# Mineral and Protein-Bound Water and Latching Action Control Mechanical Behavior at Protein-Mineral Interfaces in Biological Nanocomposites

Authors: Pijush Ghosh; Dinesh R. Katti; Kalpana S. Katti
Journal: Journal of Nanomaterials
 (2008)

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## Abstract

The nacre structure consists of laminated interlocked mineral platelets separated by nanoscale organic layers. Here, the role of close proximity of mineral to the proteins on mechanical behavior of the protein is investigated through steered molecular dynamics simulations. Our simulations indicate that energy required for unfolding protein in the proximity of mineral aragonite is several times higher than that for isolated protein in the absence of the mineral. Here, we present details of specific mechanisms which result in higher energy for protein unfolding in the proximity of mineral. At the early stage of pulling, peaks in the load-displacement (LD) plot at mineral proximity are quantitatively correlated to the interaction energy between atoms involved in the latching phenomenon of amino acid side chain to aragonite surface. Water plays an important role during mineral and protein interaction and water molecules closer to the mineral surface are highly oriented and remain rigidly attached as the protein strand is pulled. Also, the high magnitude of load for a given displacement originates from attractive interactions between the protein, protein-bound water, and mineral. This study provides an insight into mineral-protein interactions that are predominant in biological nanocomposites and also provides guidelines towards design of biomimetic nanocomposites.


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## Body

## 1. Introduction

Minerals and proteins exist in
close proximity and at nanoscale dimensions in biology. Interactions at these
interfaces are vital to the functions of almost all structural materials in
nature such as teeth, seashells, and bone. Knowledge of these interfaces, in
particular, is useful in understanding the mechanical and physiochemical behavior
of natural biocomposites such as bone, teeth, nacre, and so forth consisting of mineral
and organic phases. The percentage constituent of mineral and organics is different
in the various biological materials. In
bones and teeth, the mineral content is about 60% and 90%, respectively [1],
whereas, in nacre it varies from 95% to 98% [2]. The major component of the
organic phase in nacre is proteins. Although organics are present in small
proportion, they significantly alter the mechanical behavior of biocomposites.
Nacre, for example, exhibits fracture toughness about 3 000 times more than the
pure aragonite [2]. The proteins present in the natural biocomposite nacre show
properties significantly different from any other bulk proteins. Our finite
element modeling study, also later verified by experiments, shows that proteins
present in nacre have modulus of about 15–20 GPa [3–7]. These simulations were done based on a parametric study of
varying values of elastic modulus of the organic from 5 MPa to 100 GPa. This
is about twenty times higher than the modulus of bulk proteins. The protein in
nacre exists in a 20 nm space, between aragonite platelets of 200–250 nm thickness
in an interlocked “brick and mortar” architecture [6]. Two possible factors
which could cause the nacre protein to possess such high modulus are (i) its
confinement in 20 nm space, and (ii) the mineral-protein interactions. Our
current research focuses on the effect of mineral-protein interactions. The
effect of confinement will be the focus of our future work. We have found in
our previous work that the proximity of mineral influences the mechanical behavior
of proteins [8]. We have reported that more energy is required to pull a
protein at mineral proximity than in the absence of mineral. Recently, we have
also reported the large influence of mineral on mechanical behavior of protein
through simulations of collagen-hydroxyapatite in bone [9]. Here, we present
the detailed mechanisms responsible for large changes in mechanical response
during protein unfolding in the proximity of the mineral. In this molecular
modelling simulation, the GS domain [10, 11] of the Lustrin A,
protein which is found close to mineral surface in nacre, is pulled under two
conditions: (i) in the proximity of mineral aragonite, (ii) in the absence of any
mineral proximity. All other conditions of simulation remain identical for the
two cases. As the GS domain is pulled keeping one end constrained, the protein
domain unfolds. The mechanisms involved in the unfolding pathways are
significantly different under the two conditions of pulling, resulting in large
differences in energy required for unfolding at mineral proximity.Molecular dynamics is a useful technique for
studying the folding/unfolding
behavior of proteins [12, 13]. The reversible unfolding of individual
immunoglobulin domains was successfully investigated using a combination of
steered molecular dynamics (SMD) and atomic force microscopy (AFM) [14–16]. The
SMD technique has been successful in reproducing the stretching events for
individual solvated I27 domain [17]. This technique has also been used for studying
the mechanical properties of clay minerals at nanometer length scale [18–21]. In our
previous work, [21] we have used steered molecular dynamics to understand the
deformational behavior of the beta barrel and beta planar sheet structures in
proteins. Also, in literature, the helix-coil transition of peptide deca-alanine
in vacuum was studied using molecular dynamics simulation [22]. Researchers [23] were able to investigate the different transition states of biopeptides at
different temperatures.The model protein structure used in this
study is the nacre protein Lustrin A. The primary reason for this choice is
that this protein is located close to the aragonite surface and thus can
potentially interact with the mineral. The protein Lustrin A is made of 1428
amino acid residues and has molecular weight of about 112 KDa. The complete
structure of this protein is not known. Hence, at present, this study is
confined to the response of a single-domain Glycine-Serine (GS) of the protein Lustrin A.
Molecular modeling techniques such as coarse-grained modeling [24] can be
applied in the future to study the response of the full protein when the
structure is known. Replica-exchange molecular dynamics method has been
successfully used in literature to study the folding unfolding behavior of
proteins and peptides [25]. There are various experimental techniques such as
laser tweezers [26], florescence quenching [27], and AFM [28], which are found
to be useful in characterizing the protein folding and refolding response. Most
of these techniques are fairly successful in the study of single-molecule
protein folding. These studies have
primarily looked at the molecular mechanisms in the unfolding of proteins, however
recently we have reported measurement of adhesion forces between aragonite and
nacre proteins by pulling proteins off the aragonite surface using an AFM tip
[29]. Experiments were conducted on freshly cleaved samples of nacre, and
protein molecules were pulled in the presence of aragonite at various
velocities using an AFM tip. The results indicate that protein molecules can
withstand large pulling forces of the order of 6 nN before separating from the aragonite or the AFM
tip [29].


## 2. Model Construction and Simulation Details

One of the most widely studied
nacre proteins is Lustrin A. It is a domain-based protein, and is located close to the
interface formed by the inorganic (aragonite) and organics (primarily
proteins). Lustrin A is populated by ten cysteine rich domains with nine
protein rich domains sitting in between. It has a domain made of 275 amino
acids, and significantly rich in glycine and serine. It is located close to the C-terminal and
named as GS domain in this work. For the sake of these simulations, GS domain
is the organic entity. The details about the possible role of each of the
domains in Lustrin A are discussed in our previous work [8]. From the known
primary sequence of GS-domain
random, structures were generated, minimized, and then solvated by “SOLVATE”
module of VMD. The aragonite is built by extending its unit cell [30] to 16
units each in X- and Y-direction
and 2 units in Z-direction. The solvated GS domain built earlier is placed next
to the aragonite to build the organic inorganic model used in this study. The
details of model building are discussed in our earlier paper [8]. It should also be noted that, since water has significant
role in the influence of mineral proximity, appropriate solvation of protein is
very important. In our model [8], before pulling the GS domain is completely
solvated, thus enclosing the entire domain in a water box.Steered molecular dynamics (SMD) is used to
study the influence of mineral proximity on the mechanical response of protein
(GS domain). One of the alpha-carbon
atoms present close to aragonite surface is pulled to unfold the GS domain
while fixing the other end located opposite to it. Same pulling and fixed atoms
are used when GS is pulled in the presence and absence of aragonite. Three
different magnitudes of velocity 0.25 Å/ps, 0.50 Å/ps, and 1.00 Å/ps are
applied in unfolding GS domain. Using each of these velocities, the GS domain
is pulled for a period of 250 ps. The stiffness of SMD spring used is 5 Kcal/mol/Å. Molecular dynamics software NAMD [31] is used for all simulations,
and VMD [32] is used for visual analysis. CHARMm forcefield is applied in this
work. The parameters for GS domain are obtained from standard CHARMm protein
parameter file [13]. The CHARMm bonded parameters for aragonite are used from
the literature whereas the nonbonded parameters are derived from existing
Buckingham potential. The CHARMm aragonite parameters used in this work are
shown in Table 1.Table 1
CHARMm forcefield parameters for aragonite.
BondKB (Kcal/molÅ2)roC–O19219.281.30C–O29219.281.28AngleKA (Kcal/molrad2)θoO1–C–O1214.7613119.58O2–C–O2214.7613120.19NonbondedσijεijCa–O12.711.52Ca–O22.711.52C–O12.9991.5C–O22.9991.5O1–O22.8550.35σij=(σi+σj)/2;εij=εi*εjσiεiO2.8551.6023C3.1431.7639Ca2.5651.4395


## 3. Results and Discussion

It is been found from our previous
work [8, 9] that the proximity of mineral influences the mechanical response of
proteins as observed in both seashells and bone. More energy is required to
unfold a protein when pulled in the presence of aragonite than when pulled in
its absence. It is also observed that energy required to unfold at mineral
proximity depends on the velocity of pulling. The specific reason for this
velocity dependence is not understood yet and is a part of our future research.
In the current work, studies are conducted to find mechanisms leading to large
increase in energy required for unfolding of protein at mineral proximity and
in the absence of the mineral.Protein molecules exhibit a
tertiary structure due to several cross links (hydrogen bond, disulphide
bridge, etc.) between domains and within domains. Specific hydrogen bonds give
rise to mathematically defined structures such as alpha-helix, beta-sheets. A
protein has several turns (such as hairpin) between strands. When a protein is pulled (in the absence
of mineral), all these bonds offer resistance to pulling. When the protein is
pulled in presence of mineral the resistance comes from both structural
features of the protein molecule as well as the resistance forces due to
interaction of protein with mineral.The
load-displacement characteristics of the protein domain (GS domain) pulled at
mineral proximity and without the mineral at a velocity of 1.00 Å/ps are shown
in Figure1. The ratio of the area under the load-displacement (L-D) curve in
the presence of aragonite to the area under L-D curve in the absence of
aragonite is defined as “Work Factor.” The work factors obtained at three different
velocities are shown in Table 2. From this table, it is observed that when the
protein is pulled at velocity of 0.25 Å/ps, about ten times more energy is
necessary to unfold the GS domain by same magnitude of displacement in the
presence of aragonite than in the absence of it. It is observed from Figure 1
that the L-D responses are significantly different under the two conditions.
The primary factors leading to the larger area under the L-D curve pulled at
mineral proximity areTable 2
“Work
factor” at different velocities.
Velocities (Å/ps)Work factor0.2510.500.508.501.005.00Figure 1
The L-D characteristics of the GS domain, pulled at a velocity of 1.00 Å/ps: at proximity of aragonite and in the absence of aragonite.(a)
the presence of  peaks of higher
magnitude in L-D response curve for mineral proximity;(b)
and the presence of higher value of load for a given displacement. In the following two sections, we
present the results of our study about the mechanisms arising from the above
two factors.


### 3.1. Section I

Peaks in the L-D curve are present
in both plots as indicated in Figure1. The peaks observed in the absence of
mineral aragonite have a height of about 150 to 200 pN, whereas the peaks in
the presence of aragonite are of larger magnitude and are in the range of 1500
to 2000 pN and have lower frequency of occurrence. The magnitude of peak
heights in the absence of aragonite corresponds to the opening of loops and
turns and breaking of hydrogen bonds. From the nature of peaks observed in two
cases, it can be inferred that different mechanisms are involved in the
formation of peaks when pulling protein at mineral proximity. From the
trajectory analysis of unfolding, it is observed that peaks occur until water
enters between the aragonite surface and GS strands. As pulling is continued
and water enters in between, no additional peaks are observed in the L-D curve.
This observation indicates that direct interaction between aragonite surface
and GS strands may result in the formation of these peaks. We have investigated
the peaks up to 50Ådisplacement. This displacement corresponds to the point
after which significant water enters in between the aragonite surface and GS
strand. From the trajectory analysis, it is
observed that when the GS strand is pulled at mineral proximity, a mechanism
similar to the physical phenomenon of “latching” is observed to take place
between aragonite surface and GS strand in proximity. The “latch” observed here
is not a physical latch but a result of strong nonbonded interactions between
an atom or a group of atoms of amino acid residues (attached to GS strand) and
aragonite surface. These latches are formed when the interacting group(s) come(s)
close to the aragonite surface, but as pulling is continued they are observed
to break. These latches thus go through a process of “forming" and “breaking"
as the GS strand is pulled along the aragonite surface, giving rise to the
peaks in the L-D curve.Further, we zoomed into a small section,
circled in Figure2(a) where, a “latch” is observed. The “forming" and
“breaking" phenomenon of a latch is represented in Figures 2(b) and 2(c).
The aragonite layer close to the surface is shown in the figures. The L-D
characteristics of one of the peaks are shown in Figure 2(d). As the strand is
pulled in the direction of the arrow in the figure, a group of atoms (marked as
blue balls) comes closer to the surface, builds a strong interaction, arrives
between the carbonate groups of aragonite, and gives rise to what we called as
a “latch." The formation of the “latch” corresponds to the base point of the
peak as marked in Figures 2(b) and 2(d). Once the latch is formed, it
offers resistance to pulling, causing the magnitude of load to rise sharply.
The load curve keeps rising as pulling is continued until the load level where
the latch can no longer sustain the pulling force results. At that point, the
latch breaks, and thus the force decreases. The apex of a peak therefore
corresponds to the point where a latch breaks (Figure 2(c)).Figure 2
The mechanism of “latching” and formation of peaks: (a) latching site (b) formation of latch (c) breaking of latch (d) peaks resulting from latching mechanism.Aragonite exhibits orthorhombic crystal
symmetry. The surface formed by (001) is rich in negatively charged oxygen
atoms belonging to carbonate group. The positively charged calcium atoms are
located very close to the surface. The charges on the surface oxygen and
calcium atoms are found to be −0.9995 and +2.000, respectively. The blue atom
group constitutes mainly of hydrogen and/or oxygen atoms. The hydrogen and
oxygen atoms of amino acid strongly interact with oxygen and calcium of aragonite,
respectively, forming these latches. As polar water molecules enter between the
aragonite surface and GS strand, these interacting groups can no longer come
close enough to form the latches and thus no sharp peaks are observed. In
Figure1, no peaks are observed beyond a displacement of about 60 Å. The peaks
within the same L-D curve (at mineral proximity) are different in terms of
height, base width, shoulder characteristics, etc. This heterogeneity in peak
characteristics depends on the number of latches formed at a given time, the
atoms involved (blue group) in the “latching” action and on the presence of
other interacting groups closer to the aragonite surface.Thus, a peak in the L-D curve starts rising
when a side chain group builds up a strong nonbonded interaction with aragonite
surface. It falls when this interaction is broken upon further pulling. Thus,
the energy stored in the rising half of the peak is due to the resistance
offered by the latch and is thus expected to be approximately equal to the
nonbonded energy holding these two interacting groups together. The nonbonded
energy which is the summation of van der Waals and electrostatic energy between
the interacting group of amino acid side chains, and the aragonite section
(shown in Figure2(b)) is determined. The variation of the electrostatic and
van der Waals energy with time (frame) is shown in Figures 3(a) and 3(b). In
Figure 2(d), it is observed that Peak 0 starts rising at time frame 85 (17 ps).
The electrostatic and van der Waals energy corresponding to this time frame as
observed in Figures 3(a) and 3(b) is 2000 pN-Åand 700 pN-Å, respectively. The
area bound by the rising half of the Peak 0 (X, Y and Z in Figure 2(d)) is 2500 pN-Å, which is close to the total nonbonded interaction (2700 pN-Å) energy
between the interacting groups. From a similar analysis of Peak 1 (Figure 4(a)),
where the peak starts at time frame of 135 (27 ps), the total attraction energy
(900 pN-Å) is found to be very close to the area bound by the rising part of the
curve (1100 pN-Å). The variation of the electrostatic and van der Waals energy
with time (frame) for Peak 1 is shown in Figures 4(b) and 4(c).For other major peaks, these values are also
in close agreement. Thus it is quantitatively shown that the large peaks
observed in the proximity of the mineral are a result of direct nonbonded
interactions between the side chain atoms and the complementary atoms on the
mineral surface.The nonbonded energy between the interacting groups giving rise to Peak 0: (a) electrostatic energy (b) van der Waals energy.
(a)(b)Peak 1 (a) load-displacement plot (b) electrostatic (c) van der Waals energy.
(a)(b)(c)


### 3.2. Section II

As observed in Figure1 and also
mentioned earlier, one of the factors which contribute to the work factor is
the higher magnitude of load at any given displacement, when pulled at mineral
proximity. The peak load observed in the figure is about 13 nN, a high
magnitude, predominantly as a result of mineral protein interactions. This high
magnitude of force resulting from constant velocity pulling simulations is
possible without fracture of the single protein strand being pulled. Flow
induced fracture of single polymer chains is reported to be between 2.5 to 13.4 nN [33, 34] and the fracture strength is dependent upon the polymer and rate of
loading. In this section, the potential mechanisms
involved are described.  As the GS domain
is pulled, the water molecules present closer to the aragonite surface orient
and arrange themselves in a definite pattern and further undergo very little
movement. On moving away from aragonite surface, the water molecules are
structurally altered, and behave like “normal solvent water" attached to the
protein.A clear difference in behavior is observed
between water molecules closer to the surface and away from the surface both in
terms of structure and movement during pulling. We have therefore categorized
the water into two types. The water molecules close to the aragonite surface are
named here as Aragonite-Bound Water (A-BW), and water molecules moving with the
protein are named
as Protein-Bound Water (P-BW). The schematic representation of these two types
of water molecules is shown in Figure5.
At low displacement of pulling, very few water molecules are observed to
be present between aragonite and GS strands. However, this number increases as
pulling is continued. The strong hydrogen-bonded interaction between carbonate surface of
aragonite and polar water molecules causes some of the water molecules to lie
close to the aragonite surface. The water molecules form a periodic pattern
which is primarily due to the periodicity of the aragonite surface in the X-Y
plane. The P-BW shows no definite pattern, with the water molecules surrounding
the protein and moving freely with the protein. As the GS is pulled, the A-BW
remains tightly bound to the aragonite surface and exhibits almost no movement.
The P-BW on the other hand moves freely with the GS strand. On careful
observation, it is observed that there is a 3-4Åthick layer of water (about
two water molecules thick) which behaves as a transition layer between A-BW and
P-BW. In this layer, the slipping of
P-BW is observed to happen with respect to the A-BW. The P-BW forms a sheath
around the GS strands. When GS is pulled, the strands move with sheath of water
around it. P-BW thus moves with GS strands and A-BW remains bound to the
surface. A schematic representation of this mechanism is shown in Figure 6. This
figure illustrates that when GS is pulled, the interaction contributing to
resistance to displacement includes the following:Two “types” of water molecules observed during pulling of GS domain (a) simulation (b) schematic representation. A-BW: aragonite bound water and P-BW: protein bound water.
(a)(b)Figure 6
Schematic representation of movement of the GS domain and protein-bound-water (P-BW) when GS domain is pulled.(i)
the net attractive interactions
of GS-(A-BW) and GS-aragonite;(ii)
the net attractive interaction
of (P-BW)-(A-BW) and (P-BW)-aragonite and GS-(P-BW).To study the
contribution of these different interactions to the L-D characteristics of GS
domain, aragonite sections of smallerX-Y dimension and part of GS strand close
to it are selected. We have used two such sections for the analysis of the
interaction energy. One of them is shown in Figure 7. This section extends to
20Åin the X-direction and 50Åin the Y-direction. Figures 7(a) and 7(b) show
this section at 180 ps and 250 ps, timeframes, respectively. The white region
between A-BW and P-BW, in reality, contains water molecules, however these are not shown here
for clarity. This region is the layer of transition or slippage as mentioned
earlier. Figure 7(b) shows the movement of P-BW (green atoms) as the strand is
pulled. The interaction energy for each of the pairs is shown in Table 3. The
electrostatic interaction energy is dominant compared to the van der Waals
energy. The attractive nonbonded interaction energy between mineral aragonite
and A-BW is −18×104pN-Å. Thus,
the A-BW is tightly bound to the aragonite surface and does not move when the
GS domain is pulled alongside the mineral.Table 3
Values of
interaction energies between different pairs of constituents of
aragonite-water-GS domain.
Interaction pairElectrostatic energy (pN-Å)van der Waals energy (pN- Å)Aragonite—(A-BW)−17×104−1×104Aragonite—(P-BW)−8×104−0.85×104Aragonite—GS−0.60×104−0.125×104GS—(A-BW)−0.20×104−0.03×104GS—(P-BW)−1.20×104−0.10×104(P-BW)—(A-BW)(−0.50to0.50)×104−0.20×104Snapshots taken during the steered molecular dynamics simulation of the pulling of the GS domain in the presence of aragonite mineral proximity at (a) 180 picoseconds and (b) 250 picoseconds.
(a)(b)The attractive interaction energy between
aragonite and P-BW is−8.85×104pN-Å, a very significant magnitude.
The P-BW and the GS domain have an attractive interaction energy of −1.30×104pN-Å. In addition, aragonite and A-BW also have attractive interactions with
the GS domain, although smaller magnitudes of −0.725×104pN-Åand −0.230×104pN-Å, respectively. The dominant resistance to pulling of the GS is the
attractive interaction between GS domain and the P-BW, which in turn has
significant interaction with the mineral. Although the direct interaction
between the mineral and GS is small because of the distance, water plays a
critical role in building a  “bridge”
between the mineral and the protein, facilitating development of a large
resistance to pulling in the presence of mineral. As a result, the load
experienced at the pulling end is high. This therefore results in higher
magnitude of load at a given displacement in the L-D curve.


## 3.1. Section I

Peaks in the L-D curve are present
in both plots as indicated in Figure1. The peaks observed in the absence of
mineral aragonite have a height of about 150 to 200 pN, whereas the peaks in
the presence of aragonite are of larger magnitude and are in the range of 1500
to 2000 pN and have lower frequency of occurrence. The magnitude of peak
heights in the absence of aragonite corresponds to the opening of loops and
turns and breaking of hydrogen bonds. From the nature of peaks observed in two
cases, it can be inferred that different mechanisms are involved in the
formation of peaks when pulling protein at mineral proximity. From the
trajectory analysis of unfolding, it is observed that peaks occur until water
enters between the aragonite surface and GS strands. As pulling is continued
and water enters in between, no additional peaks are observed in the L-D curve.
This observation indicates that direct interaction between aragonite surface
and GS strands may result in the formation of these peaks. We have investigated
the peaks up to 50Ådisplacement. This displacement corresponds to the point
after which significant water enters in between the aragonite surface and GS
strand. From the trajectory analysis, it is
observed that when the GS strand is pulled at mineral proximity, a mechanism
similar to the physical phenomenon of “latching” is observed to take place
between aragonite surface and GS strand in proximity. The “latch” observed here
is not a physical latch but a result of strong nonbonded interactions between
an atom or a group of atoms of amino acid residues (attached to GS strand) and
aragonite surface. These latches are formed when the interacting group(s) come(s)
close to the aragonite surface, but as pulling is continued they are observed
to break. These latches thus go through a process of “forming" and “breaking"
as the GS strand is pulled along the aragonite surface, giving rise to the
peaks in the L-D curve.Further, we zoomed into a small section,
circled in Figure2(a) where, a “latch” is observed. The “forming" and
“breaking" phenomenon of a latch is represented in Figures 2(b) and 2(c).
The aragonite layer close to the surface is shown in the figures. The L-D
characteristics of one of the peaks are shown in Figure 2(d). As the strand is
pulled in the direction of the arrow in the figure, a group of atoms (marked as
blue balls) comes closer to the surface, builds a strong interaction, arrives
between the carbonate groups of aragonite, and gives rise to what we called as
a “latch." The formation of the “latch” corresponds to the base point of the
peak as marked in Figures 2(b) and 2(d). Once the latch is formed, it
offers resistance to pulling, causing the magnitude of load to rise sharply.
The load curve keeps rising as pulling is continued until the load level where
the latch can no longer sustain the pulling force results. At that point, the
latch breaks, and thus the force decreases. The apex of a peak therefore
corresponds to the point where a latch breaks (Figure 2(c)).Figure 2
The mechanism of “latching” and formation of peaks: (a) latching site (b) formation of latch (c) breaking of latch (d) peaks resulting from latching mechanism.Aragonite exhibits orthorhombic crystal
symmetry. The surface formed by (001) is rich in negatively charged oxygen
atoms belonging to carbonate group. The positively charged calcium atoms are
located very close to the surface. The charges on the surface oxygen and
calcium atoms are found to be −0.9995 and +2.000, respectively. The blue atom
group constitutes mainly of hydrogen and/or oxygen atoms. The hydrogen and
oxygen atoms of amino acid strongly interact with oxygen and calcium of aragonite,
respectively, forming these latches. As polar water molecules enter between the
aragonite surface and GS strand, these interacting groups can no longer come
close enough to form the latches and thus no sharp peaks are observed. In
Figure1, no peaks are observed beyond a displacement of about 60 Å. The peaks
within the same L-D curve (at mineral proximity) are different in terms of
height, base width, shoulder characteristics, etc. This heterogeneity in peak
characteristics depends on the number of latches formed at a given time, the
atoms involved (blue group) in the “latching” action and on the presence of
other interacting groups closer to the aragonite surface.Thus, a peak in the L-D curve starts rising
when a side chain group builds up a strong nonbonded interaction with aragonite
surface. It falls when this interaction is broken upon further pulling. Thus,
the energy stored in the rising half of the peak is due to the resistance
offered by the latch and is thus expected to be approximately equal to the
nonbonded energy holding these two interacting groups together. The nonbonded
energy which is the summation of van der Waals and electrostatic energy between
the interacting group of amino acid side chains, and the aragonite section
(shown in Figure2(b)) is determined. The variation of the electrostatic and
van der Waals energy with time (frame) is shown in Figures 3(a) and 3(b). In
Figure 2(d), it is observed that Peak 0 starts rising at time frame 85 (17 ps).
The electrostatic and van der Waals energy corresponding to this time frame as
observed in Figures 3(a) and 3(b) is 2000 pN-Åand 700 pN-Å, respectively. The
area bound by the rising half of the Peak 0 (X, Y and Z in Figure 2(d)) is 2500 pN-Å, which is close to the total nonbonded interaction (2700 pN-Å) energy
between the interacting groups. From a similar analysis of Peak 1 (Figure 4(a)),
where the peak starts at time frame of 135 (27 ps), the total attraction energy
(900 pN-Å) is found to be very close to the area bound by the rising part of the
curve (1100 pN-Å). The variation of the electrostatic and van der Waals energy
with time (frame) for Peak 1 is shown in Figures 4(b) and 4(c).For other major peaks, these values are also
in close agreement. Thus it is quantitatively shown that the large peaks
observed in the proximity of the mineral are a result of direct nonbonded
interactions between the side chain atoms and the complementary atoms on the
mineral surface.The nonbonded energy between the interacting groups giving rise to Peak 0: (a) electrostatic energy (b) van der Waals energy.
(a)(b)Peak 1 (a) load-displacement plot (b) electrostatic (c) van der Waals energy.
(a)(b)(c)


## 3.2. Section II

As observed in Figure1 and also
mentioned earlier, one of the factors which contribute to the work factor is
the higher magnitude of load at any given displacement, when pulled at mineral
proximity. The peak load observed in the figure is about 13 nN, a high
magnitude, predominantly as a result of mineral protein interactions. This high
magnitude of force resulting from constant velocity pulling simulations is
possible without fracture of the single protein strand being pulled. Flow
induced fracture of single polymer chains is reported to be between 2.5 to 13.4 nN [33, 34] and the fracture strength is dependent upon the polymer and rate of
loading. In this section, the potential mechanisms
involved are described.  As the GS domain
is pulled, the water molecules present closer to the aragonite surface orient
and arrange themselves in a definite pattern and further undergo very little
movement. On moving away from aragonite surface, the water molecules are
structurally altered, and behave like “normal solvent water" attached to the
protein.A clear difference in behavior is observed
between water molecules closer to the surface and away from the surface both in
terms of structure and movement during pulling. We have therefore categorized
the water into two types. The water molecules close to the aragonite surface are
named here as Aragonite-Bound Water (A-BW), and water molecules moving with the
protein are named
as Protein-Bound Water (P-BW). The schematic representation of these two types
of water molecules is shown in Figure5.
At low displacement of pulling, very few water molecules are observed to
be present between aragonite and GS strands. However, this number increases as
pulling is continued. The strong hydrogen-bonded interaction between carbonate surface of
aragonite and polar water molecules causes some of the water molecules to lie
close to the aragonite surface. The water molecules form a periodic pattern
which is primarily due to the periodicity of the aragonite surface in the X-Y
plane. The P-BW shows no definite pattern, with the water molecules surrounding
the protein and moving freely with the protein. As the GS is pulled, the A-BW
remains tightly bound to the aragonite surface and exhibits almost no movement.
The P-BW on the other hand moves freely with the GS strand. On careful
observation, it is observed that there is a 3-4Åthick layer of water (about
two water molecules thick) which behaves as a transition layer between A-BW and
P-BW. In this layer, the slipping of
P-BW is observed to happen with respect to the A-BW. The P-BW forms a sheath
around the GS strands. When GS is pulled, the strands move with sheath of water
around it. P-BW thus moves with GS strands and A-BW remains bound to the
surface. A schematic representation of this mechanism is shown in Figure 6. This
figure illustrates that when GS is pulled, the interaction contributing to
resistance to displacement includes the following:Two “types” of water molecules observed during pulling of GS domain (a) simulation (b) schematic representation. A-BW: aragonite bound water and P-BW: protein bound water.
(a)(b)Figure 6
Schematic representation of movement of the GS domain and protein-bound-water (P-BW) when GS domain is pulled.(i)
the net attractive interactions
of GS-(A-BW) and GS-aragonite;(ii)
the net attractive interaction
of (P-BW)-(A-BW) and (P-BW)-aragonite and GS-(P-BW).To study the
contribution of these different interactions to the L-D characteristics of GS
domain, aragonite sections of smallerX-Y dimension and part of GS strand close
to it are selected. We have used two such sections for the analysis of the
interaction energy. One of them is shown in Figure 7. This section extends to
20Åin the X-direction and 50Åin the Y-direction. Figures 7(a) and 7(b) show
this section at 180 ps and 250 ps, timeframes, respectively. The white region
between A-BW and P-BW, in reality, contains water molecules, however these are not shown here
for clarity. This region is the layer of transition or slippage as mentioned
earlier. Figure 7(b) shows the movement of P-BW (green atoms) as the strand is
pulled. The interaction energy for each of the pairs is shown in Table 3. The
electrostatic interaction energy is dominant compared to the van der Waals
energy. The attractive nonbonded interaction energy between mineral aragonite
and A-BW is −18×104pN-Å. Thus,
the A-BW is tightly bound to the aragonite surface and does not move when the
GS domain is pulled alongside the mineral.Table 3
Values of
interaction energies between different pairs of constituents of
aragonite-water-GS domain.
Interaction pairElectrostatic energy (pN-Å)van der Waals energy (pN- Å)Aragonite—(A-BW)−17×104−1×104Aragonite—(P-BW)−8×104−0.85×104Aragonite—GS−0.60×104−0.125×104GS—(A-BW)−0.20×104−0.03×104GS—(P-BW)−1.20×104−0.10×104(P-BW)—(A-BW)(−0.50to0.50)×104−0.20×104Snapshots taken during the steered molecular dynamics simulation of the pulling of the GS domain in the presence of aragonite mineral proximity at (a) 180 picoseconds and (b) 250 picoseconds.
(a)(b)The attractive interaction energy between
aragonite and P-BW is−8.85×104pN-Å, a very significant magnitude.
The P-BW and the GS domain have an attractive interaction energy of −1.30×104pN-Å. In addition, aragonite and A-BW also have attractive interactions with
the GS domain, although smaller magnitudes of −0.725×104pN-Åand −0.230×104pN-Å, respectively. The dominant resistance to pulling of the GS is the
attractive interaction between GS domain and the P-BW, which in turn has
significant interaction with the mineral. Although the direct interaction
between the mineral and GS is small because of the distance, water plays a
critical role in building a  “bridge”
between the mineral and the protein, facilitating development of a large
resistance to pulling in the presence of mineral. As a result, the load
experienced at the pulling end is high. This therefore results in higher
magnitude of load at a given displacement in the L-D curve.


## 4. Conclusions

In our previous work, we have shown
that the proximity of mineral influences the mechanical response of protein.
More energy is required to unfold a protein in presence of aragonite than
without it. The L-D curve in the presence of aragonite shows different
responses from the one when pulled without mineral proximity. In the presence
of aragonite, larger peaks are observed, and the magnitude of load at any given
displacement is significantly higher. In this work, we have found quantitatively
the mechanisms leading to the difference in load displacement response of
protein at mineral proximity. The following hold.(i)
At the early stage of pulling, the peaks
in the L-D plot at mineral proximity are quantitatively correlated to the
interaction energy between the atoms involved in the latching phenomenon of
amino acid side chain to aragonite surface.(ii)
The role of water in mineral and protein
interaction is very significant.(iii)
The water closer to the mineral surface
is highly oriented and does not move while the protein strand is pulled. Water
layer around the strands moves with the strand as the protein is pulled.(iv)
The high magnitude of load for a given
displacement originates from attractive interactions between the protein,
protein-bound water, and the mineral. Here, for the first time,
quantitative description of the mechanics responsible for the large differences
in the magnitude of force needed to unfold proteins in the proximity of the
mineral in nacre is provided. This work provides clues as to possible reasons
for extraordinary properties of organic phase such as high elastic modulus and large deformation
before failure observed in nacre.



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*Source: 582973-2008-09-14.xml*