinaceous
environment (3,30). By extension, the detailed analysis of
fluctuations in the hydrogen-bond coordination and the
polarization of putative proton relay groups should provide
useful insight into the factors leading to the modulation and
the blockage of proton movement in protein interiors.
We examined the structure and fluctuations of a
methanol-containing water chain in a gA channel analog. A
priori, full determination of the molecular origin for the
attenuation of proton conductance by methanol is a
challenging problem combining the movement of H+ with
that of MeOH through the pore. In addition to possible
gating or blockage of proton movement by a stationary
molecule of methanol, the fractional channel occupancy
and the rate of transport of methanol itself must be taken
into account. Our preliminary results indicate that both
unprotonated and protonated forms of methanol fit in the
channel interior and are stabilized (solvated) by hydrogen
bonds with water and with the channel wall. However, the
present results also suggest that whereas MeOH is a
suitable bonding defect, its protonated counterpart would
not qualify as an ionic defect because it fails to form a
continuous hydrogen-bonded chain while in the single-file
region. The methyl group of permeating methanol
molecules would constitute a plug blocking the relay of
protons.
7. ACKNOWLEDGMENTS
This work was supported in parts by NIH (grant
RO1-GM59674) and by an RTC fellowship to CHY from
the Research Institute of the Hospital for Sick Children.
RP is a CRCP Chairholder.
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Key Words: Molecular Dynamics, Ion Channels,
Grotthuss Mechanism, Proton Wire, Review
Send correspondence to: R. Pomès, Structural Biology
and Biochemistry, Hospital for Sick Children, 555
University Ave., Toronto, Ontario, Canada M5G 1X8; Tel:
1-416 813-5686; Fax: 1-416 813-5022; E-mail:
pomes@sickkids.ca