Solvent penetration in photoactive yellow protein R52Q mutant: A theoretical study

Daniel J. Sindhikara
a
, Norio Yoshida
a,b
, Mikio Kataoka
c
, Fumio Hirata
a,b,

a
Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki 444-8585, Japan
b
Department of Functional Molecular Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan
c
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma Nara 630-0192, Japan
a b s t r a c t a r t i c l e i n f o
Article history:
Received 13 February 2011
Received in revised form 31 March 2011
Accepted 9 April 2011
Available online 27 April 2011
Keywords:
PYP
3D-RISM
Hydronium
An R52Q mutation of photoactive yellowprotein creates a cavity mimicking an intermediate state in the wild-
type. This mutation, of a positively charged arginine to a neutral glutamine, does not affect the spectral tuning
of photoactive yellow protein. Contradicting explanations for this phenomenon suggest either water or
hydronium occupation in the cavity. To solve this controversy, we have performed 3D-RISM calculations on
the mutant structure examining the solvation structure. Our results showthat while there is a high probability
of hydronium in the cavity compared to bulk, there is still a relatively low occupation when considering
realistic concentrations. Thus our ndings suggest that while the cavity is clearly accessible by hydronium, it is
mostly occupied by water molecules. We expect high-resolution neutron crystallographic analysis of the
mutant to conrm our prediction.
2011 Elsevier B.V. All rights reserved.
1. Introduction
It is evolutionarily advantageous for creatures to be able to
recognize and avoid dangerous conditions. Purple phototropic
bacteria such as Halorhodospira halophila are vulnerable to photo-
destruction but exhibit negative phototaxis for blue-light to circum-
vent their demise. This is done with the aid of the uniquely water-
soluble photoreceptor protein, photoactive yellow protein (PYP) [1].
The chromophore for PYP is p-Coumaric acid (pCA), covalently bound
to its cysteine-69 via a thioester bond. Absorption of a photon induces
a multi-step photocycle including photoisomerization and subse-
quent protonation of pCA's phenolic oxygen [28]. In the long-lived
(130 ms) intermediate, PYP
M
, the sidechain of R52 is exposed to
solvent resulting in the formation of a cavity in the vicinity of the
chromophore [8]. It was found that an R52Q mutation (which mimics
the cavity) increases the pKa of the chromophore by one pKa unit but
does not affect the absorption spectrum [9,10]. This is somewhat
surprising considering that R52 is expected to be positive in order to
stabilize the negatively charged chromophore while Q52 is neutral
[11]. Thus this apparent change in local charge does not affect the
absorption spectrum. In 2007, Changenet-Barret, studying R52Q,
found that the positive charge of R52 does not likely counteract the
photo-induced charge shift in the chromophore [12]. Shimizu et al.
crystallized an R52Q mutant to investigate the role of R52 in the pKa
shift [13]. They found water molecules in the cavity and thus proposed
that the pKa shift is caused by the allowed passage of water molecules
to the vicinity of the chromophore (by way of the cavity). Thus, via
analogy to the mutant, it was proposed that the PYP
M
intermediate
changes the chromophore pKa by allowing exposure to solvent [13].
But how does one rectify the lack of effect of mutating a positively
charged sidechain to a neutral one? In 2009, Yamaguchi et al.
performed a combined X-ray and high-resolution neutron crystallo-
graphic analysis of PYP [14]. This study, which successfully detected a
low-barrier hydrogen bond between the chromophore and E46,
despite having found 87% of hydrogen/deuteriumatoms, found R52 to
be unprotonated. If R52 were indeed unprotonated, much of the
problem of the change-in-charge problem for R52Q would be
rectied. However, it would also introduce a new problemof having a
high amount of buried negative charge in PYP. Yamaguchi et al.
explained that this instability might be simply counteracted by the
strong bond-strength of the low barrier hydrogen bond between pCA
and E46 [14].
Much of the change-in-charge dilemma occurs under the assump-
tion that the solvent penetration resultant from the R52Q mutation is
only water. If, rather, hydronium occupied the cavity, the local charge
would be preserved. Conrmation of a signicant hydroniumoccupation
would contradict the claims of Yamaguchi et al. that the WT R52 is
unprotonated.
Here, we attack this controversy directly by investigating the
solvent penetration into PYP. To get such atomic-level information,
we chose to use computational methods. While standard atomic-level
molecular dynamics is often useful for exploration of solute conforma-
tional sampling, 3D probability distributions of solvent (non-bulk)
require extensive sampling outside the reach of standard techniques.
3D-RISM, rather, is anexcellent methodfor identifying solvent molecule
distributions in the presence of static solutes [1517]. This theory
Journal of Molecular Liquids 164 (2011) 120122
Corresponding author at: Department of Theoretical and Computational Molecular
Science, Institute for Molecular Science, Okazaki 444-8585, Japan.
E-mail address: hirata@ims.ac.jp (F. Hirata).
0167-7322/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molliq.2011.04.007
Contents lists available at ScienceDirect
Journal of Molecular Liquids
j our nal homepage: www. el sevi er. com/ l ocat e/ mol l i q
analytically evaluates the solvent distribution both around the
solute molecule and inside the cavity of the solute molecule [1829].
Essentially, it integrates fully through solvent congurational space
and provides the solvent congurational distribution function.
2. Materials and methods
The initial structure for the mutant calculation was taken from the
PDB (code 2D02) [6]. For all standard residues, parameters from the
AMBER99 force eld were used [30]. The protonation states were
enforced according to the originating article, [6] of which three
residues were not present in the AMBER99 force eld (Y42
unprotonated, E46 protonated, and Q52 protonated). For these
residues, atomic partial charges were taken from Mongan et al.'s
constant pH dynamics paper (2004) [31]. For the wild-type study, the
structure was taken from the 2009 X-ray/neutron crystallography
study PDBs 2ZOH/2ZOI [14]. The charges for the chromophore, pCA-,
which is covalently bound to C69, were derived using RESP [3238]
from a quantum mechanical calculation (MP2, 631 g (d,p) in
vacuum) [39,40] using Gaussian 03 [41]. The quantum system
includes the entire pCA molecule along with the cysteine sidechain
capped by a methyl group. All the rest of the missing force eld
parameters were taken from GAFF [42]. The 3D-RISM/minimization
calculation was performed in TINKER 5.0 (modied to include RISM)
[4347]. We employed 3D-RISMto PYP considering the protein to be a
solute in innite dilution with a hydronium and chloride concentra-
tion each of 0.01 M at 298 K with a dielectric constant of 78.5. Solvent
force eld parameters for water were taken from the SPC model [48]
with a LennardJones correction for the hydrogen, [49] for hydronium
from the MS-EVB model, [50] and for chloride from OPLS [51].
3. Results and discussion
According to the mutant crystallographystudy, twowater molecules
reside in the cavity created by the deletion of the guanidino groups.
Here we will use the same terminology for those twoapparent waters as
in the study: wat27 and wat38 [6]. Fig. 1 shows the solvent spatial
distributions in the cavity, calculated from 3D-RISM theory, which are
represented as isosurfaces. These results suggest that there is relatively
high hydronium penetration into the cavity, especially in the vicinity
of the purported water 38. Fig. 2 shows the solvent penetration in the
context of the entire protein surface.
In order to specically identify the molecules detected by
crystallography, radial distributions fromthe purported water centers
were analyzed. The radial distribution function is evaluated by
orientational averaging of the spatial distribution function. Fig. 3
shows these distributions for wat27 and wat38 (Fig. 3A and B
respectively).
Near wat27, the distributions for water and hydronium ions are
similar with each other. Near wat38, however, the distribution of
hydronium atoms is higher than that of water. This indicates that the
cavity has higher afnity to hydronium ion than to water but is
accessible by either. However, at physiological pHs, the concentration
of hydronium is so low (e.g. 0.0025 M at pH 6.0) that the site
occupation will be nearly always water (a g(r) above 10
7
would show
hydronium dominance). Thus it is highly likely that the oxygen atoms
detected by Shimizu et al. were indeed from water molecules [6].
Furthermore, since our data shows nosignicant excess positive solvent
Fig. 1. 3D distribution of solvent atoms in the cavity. Non-hydrogen protein atoms represented with a ball-in-stick model. Reported waters from crystallography are green spheres.
Surfaces show areas with g
Hyd
N3.5 and g
Oxy
N5.0. A) Water distribution, (silver surface hydrogen, red surface oxygen). B) Hydronium distribution (silver surface hydrogen,
red surface oxygen).
Fig. 2. Solvent distribution in the context of the entire protein surface. Solvent hydrogen
surfaces with gN3.5 are shown. The water hydrogen distribution is shown in yellow,
hydronium in purple. While hydronium distributions are high throughout the cavity
entrance, signicant water distributions exist near the experimentally purported
waters (oxygen, pictured in green). The electrostatic potential is projected onto the
solvent accessible surface of the protein.
121 D.J. Sindhikara et al. / Journal of Molecular Liquids 164 (2011) 120122
charge in this region, it complements the ndings of Yamaguchi et al.
that R52 is unprotonated [14]. To compare with the wild type, we also
calculated the radial distribution functions froma geometrically similar
location between the two species (Fig. 3 inset). The sidechain of R52
clearly sterically restricts water access completely in these two
locations.
4. Conclusion
Through the use of 3D-RISM calculations on photoactive yellow
protein, we have identied water molecules in the cavity created by
the R52Q mutation. Despite solvent-exposed negative charge and
some hydronium afnity, we did not nd hydronium correlation high
enough to expect signicant population in near-neutral pH. These
ndings settle the debate about the solvent charge in the mutant and
support the claims by the Kataoka group about the protonation
state of R52. We suggest a future neutron-scattering experiment to
conrm our results. We further suggest that our technique should be
applied to similar conicting explanations.
Acknowledgments
These works are supported by the Grant-in Aid for Scientic
Research on Innovative Areas Molecular Science of Fluctuations
toward Biological Functions from the MEXT in Japan. We are also
grateful to Next Generation Integrated Nanoscience Simulation
Software, the project of the ministry. Molecular graphics images
were produced using VMD [52,53].
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Fig. 3. Radial distribution of solvent atoms from purported water locations. Oxygen
distributions shown in red, hydrogen in black. Water atoms shown using lines,
hydronium in squares. A) wat27, B) wat38. The insets show the distributions from the
wild type.
122 D.J. Sindhikara et al. / Journal of Molecular Liquids 164 (2011) 120122