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Protein Adaptation to High Hydrostatic Pressure:
Computational Analysis of the Structural Proteome
Samvel Avagyan1,2, Daniel Vasilchuk1,3§ and George I. Makhatadze1,2,3,*
1Center for Biotechnology and Interdisciplinary Studies, 2Department of Biological Sciences,
3Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New
York 12180
*Corresponding Author: George I. Makhatadze, Center for Biotechnology and Interdisciplinary
Studies,, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180 USA Phone: (518) 276-
4417 E-mail: [email protected]
Keywords: Piezophilic adaptation; Protein stability; Protein Volume; Structural
Proteome; Pressure Perturbation Calorimetry; Structural Modeling; Osmolytes
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ABSTRACT
Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles,
organisms that live under high hydrostatic pressure. However, the mechanisms by which
piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One
proposed hypothesis is that the volume changes of unfolding (ΔVTot) for proteins from piezophiles
is distinct from those of non-piezophilic organisms. Since ΔVTot defines pressure dependence of
stability, we performed a comprehensive computational analysis of this property for proteins from
piezophilic and non-piezophilic organisms. In addition, we experimentally measured the ΔVTot of
acylphosphatases and thioredoxins belonging to piezophilic and non-piezophilic organisms.
Based on this analysis we concluded that there is no difference in ΔVTot for proteins from
piezophilic and non-piezophilic organisms. Finally, we put forward the hypothesis that increased
concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from
piezophilic organisms and provide experimental thermodynamic evidence in support of this
hypothesis.
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1. INTRODUCTION
Single-celled organisms represent the dominant form of life on earth 1. Exploration has
led us to appreciate not only the diversity of life on earth, but also the diversity of the environments
in which these organisms live 2. Until recently, it was thought that life could not exist in extreme
conditions such as the bottom of the ocean or deep subsurface environments 2-5. However,
collection of pristine samples from such environments, and exploration of deep caves and mines
has shown that many life forms, including bacteria, archaea, and eukarya exist in places previously
thought to be uninhabitable 2,6. Examining of the genomic data from these samples has led to
estimate that the vast majority of life exists in the depths of the oceans and buried deep in the
subsurface crust 1,6. Such studies have suggested that there is a trove of species living in the deepest
parts of the earth’s crust waiting to be discovered. For example, it is known that the crust of the
earth is not uniform and large cracks, or fissures, penetrate kilometers into the earth 2. These
fissures are full of water, and because they are so deep, the heat from the mantle of the earth is
able to warm these waters 2. Minerals are then able to dissolve into these waters providing the
primary ingredients to sustain life 6. Interestingly, similar conditions have been proposed to be
drivers of life Jupiter’s moon Europa. On Europa, it has been suggested that there is sufficient
geological and tidal activity in its ocean to provide energy for life 7. Should this hypothesis hold
true it would be possible for life forms to exist in the liquid phase of its oceans, which reach depths
of up to 170 km 7.
While extra-terrestrial life remains a hypothesis, isolation and culturing of organisms living
in extreme conditions on earth has shown that these organisms are very sensitive to changes in
their environment 8-12. For example, when an organism that is adapted to more “mild”
(atmospheric pressure) conditions is subjected to the extreme condition (high pressure) it is unable
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to grow. 8. This is because changes in pressure, much like changes in temperature, have deleterious
effects on biomacromolecules, including changes in the fluidity of membranes, the dissociation of
protein-ligand complexes, and denaturation of proteins 9.
A response of a system to changes in pressure is described by the Le Chatelier’s principle
which suggests that a when a system in equilibrium is perturbed, it will re-equilibrate to a new
state which minimizes the effect of the perturbation. Thermodynamically this can be written as:
(∂∆G
∂P)
T= ∆VTot 1.
where G is the Gibbs energy and VTot is the volume changes, P is the pressure and T is the
temperature. In the case of pressure unfolding of proteins, the system is the protein in an aqueous
environment, the perturbation is the application of the pressure, and the equilibrium is between
folded and unfolded states. The majority of proteins have a negative VTot of unfolding 13-15, i.e.
the volume of the unfolded state is smaller than the volume of the native state. Thus, the increase
in pressure will lead to protein unfolding. Interestingly, there are examples of proteins that have a
positive VTot which suggests that the folded state will become more stable at increased pressure
15. Based on these observations, one of the possible mechanisms of adaptation to high hydrostatic
pressure is that proteins from organisms that live under elevated pressure evolved to have less
negative, or even positive, volume changes upon unfolding,. This hypothesis has been tested in
the present work.
We have previously established a method to compute the ΔVTot for proteins using its three-
dimensional structure 15,16. To better understand the molecular mechanisms of adaptation to high
pressure, we have applied this formalism to ΔVTot analysis of the proteome of organisms living
under diverse environmental conditions which include psychro-piezophiles (high-pressure, low-
temperature adapted organisms), thermo-piezophiles (high-temperature, high-pressure adapted
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organisms,) thermophiles (high-temperature adapted organisms), and psychrophiles (low-
temperature adapted organisms). We also analyzed the ΔVTot on a protein-by-protein basis, by
finding homologous matches across the four groups mentioned above, and compared them to the
proteins from mesophilic organisms. Finally, we experimentally measured and computed ΔVTot
for two protein families, acylphosphatase and thioredoxin, using representative structures from
these classes of organisms.
2. Materials and Methods
Obtaining and Preparing Structures from PDB
Groups of Archaea and Prokarya belonging to psychrophiles, psychro-piezophiles,
thermophiles, and thermo-piezophiles were identified that contained structures deposited to the
Protein Data Bank (PDB). Effort was taken to ensure that a diverse group of organisms was
represented in the study, thus both bacterial and archaeal species were included in the four groups.
Some organisms such as Thermus thermophilus is over represented in the Protein Data Bank,
having more than 10,000 deposited structures; in such cases, the organism was left out of the initial
analysis. This was done to minimize bias towards any single organism. Only structures solved by
X-ray crystallography were included into analysis.
Structures were obtained using the RESTful web service of the PDB. An XML document
containing the PDB Tree Entity ID for an organism, chain type containing only proteins (yes
Protein, no DNA, no RNA, no RNA/DNA hybrids), and an X-ray resolution of < 2.7 Å was
uploaded to http://www.rcsb.org/pdb/rest/search. The search results returned a list of PDB IDs
matching the aforementioned criteria. All structures were analyzed as monomers. In cases of
homomultimers, chain A was selected to further analysis. In cases of heteromultimers, the first
- 6 -
representative chain of each sequence was chosen to undergo further analysis. Any structures that
were discontinuous due to missing amino acids were discarded. To ensure low redundancy in the
analysis, proteins with multiple structures were identified and the longest, continuous, structure
was chosen, the rest were discarded. Sequences containing modified or unnatural amino acids
were discarded. The only exception was for sequences containing selenomethionine. The
Modeller software package was used to model in missing sidechain atoms 17. Complete list of
PDB id is provided.
Generating the Native and Unfolded Sate Ensembles
To generate the native state ensemble, a native-state, all-atom, explicit solvent MD was
performed using GROMACS, as previously described 15,18. The CHARMM27 force field and the
TIP3P water model were used to model the protein water system 19,20. The obtained crystal
structures were solvated in a dodecahedron such that the all of the protein atoms were at least 10
Å away from the edges of the box. Charges on the protein were neutralized by the addition of 0.1
M of NaCl. An initial energy minimization steepest descent was performed for 1000 integration
steps or until the maximum force < 1,000 kJ/mol/nm was reached. This was followed by a 200 ps
of constant volume equilibration, then 100 ps of constant pressure equilibration. Lastly, a 50 ns
production run was generated at a temperature of 300 K and 1 bar of pressure. Pressure was
monitored using the Parinello-Rahman barostat, having a 2 ps relaxation time and a
compressibility of 4.6·10-5atm-1 21. Temperature was monitored with V-rescale, having a relaxation
time of 0.1 ps 18. High frequency bond vibrations were constrained using the LINCS and SETTLE
algorithms 22. Electrostatic interactions were modelled using a smooth Particle Mesh Ewald
(PME), with a PME order paramour of four, and a Fourier spacing of 0.16 23. At the end of the
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production run, 50 structures were extracted from the production trajectory at a sampling
frequency of one structure per nanosecond in order to obtain the native state ensemble.
The unfolded state ensemble containing 1000 structures for each protein was generated
using Trajectory Directed Ensemble Sampling (TraDES) 24. Secondary structure propensity was
removed using the all-coil flag in TraDES software (-c T). The generated structures were
subsequently energy minimized in implicit solvent as described above.
Calculating Volume of Proteins
Calculation of protein volume in solution has been extensively described by us in detail
and will be described here briefly 15,16. The total volume of a protein in solution can be described
by equation, where VTot refers to total volume of a protein in solution, VSE refers to the solvent
excluded volume, and VHyd refers to the hydration volume.
VTot = VSE + VHyd 2.
The solvent excluded volume consists of the protein’s void (VVoid) and van der Waals
volumes (VvdW):
VSE = VVoid + VvDW 3.
The void volume is due to the fact that the atoms of a protein do not pack at 100%
efficiency, thus there will be unoccupied space that is also shielded from the solvent; this space is
the void volume 15,25. The van der Waals is volume is derived from the van der Waals radii of
atoms. The hydration volume, VHyd, is the result of the interaction of the protein molecules with
its surrounding water molecules. This interaction will affect the density of water at the surface of
the protein when compared to the density of the bulk water 15. As a result, the hydration volume is
dependent on the surface area that is interacting with water 15. While it has been established that
the contribution of hydration volume will always be positive, the magnitude is dependent on the
- 8 -
total exposed polar and the non-polar surface 15. The contribution of each type surface area are
defined by the non-polar (kNP) and polar (kP) coefficients. kNP=0.38 Å-5 and kP=0.03 Å-5 and were
derived empirically from a linear fit to the dependence of hydration volume on each type of surface
area model compounds 15, i.e.:
VHyd = (kNP × MSANP) + (kP × MSAP) 4.
The change in volume upon unfolding ΔVTot can be calculated as the sum in the change in void
volume (ΔVVoid) and the change in hydration volume (ΔVHyd) upon unfolding 15.
ΔVTot = VVoid,U − VVoid,N + VHyd,U − VHyd,N = ΔVVoid + ΔVHyd 5.
where the ΔVVoid and ΔVHyd are calculated by subtracting the respective unfolded state values from
the native state values. The VvdW volumes do not change upon protein unfolding, therefore they
are not considered when calculating ΔVTot 15.
Protein expression, purification and quantification
Modern thioredoxin (Trx) genes from the following organisms: Escherichia coli (EC),
Methanococcus jannaschii (MJ), Pyrococcus yayanosii (PY), Shewanella benthica (SB), Thermus
thermophilus (TT) have been expressed and purified as described previously 26. In addition,
ancestral Trx protein sequences corresponding reconstructed using Ancestral Sequence
Reconstruction (ASR) were used 27. The recombinant ancestral Trx genes from the last bacterial
common ancestor (LBCA), last archaeal common ancestor (LACA), archaeal-eukaryotic common
ancestor (AECA), last common ancestor of the cyanobacterial deinococcus and thermos groups
(LPBCA), last eukaryotic common ancestor (LECA), last common ancestor of γ-proteobacteria
(LGPCA), and the last common ancestor of animals and fungi (LAFCA), were expressed and
purified as described 26,27. Modern acylphosphatase (Acp) sequences from the following
organisms Bacillus subtilis (BS), Marinitoga piezophila (MP), Thermus thermophilus (TT),
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Sulfolobus tokodaii (ST) with engineered His-tags at the N-terminus were codon-optimized for
expression in E. coli and cloned into the pGia vector (Blue Heron, Bothell, WA).
Protein expression was done BL21 (DE3) E. coli strain in LB media (18 g tryptone, 10 g
yeast extract, and 5 g sodium chloride, per 1 L) 37°C. Overexpression of protein was induced
when OD600 reached ~0.8 o.u., using isopropyl-β-D-1-thiogalactopyranoside (IPTG) with a final
concentration of 1 mM. Cells were harvested by centrifugation 6-10 hours post induction. The
supernatant was discarded and the remaining pellets were stored at -20oC prior to further
purification. Frozen cell pellets were thawed and lysed using a French press cell. The resulting
lysate was diluted twice with 20 mM sodium phosphate, 500 mM sodium chloride, and 30 mM
imidazole, pH 7.4. Cellular debris was removed by centrifugation and the resulting supernatant
was passed through a Nickel-Nitrilotriacetic Acid (Ni-NTA) Agarose column, equilibrated with
20 mM sodium phosphate, 500 mM sodium chloride, and 30 mM imidazole, pH 7.4 buffer. The
stationary phase was washed with the binding buffer until the flow-through OD280 decreased to a
value of ~0.01 optical units. Protein was eluted using the elution buffer (20 mM sodium phosphate,
500 mM sodium chloride, 500 mM imidazole, pH 7.4). The eluted solution was concentrated to
approximately 30 ml using centrifugal filter units (Millipore, Billerica, MA) with a 3.5 kDa
molecular weight cutoff (MWCO) and loaded onto Sephadex G-50 column (2.5x170 cm)
equilibrated with a pH 7.4 buffer (20 mM sodium phosphate, 500 mM NaCl). Fractions containing
protein were combined and concentrated down to 10-15 ml using centrifugal units. The purity of
protein was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE). The protein solution was aliquoted into 2 ml Eppendorf tubes, flash-frozen, and stored at
-20oC.
Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC)
- 10 -
Before carrying out DSC and PPC experiments, protein samples were dialyzed extensively
using a Fisherbrand dialysis membrane with a 3.5 kDa MWCO. For acidic pH ranges, 30 mM
glycine-HCl (pH 2.0-3.4) and 30 mM sodium acetate (pH 3.5-5.0) buffers were used. Buffers
were chosen to minimize both enthalpy and volume changes upon ionization 28-30. After overnight
dialysis, the samples were centrifuged for at least 15 minutes at 4oC and 14,000 rpm (20,800xg) in
an Eppendorf 5417R microcentrifuge, in order to pellet any insoluble material present after
dialysis. A Hitachi U-2900 dual-beam spectrophotometer was employed to measure near
ultraviolet-visible (UV-Vis) absorption spectra of the proteins using 10 mm path-length
rectangular quartz cuvettes. Protein concentration was determined after accounting for light
scattering as described 31. The molar extinction coefficients (ε280nm), and partial specific volumes
(ῡpr) for Trx and Acp variants are given in Table S1. Extinction coefficients for Trx and Acp were
calculated as described previously 32. Partial specific volumes for the Trx and Acp variants were
calculated from amino acid sequence as previously described 33.
DSC experiments were performed using a VP-DSC instrument (MicroCal Inc./GE
Healthcare) with Trx and Acp protein concentrations of 0.3-1.5 mg/ml, and a scan rate of
90oC/hour. Reversibility of the protein unfolding transitions was determined by stopping the
protein-unfolding scan just after the completion of the transition, allowing the cells cool down,
and observing another transition as the temperature was increased once again. The studied proteins
had 80-90 % reversibility, as calculated by dividing the fitted calorimetric unfolding enthalpy
values of the second scan (re-melt) by that of the first scan. The experimental DSC data were
analyzed according to a two-state model using an in-house written NLREG script as described 34.
Summary of experimentally measured enthalpies of unfolding, H, and transition temperatures,
Tm, are shown in Figure S1.
- 11 -
PPC was performed using an additional pressure-cap attachment with the VP-DSC
instrument, as has been described previously 35-37. Experimental scans were carried out at protein
concentrations of 2.0-5.0 mg/ml. The coefficient of thermal expansion of a protein as a function
of temperature, αpr (T), was measured directly as the difference between the heat produced by the
protein in the sample cell, and that of the buffer in the reference cell, using pressure jumps of ~0.55
MPa. Duplicate measurements of heat changes upon compression and decompression were
performed every five degrees in the range of 2-97 oC. Data was collected every two degrees in the
unfolding transition temperature ranges to better describe the transition profile. Prior to running
the experiments, baseline experiments with sample-reference cells containing buffer-buffer,
buffer-water, and water-water were performed. Heat effects arising from the buffer component
and water were accounted for as follows 36:
𝑎𝑝𝑟 = 𝛼𝐻2𝑂 −∆𝑄𝑏𝑢𝑓−𝐻20
𝑇∙∆𝑃∙𝑉𝑐𝑒𝑙𝑙−
∆𝑄𝑝𝑟−𝑏𝑢𝑓
𝑇∙∆𝑃∙𝑚𝑝𝑟∙ῡ𝑝𝑟 6.
Where αH2O is the coefficient of thermal expansion for water, ∆Qbuf-H20 and ∆Qpr-buf describe the
heat changes for the buffer and protein solutions, ∆P is the change in pressure of the system, Vcell
is the volume of the calorimetric sample cell, and mpr is the mass of the protein in solution. The
overall experimental αprexp(T) profile was fit as follows using in house written NLREG scripts 36:
𝛼𝑝𝑟𝑒𝑥𝑝(𝑇) = FN(T) ∙ αN(T) + FU(T) ∙ αU(T) +
𝐾𝑒𝑞(𝑇)
(1+𝐾𝑒𝑞(𝑇))2
∆𝐻(𝑇)∙∆𝑉𝑃𝑟
(1+𝐾𝑒𝑞(𝑇))2∙ῡ𝑃𝑟 7.
In which FN(T), FU(T) describe the temperature dependencies of the fraction of folded and
unfolded protein, αN(T) and αU(T) describe the temperature dependencies of the folded and
unfolded thermal expansion profiles, and Keq(T) is the temperature dependence of the equilibrium
constant of protein unfolding. Summary of the experimentally measured enthalpies of unfolding,
- 12 -
H, the volume changes upon unfolding, V, and the transition temperatures, Tm, for
acylphosphatase and thioredoxin protein families are shown in Figure S1. Values linearly
extrapolated to 25°C were used for comparison with the computed values in Figure 3.
Thermodynamic parameters obtained from DSC and PPC experiments were used to
compute the stability of a protein using the following equation 38:
ΔG = ΔH (T − Tm) Tm⁄ + Δc𝑝(T − Tm) − Δc𝑝Tln(T Tm⁄ ) +
ΔV (p − po) + ∆α(T − Tm)(p − po) + (∆β 2⁄ )(p − po)2
8.
where H is the enthalpy of unfolding at Tm, V is the volume change upon unfolding at Tm, and
po is the reference pressure (0.1 MPa). The Δcp is the change in heat capacity (calculated from the
temperature dependence of enthalpy of unfolding see Figure S1), ∆α is the change in thermal
expansivity (calculated from the temperature dependence of the volume changes upon unfolding
see Figure S1), the Δβ is the change in compressibility upon unfolding, taken from 39. A pressure-
temperature (P-T) stability phase diagram can be generated by setting the ΔG(p, T) = 0 and defines
the boundaries of stability for the protein (Figure 4) The area under the line corresponds to the
pressure and temperature ranges in which the protein will be largely in the native state. The area
above of the line describes the conditions in which the protein is largely in the unfolded state 40.
2. RESULTS
Computational analysis was performed on the protein structures deposited to the Protein
Data Bank (PDB) from the four groups of organisms: thermophiles, psychrophiles, thermo-
piezophiles, and psychro-piezophiles. Proteins ranging in size from 50 to 500 amino acid residues
- 13 -
were selected and the final number of structures for each group was as follows: 254 for thermo-
piezophiles, 421 for thermophiles, 52 for psychro-piezophiles and 84 for psychrophiles. In
addition, 171 structures from mesophiles that were analyzed previously 15 were included. The
complete list of PDB IDs for each group can be found in Table S2. Despite the relatively small
number of structures in the data sets, they possess features that have been identified from a larger
analysis of sequences of proteins. For example, the amino acid composition of thermophilic
proteins in our data set shows an increased fraction charged amino acids and decreased fraction of
polar, while the psychrophilic proteins have an have increased fraction of polar residues and a
decreased fraction of charged residues (see Figure S2), an observation reported previously by
Claverie and colleagues as CvP-bias 41,42.
To compare the volume change of unfolding the ΔVTot of each protein from the compiled
datasets was calculated as described in Materials and Methods. Figure 1A shows a comparison of
ΔVTot as a function of the number of amino acid residues. It is evident that although ΔVTot becomes
more negative with increase in protein size, this trend is similar for the five sets of proteins
(thermophiles, psychrophiles, mesophiles, thermo-piezophiles, and psychro-piezophiles.) Smaller
proteins have a ΔVTot that is close to zero while larger proteins have increasingly more negative
ΔVTot. Furthermore, as protein size is increased the variance of the data increases. Figure 1B
compares the ΔVTot values for the five datasets in a form of a box-plot. The median ΔVTot values
are -310 Å3, -279 Å3, -221 Å3, -273 Å3 and -236 Å3 for thermophiles, psychrophiles, mesophiles,
thermo-piezophiles, and psychro-piezophiles, respectively. A two-way Analysis of Variance
performed on the five datasets returned a P-value of 0.39, which indicates no detectable difference.
Furthermore, the Student’s t-test pairwise p-values range between 0.09 and 0.95, also indicating
that there is no significant difference in the ΔVTot for the proteins in these five groups.
- 14 -
The proteome-wide calculations of ΔVTot indicated that there is no discernible difference
across thermophiles, psychrophiles, mesophiles, thermo-piezophiles, and psychro-piezophiles. To
further illustrate this point, we computed ΔVTot of individual homologous structures from the five
groups. To this end, we analyzed six different protein families, for which structures are available
across the five groups. Comparison of computed ΔVTot is shown in Figure 2 (see also Figure S3
for expanded set of data). Despite variance in the individual ΔVTot values, there is no clear trend
which suggests that proteins belonging to a particular group (i.e. piezophilic organisms vs non-
piezophilic) have distinct a ΔVTot.
Finally, we have experimentally measured thermodynamics properties including the ΔVTot
for representative members of two protein families: acylphosphatase, Acp, and thioredoxin, Trx,
(see Materials and Methods section and Figure S1). The acylphosphatases studied were
mesophilic Bacillus subtilis (Acp-BS), thermophilic Thermus thermophilus (Acp-TT) and
Sulfolobus tokodaii (Acp-ST), and thermo-piezophilic Marinitoga piezophila (Acp-MP) and
Pyrococcus yayanosii (Acp-PY) organisms. The thioredoxins studied were from the psychro-
piezophilic Shewanella benthica (Trx-SB), mesophilic Escherichia coli (Trx-EC), and
thermophilic Thermus thermophilus (Trx-TT) and Methanocaldococcus jannaschii (Trx-MJ)
organisms. In addition, we included six thioredoxins that were obtained using ancestral sequence
reconstruction (ASR). The details for ASR are described in Perez-Jimenez et al. 27 and based on
their constructed phylogenetic tree, the sequences are dated to be between 1.6 and 4.2 Gyr old.
The experimental ΔVTot values for all these proteins at 25°C are shown in Figure 3.
The three-dimensional structures for the Acp and Trx proteins studied experimentally are
available (see Table S1 for PDB codes) and were used to compute the ΔVTot using the procedure
identical to the one described above. The computed and experimentally determined ΔVTot values
- 15 -
are compared in Figure 3 and show a good agreement between the two data sets. This, together
with our previously published evidence 15, further supports the validity of our computational
approach to calculate the volume changes upon protein unfolding.
In addition, the experimental data shown in Figure 3 shows two important features. First,
there is no trend suggesting a difference for the absolute values of the experimentally measured
ΔVTot for proteins from piezophilic versus non-piezophilic organisms. Second, ΔVTot values for
ancestral thioredoxins show similar variance as the ΔVTot of extant proteins suggesting that this
parameter is not conserved during the evolution. The experimental results analyzed together with
the proteome-level calculated ΔVTot values led us to conclude that the ΔVTot of proteins from
piezophilic organisms is comparable to the ΔVTot of proteins from non-piezophilic organisms.
3. DISCUSSION
The conclusion we reached above refutes our original hypothesis that the modulation of
volume changes upon unfolding of proteins can be used as a mechanism to cope with high
hydrostatic pressure. We find no evidence in support for this hypothesis and thus conclude that
modulating ΔVTot is not a mechanism with which piezophilic organisms evolved to stabilize their
proteins against high hydrostatic pressure. Yet it is known that life exists in the deepest parts of
the ocean where hydrostatic pressure reached 100-120 MPa. 3-5,11,12,43-45. This suggests that there
must be non-volume based mechanisms that stabilize proteins against pressure denaturation.
Proteins with higher thermodynamic stability will have expanded pressure range where they
remain native. Conversely, proteins that do not have high stability will be more susceptible to
high hydrostatic pressure. This can be seen from the P-T diagrams constructed using experimental
data for Acp and Trx protein families (Figure 4A-B). Thus, factors that increase the net protein
- 16 -
thermostability play a role. These factors, such as increase in number of hydrogen bond, optimized
electrostatic interactions, changes in conformational entropy of the native and unfolded states,
increase in secondary structure propensities, etc have been well documented 46-49. Yet, it has been
shown that different proteins adopt different strategies to increase their thermostability 50. There
is however, one mechanism of thermostabilization that can act on all proteins and may be relevant
for piezophilic organisms - the stabilizing effect of protective osmolytes. Stabilizing osmolytes,
such as trimethylamine oxide (TMAO), glutamate, betaine, alanine, β-hydroxybutyrate (β-HB),
mannosylglycerate, di-myo-inositol phosphate and N-Acetyl-β-lysine, are present in high
concentrations in many piezophilic organisms 9,51,52. These molecules were shown to increase
protein stability (see e.g. references 53-60) and this increase in stability is not accompanied by
volume changes 61. Such increase in stability may have a physiological role in piezophiles. For
example, Photobacterium profundum is a well-studied facultative piezophile that grows optimally
at 200-300 bars of pressure but is also able to grow at atmospheric pressures. Growth studies of
this organism at atmospheric and high pressures have shown that P. profundum concentrates
osmolytes such as betaine and glutamate (both of which are stabilizing osmolytes) when incubated
at high pressures 62. The presence of elevated concentration of osmolytes is not unique to single-
celled organisms. Deep-sea animals such as muscles, skates, and teleosts have also been observed
having high levels of TMAO or another type of osmolyte, such as scyllo-inositol, β-alanine, or
betaine 51,52,63. Moreover, a correlation between the concentration of TMAO and the habitat depth
(i.e. increase in hydrostatic pressure) of certain fish is well documented 64,65. Thus, a likely
mechanism for the piezophilic adaptation can be the net increase in stability due to increase in the
concentration of osmolytes. Addition of osmolytes can produce a significant modulation in the P-
T stability diagram. For example, the modulation in P-T stability of a model protein RNAse A by
- 17 -
various osmolytes (see Figure 4C) significantly increases the tolerance to high hydrostatic pressure
and is expected to produce qualitatively similar stabilizing effects on any protein. Such mechanism
is much easier to implement from an evolutionary perspective as it will work on a systemic level
and will stabilize all proteins. Moreover, osmolytes usually have stabilizing effects on protein
interactions with the ligands or substrates again providing net increase in stability and thus increase
in pressure tolerance on functional level beyond just protein structure.
- 18 -
Supplementary Information
The Supplementary Information includes Tables with PDB codes, and additional figures (2
separate PDF files)
Acknowledgements
Supported by grants CHEM-1506468 and CHEM-1803045 from the US National Science
Foundation (awarded to G.I.M) and the RPI Presidential Fellowship awarded to S.A. This work
used the Extreme Science and Engineering Discovery Environment (XSEDE) comet (SDSC) and
stampede2 (TACC) using allocation TG-MCB140107, which is supported by National Science
Foundation grant number ACI-1548562.
Conflict of Interest
The authors declare no conflict of interest.
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FIGURE LEGENDS
Figure 1. Comparison of the computed volume changes show no difference between
piezophilic and non-piezophilic proteins. Panel (A). Dependence of the VTot on
number of amino acid residues from Thermophiles (, gray circles), Thermo-
piezophiles (, red down triangles), Mesophiles (, black squares) Psychrophiles
(, blue diamonds) and Psychro-piezophiles (, green up tringles). See Table S2
for list of proteins PDB codes. Panel (B). Box-plot comparison of the data presented
in Panel A using the same color-coding.
Figure 2. Comparison of the computed volume changes for the homologous proteins from
piezophilic and non-piezophilic proteomes. Panel (A). Enoyl-CoA Hydratase (EC
4.2.1.17); Panel (B). Dihydrodipicolinate Synthase (EC 4.2.1.52); Panel (C). Lactate
Dehydrogenase (EC 1.1.2.3); Panel (D). Adenylate Kinase (EC 2.7.4.3); Panel (E).
Malate Dehydrogenase (EC 1.1.1.37); Panel (F). Isopropyl Malate Dehydrogenase
(EC 1.1.1.85). The colors are: gray - Thermophiles; red – Thermo-piezophiles; black
- Mesophiles; blue - Psychrophiles; green – Psychro-piezophiles. The corresponding
PDB codes for each protein are listed on the x-axes (see also Table S3 and expanded
data in Figure S3).
Figure 3. Comparison between experimentally measured and computed volume changes of
two protein families. Panel (A). Acylphosphatase (EC 3.6.1.7); Panel (B).
Thioredoxin (EC 1.8.1.9). Shaded bars correspond to the V values at 25°C obtained
- 24 -
from the linear extrapolation of the temperature dependence of experimental values
shown in Figure S3 (see Materials and Methods for details) while colored bars are for
the computed values. The colors are gray - Thermophiles; red – Thermo-piezophiles;
black - Mesophiles; green – Psychro-piezophiles, yellow - ancestrally reconstructed
proteins.
Figure 4. Representative Pressure-Temperature (P-T) diagrams for acylphosphatase and
thioredoxin protein families and RNAse A from experimental data. Panel (A).
Acylphosphatases: Acp-BS - black; Acp-TT - gray; Acp-MP - red. Panel (B). Extant
thioredoxins: Trx-SB - green; Trx-EC - black; Trx-TT - gray; Panel (C).
Experimentally derived P-T diagrams for RNAseA in solution containing various
concentrations of osmolytes; 2M betaine - cyan; 2 M sarcosine -– pink; 2 M glutamate
- orange or no osmolyte - black. P-T diagrams on Panels A and B were calculated
using equation 8 from the experimental data shown in Figure S1. Panel C is adopted
with modifications from 61.
- 25 -
Figure 1.
Figure 2.
Thermo Thermo Meso Psychro Psychro
VT
ot (Å
3)
-1000
-750
-500
-250
0
PiezoPiezoNumber of Amino Acid Residues
50 100 150 200 250 300 350 400 450 500
V
Tot (Å
3)
-2500
-2000
-1500
-1000
-500
0
Thermophiles
ThermoPiezophiles
Mesophiles
Psychrophiles
PsychroPiezophiles
A B
5VE2 4K3W 5YLO 4JVT 2PBP
VT
ot(
Å3)
-1000
-800
-600
-400
-200
0
Enoyl-CoA Hydratase
5AFD 4PFM 3IRD 3PB0 2YXG
VT
ot(
Å3)
-1250
-1000
-750
-500
-250
0
Dihydrodipicolinate Synthase
6LDH 3PQE 2V6M 1HYG
VT
ot(
Å3)
-1200
-1000
-800
-600
-400
-200
0
Lactate Dehydrogenase
1S3G 4K46 2CDN 1ZIP
VT
ot(
Å3)
-600
-500
-400
-300
-200
-100
0
Adenylate Kinase
2V65 1B8P 4JCO 4KDE 2X0I
VT
ot(
Å3)
-1000
-800
-600
-400
-200
0
Malate Dehydrogenase
2UXQ 3VKZ 1CM7 1VLC
VT
ot(
Å3)
-1250
-1000
-750
-500
-250
0
Isopropyl Malate Dehydrogenase
A B C
D E F
- 26 -
Figure 3.
BS MP PY TT ST
DV
(cm
3/m
ol)
-200
-150
-100
-50
0SB EC TT MJ LPBCA LGPCA LACA LAFCA AECA LBCA
DV
(cm
3/m
ol)
-200
-150
-100
-50
0
A B
- 27 -
Figure 4.
C
Temperature (oC)
0 20 40 60 80
Pressu
re (MP
a)
0
100
200
300
400
500
A
Temperature (oC)
0 20 40 60 80
Pre
ssu
re (
MP
a)
0
100
200
300
400
B
Temperature (oC)
0 20 40 60 80
Pre
ssu
re (
MP
a)
0
100
200
300
400
2M Glu
2M Bet
2M Sar
0M
Acp-BS
Acp-MP
Acp-TT
Trx-EC
Trx-TT
Trx-SB