UNIVERSITEIT VAN AMSTERDAM

A Coarse-Grained Model for Self-Assembling collagen-silk-like

block-copolymer
Bruno Barbosa Rodrigues
Advisor: Peter G. Bolhuis
Co-Advisor: Marieke Schor
July 2009
Abstract
We present the results of the sequence design of the adapted off-lattice minimalist model based on
the original Head-Gordon (HG) for a monodisperse, biodegradable, biocompatible and hydrophilic
collagen-like sequence. This sequence is part of a block copolymer made of two hydrophilic collagen-
like blocks anking a hydrophobic silk-like block. We start with atomistic simulations of short pep-
tides to extract the bond distances, bend and dihedral angles distributions to t the coarse-grained
(CG) force eld (FF) and adapt the HG model for this collagen-like sequence. Due to the high con-
centration of proline and charged groups at low pH in the sequence, different distributions were found
for the dihedral angles according to the relative position of proline in the chain. Thus, we extended
the original three-letters minimalist model combined with a previous adapted shifted and rescaled
non-bonded potential developed for the silk-block sequence. We compared the results of the radius
of gyration (R
g
) for different number of residues with the experimental value measured for a 400
residues long sequence, coming up with a scaling law between the R
g
and the number of residues in
the collagen-like sequence. We concluded that the exponent is close to the Flory exponent.
Keywords: Collagen-silk-like block copolymers, protein bers, Molecular Dynamics, Coarse-
Grained model.
Contents
1 Introduction p. 1
2 Methods p. 4
3 Results p. 13
4 Conclusions and Future Perspectives p. 19
Acknowledgements p. 20
Appendix A -- Dihedral Angles p. 21
Bibliography p. 23
1
1 Introduction
Protein based block copolymers that form bers upon a certain stimulus (e.g. change in pH)[16]
are potentially very useful as biocompatible nanomaterials. One example is a block copolymer made
of two hydrophilic collagen-like blocks anking a hydrophobic silk-like block. The collagen block
is responsible for limiting the growth direction of the silk block and drives it to form bers with
improved transversal strength. The sequence of the collagen block is chosen in a way that, unlike
the most common gelatin in nature that forms a gel in the presence of water, it remains soluble and
unstructured under various conditions of pH and temperature. Previously, a coarse grained model
of the very regular, highly structured silk-like block has been developed [14]. However, a different
strategy is needed to develop such a model for the unstructured collagen-like block.
In the rst section of this chapter we give an overview on the biocompatible materials and their
applications; the second section is dedicated to the protein polymers; the two last sections are ded-
icated to introduce the computer simulation techniques as tools to understand and predict protein
dynamics, the limitations and possible solutions, ending up with an overview of this work in the last
section.
1.1 Biocompatible Materials
Nature produces a wide range of different materials, but all of them for its own purposes, like
actin, which is the main constituent of cytoskeleton, or collagen, the major component of extracel-
lular matrix. Genes can encode identical amino acid sequences (also called primary structures) with
absolute control over molecular weight, composition, sequence, and stereochemistry. Control of this
protein production can lead us to build materials that could t human desires[4] such as chemo-
mechanical bers[5], tissue engineering [6, 7] and drug delivery [8, 9].
Biocompatible materials are synthetic or natural materials intended to interface with biological
systems in intimate contact with living tissue. One way to build such high value materials is via ge-
netic and protein engineering, both components of a new polymer chemistry that provide the tools for
producing macromolecular polyamide copolymers of diversity and precision far beyond the current
capabilities of synthetic polymer chemistry.
2
1.2 Protein Polymers
Control and understanding of the behavior of biocompatible materials requires one to look deep
into the atomic compostion of those materials. The function of a protein is intrinsically connected to
its spatial conformation (or tertiary structure) and the process that drives the protein towards this state
is called protein folding.
Proteins can be designed as blocks of repetitive amino acid sequences, also called block copoly-
mers. This term was introduced by Capello in 1990s [2] who built diblock copolymers containing
silk-like and elastin-like amino acid sequences. From that time on, many other block copolymers
have been produced [3], coming from diblock to multiblock sequences. The self-assembly charac-
teristic of the proteins can be used to tune organization in the molecular level [10], wich can build
organized nanoscopic objects that end up in macroscopic materials with interesting properties.
Figure 1.1: Triblock copolymers consisting of a hydrophobic pH-responsive, silk-like block anked
by hydrophilic, non-responsive collagen blocks self-assembling into m long brils. The collagen
block limits the assembly direction of the silk block [11].
The system under study on this dissertation is a collagen-like polypeptide [12] that anks a silk-
like sequence [13] which self-assembles into a roll forming stacks under low pH [14] (g. 1.1).
The triblock was produced experimentally by gene encoding of the yeast Pichia pastoris [15, 16]. The
collagen-like part [12] has a hydrophilic sequence that, although the normal collagens that form gels,
it remains soluble under many conditions of pH and temperature. With this random characteristic, the
collagen-like part can drives the silk-block towards the growth direction forming biocompatible bers
that can have improved their transversal strength upon a stimulus (e.g. a change in pH).
1.3 Computer Simulations
During the last two decades, computer simulations have gradually been recognized as giving
complementary information about the properties of such new materials or to understand conforma-
tional transitions in proteins [17, 18]. While most of the relevant dynamics in proteins, like folding
processes, occur on time scale of miliseconds and involve large molecular aggregates, atomistic simu-
3
lations can only address hundreds of nanoseconds in simulations for small proteins in explicit solvent,
which means at least four and one orders of magnitude lower in time and in degrees of freedom, re-
spectively.
In this framework, many techinques were developed to overcome the system size and simulation
time. Simplication of the systemunder study by integrating out details that are not actually important
to the overall result can lead us to address longer length and time scales. In this work we focused on
adapting the Head-Gordon (HG) [25, 26] model to apply for the collagen-like part of the collagen-
silk-collagen block-copolymer. With this improvement, it is possible to simulate big systems long
enough to predict the properties of the collagen-like block, and understand its role on driving the
growth direction of the silk-ber.
1.4 Outline of this Dissertation
The HG, developed to study protein oding and aggregation, was adapted for the collagen-like
sequence in such a way that the dihedral angles are treated either with a cosine and a harmonic
expansion. The resulting t of the parameters can be combined with previous results for the silk-
like block copolymer in order to enable a complete description of the collagen-silk-collagen block-
copolymer.
In Chapter 2 we describe the methods in molecular dynamics applied for the system under study,
as well as details concerned to the simulation procedure. We also introduce the adapted Head-Gordon
model to coarse-grain the polypetide lumping together an entire amino acid in one bead. In Chapter
3 we investigate the results of the atomistic as well as the coarse-grained simulations, present the
output distributions that were used to t the adapted HG model and show the scaling law between
the radius of gyration and the number of residues, appearing to be in very good agreement with the
experimental value. Thus, in Chapter 4 we conclude the work and give the future perspectives.
4
2 Methods
In this chapter we discuss the atomistic and CG Molecular Dynamics (MD) simulation te-
chiniques which were used to study the collagen-silk block-copolymers. We performed the atom-
istic simulations using GROMACS version 4.0 [30], which provided the distributions for the bond
distances, bend and dihedral angles between the three and four consecutive C atoms respectively.
With these parameters, we tted the adapted HG model that permitted the achievement of longer time
and length scales. The CG simulations were performed with the CM
3
D code[31].
2.1 Molecular Dynamics
The key of MD simulations is to integrate Newtons Law of motion for the N interacting particles
m
d
2
r
i
dt
2
=

j=i
F
_
r
i j
_
(2.1)
with accuracy and in such a way that the pairwise additive interactions do not scale as N
2
. Speed
up of the evaluation of both short-range and long-range forces is possible and for that we have some
techniques. Following the standard procedure to calculate the force by the derivative of a potential,
we can integrate Newtons equation of motion and many algorithms are available. A very simple
is the leap-frog integrator [32, 33] which is a Verlet-like second-order algorithm that evaluates the
velocities at half-integer time steps and uses these velocities to compute the new positions:
r(t +t) = r(t) +tv(t +t/2) (2.2)
v(t +t/2) = v(t t/2) +t
f (t)
m
(2.3)
The more sosticated Gear predictor-corrector algorithm falls into the general nite difference
pattern, where the estimate of the positions, velocities etc. at time t +t may be obtained by Taylor
expansion about time t. These values are estimated and do not represent the true trajectory. After
calculating the forces at the new position r
p
(t +t), the trajectories are corrected and the predicted
step is fed with the new information to iterate the corrected trajectory and r
c
(t +t) is now a better
approximation to the true position.
5
2.2 Atomistic Models
As described in the previous section, the key point of MD simulations is to solve Newtons equa-
tions of motion. But usually, the systems are dened by the potential energy rather than the forces,
which can then be easily calculated by the negative gradient of the potential: F(r
i j
) = V(r
i j
).
For that, many potential energy functions were developed to simulate protein systems, the so-called
force-elds (FF) [35]. The basic idea of a FF relies on mapping all the possible physical interactions
in the system and put them into a potential, like presented in eqs. 2.4 to 2.9:
V =V
noncov
+V
cov
= (V
LJ
+V
C
) +(V
bond
+V
bend
+V
dih
) (2.4)
V
LJ
(r
i j
) = 4
i j
_
C
(12)
i j
_

i j
r
i j
_
12
C
(6)
i j
_

i j
r
i j
_
6
_
(2.5)
V
C
(r
i j
) =
1
4
0
q
i
q
j

r
r
i j
(2.6)
V
bond
(r
i j
) =
1
2
k
(bond)
i j
_
r
i j
b
i j
_
2
(2.7)
V
bend
(
i jk
) =
1
2
k
bend
i jk
_

i jk

0
i jk
_
2
(2.8)
V
dih
(
i jkl
) =
1
2
[C
1
(1+cos()) +C
2
(1cos(2))
+ C
3
(1+cos(3)) +C
4
(1cos(4))] (2.9)
where the potential is divided in bonded (or covalent) and non-bonded (non-covalent). The non-
bonded interactions contain a repulsion term, a dispersion term, and a Coulomb term. The repulsion
and dispersion terms are combined in the Lennard-Jones (or 6-12 interaction). In addition, (partially)
charged atoms act through the Coulomb term. Bonded interactions are based on a xed list of atoms.
They are not exclusively pair interactions, but include 3- and 4-body interactions as well. There are
bond stretching (2-body), bond angle (3-body), and dihedral angle (4-body) interactions given by eqs.
2.7, 2.8 and 2.9, respectively.
There are many FF codes available nowadays, the most common are: AMBER [36], CHARMM
[37], GROMOS [38] and OPLS-AA [39]. Their potential energy is parametrized against experiments
and ab initio quantum mechanical calculations.
2.2.1 Setup atomistic simulations
The Atomistic simulations can reveal several details of the system, as it treats both the protein
under consideration and the water. However, it is very difcult to reach long time and length scales
within this framework. In this case, the atomistic simulations were carried out only to extract the pa-
6
rameters to t the Coarse-Grained model. A 30 residues collagen-like sequence was also constructed
for comparison with the CG model, but a simulation of the whole collagen-like sequence would not be
affordable under the computational resources available nowadays. To extract the required properties
of the degrees of freedom of the amino acids sequence , we divided the collagen-like protein in short
peptides and simulated them separetely to achieve a good sampling of the phase space.
The rst part of the work is concerned on dividing the collagen-like sequence in small blocks of 8
to 12 amino acids (see table 2.1). Checking the periodicity of the sequence and looking for blocks that
repeat very often, we found out how to minimize the number of short sequences and, consequently,
the computational effort and the amount of data to be analyzed. We ended up with 12 unit blocks
that together are able to reconstruct the whole sequence. All the sequences were constructed using
MOLMOL [40] software. The 12 blocks are labeled in such a way that it is possible to reconstruct
the original sequence (g. 2.1) by just sticking together all the short peptides in a repetitive sequence
as follows: A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A2 A3 A4 A5 A6 A7 A11 A12.
Figure 2.1: The whole collagen sequence dened by the 20-letters amino acid code. Different collors
relate to each one of the 12 short peptides.
The peptides (including the 30 residues one) were solvated with water molecules in a rhombic
dodecahedral periodic box. Details concerned to the number of atoms in the peptides as well as water
molecules and box sizes can be found in table 2.1. It has been observed that the presence of lysine
(K) and glutamine (Q) induces a positive net charge as these amino acids have a cationic form at low
pH [41]. To neutralize the system some water molecules were randomly selected and replaced by a
Cl

counterion depending on the abundance of lysine and glutamine in the peptide.

All MD simulations were performed using GROMACS package version 4.0 [30] with OPLS-AA
force eld [39] for peptides and TIP3P [42] model for water molecules. The bond vibrations, which
represent the highest frequency motion in a molecule, were constrained by the Linear Constraint
Solver (LINCS) [43] algorithm allowing us to choose a time step of 2fs with a minimum effect on
the overall behavior of the system. Ten step intervals were used for neighbor pair list updates. Long-
range electrostatic interactions were treated with Particle-Mesh Ewald summation [44, 45] with a grid
spacing of 1.2

A and minimum image convention was used for the short-range non-bonded interacting
terms.
Energy was minimized with the Conjugate Gradient algorithm [46] that uses the gradient in-
formation from previous steps to bring the systems very close to the local minimum. After energy
7
minimization, a short protein position restrained run was performed to soak the solvent in between
the peptide. The sample was then equilibrated for 1ns at a constant pressure of 1 bar with Parrinello-
Rahman [47, 48] coupling barostat with a coupling constant of 1ps and at temperature of 298K using
Nos e-Hoover thermostat [49, 50] with a coupling constant of 0.2ps. After this straightfoward proce-
dure [52], all simulations were performed in a NVT ensemble at room pressure and 298K temperature
with a box size depending on the short peptide and listed in the table 2.1.
Label Sequence Volume(nm
3
) Number of Atoms
Protein Solvent Ions Cl

A1 GAGAPGEP 27.82 71 889 0

A2 GNPGSPGNQGQP 95.61 146 3085 0
A3 GNKGSPGNPGQP 93.44 151 3,011 1
A4 GNEGQPGQPGQN 93.33 154 3,009 0
A5 GQPGEPGSNGPQ 91.62 148 2,930 0
A6 GSQGNPGKNGQP 111.30 154 3,583 1
A7 GSPGSQGSPGNQ 105.80 140 3,468 0
A8 GSPGQPGNPGQP 87.81 146 2,835 0
A9 GEQGKPGNQ 64.11 124 2,065 1
A10 GPAGEP 30.05 71 931 0
A11 GSPGQPGEKGKP 94.39 161 3,072 2
A12 GNQGPAGEGA 31.34 72 993 0
A30 GNEGQPGQPG
QNGQPGEPGS 385.69 375 12,503 6
NGPQGSQGNP
Table 2.1: Short peptides sequences simulated atomistically. Detais of the box size, number of protein
atoms, solvent and added charged atoms are presented.
The simulations were carried out on the Lisa cluster located at Sara Computing & Network Ser-
vices in parallel with up to 5 processors 2 Intel Xeon 3.4GHz Extended Memory 64 Technology, 800
MHz Front Side Bus, 4GByte of memory and Bandwidth of 800 MB/s. We performed 10ns MD
simulations for the peptides A1 - A12 which took around one day on one processor with 2 cores.
The 30 residues sequences were simulated in parallel with domain decomposition in 5 processors (10
cores), showing a performance of 6.259ns per day; MPI (Message Passing Interface) library was used
to compile GROMACS 4.0 in parallel. From the 10ns simulations we extracted the parameters of the
bonded interactios to guide us on modelling the CG eld for the system.
2.3 Coarse-Grained Models
The CG method can face the problem of big systems, as it reduces the number of interactions
by integrating out several degrees of freedom and treating the solvent implicitly. The decision of
which kind of details can be approximated and how to t the new force-elds can turn into a very
complicated problem. Since the 1970s people think about CG models for proteins [20], and from
8
that time on many approaches emerged. The CG models evoluted towards different directions, that
differ basically in the relation between the complexity of the representation versus the complexity of
the parametrization. Harmonic models represent the system by beads (usually one per amino acid)
connected by elastic springs [21], and are used basically for the analysis of the principal modes [22],
requiring a previous knowledge of an equilibrium reference conguration. Go-like models [23] also
require an a priori knowledge of the native state and lack on representing the most intriguing fact of
protein folding: the dependence on the primary sequence. A lower level of reference dependence can
be found in the Head-Gordon model [25], which represents each amino acid as one bead. Two-bead
models [27] were developed adding a second bead on the centroid of the sidechain, increasing the
independence with a reference conguration but increasing the complexity of the energy terms and
inserting correlations on dihedral angles. Four-six bead models [28, 29] represent the sidechain as
one bead but explicitly consider the coordinates of the three heavy atoms of the backbone.
We chose the Head-Gordon (HG) [25] model to coarse-grain the collagen-like block, as it was
successfully applied to the silk-like sequence before [14]. In this model, in constrast with Go [23]
model, we do not need to know anything about the tertiary structure of the native state. However, we
need to face the more difcult aspect of the protein folding problem, namely its dependence on amino
acid sequence. The C atoms trace are taken to represent the protein backbone and the structural
details of amino acids and aqueous solvent are integrated out and replaced by effective bead-bead
interactions.
2.3.1 Head-Gordon Model
The original Head-Gordon (HG) model is an improvement of previous efforts of Thirumalai and
coworkers [53] that is more general to helical, sheet and / protein topologies. The 20-
letter amino acid sequence is converted to a three-letter code dened by the avors: hydrophilic (L),
hydrophobic (B) and neutral (N). The idea of describing an amino acid as one bead can be visualized
in the gure below:
Figure 2.2: Schematic description of an amino acid as a bead in the CG model.
9
The force eld in the original HG model is dened as:
H =

1
2
k

(
0
)
2
+

_
A(1+cos) +B(1cos) +C(1+cos3) +D
_
1+cos
_
+

4
___
(2.10)
+

i, ji+3
4
H
S
1
_
_

r
i j
_
12
S
2
_

r
i j
_
6
_
where we have the bond angle between three consecutive beads with
0
=105

being the equilib-

rium angle and k

=20
H
/rad
2
. The dihedral angle between four consecutive beads can assume
different conformations depending on the region to be described, with the constants A, B, C and D
dening the shape of the distribution. The LJ potential determines the attraction-repulsion between
the beads of size with the three avors i and i separated by r
i j
: B-B interactions are attractive and
represented by S
1
= S
2
=1; S
1
=1/3 and S
2
=0 apply for L-L and L-B interactions; and N-L, N-B
and N-N interactions have the constants S
1
=1 and S
2
=0. In the original HG model the bond lengths
are constrained by the RATTLE algorithm [54]. The non-bonded potentials are plotted in the Figure
below:
Figure 2.3: Non-bonded potential between neutral, hydrophilic, hydrophobic and proline (treated as
neutral) amino acids.
2.4 Adapted HG model
Based on the original Head-Gordon model and the adapted version for the silk part [14] of
the block-copolymer, we developed a four-letters minimalist model for the collagen-like block: hy-
drophilic (L), hydrophobic (B), neutral (N) and proline (P), where we dened proline as a separated
10
Figure 2.4: Full collagen sequence transcription from the 20-letters amino acid code to the adapted
four-letters minimalist HG code based on table 2.2.
avour, due to its key role on the stiffness of the dihedral angles. In the table 2.2 below we show the
sequence mapping between 20-letter amino acid and adapted CG four letter code, which generates a
minimalist full sequence according to the g. 2.4.
Name 20 4 Name 20 4
Glycine GLY / G N Aspargine ASN / N L
Alanine ALA / A B Proline PRO / P P
Glutamic Acid GLU / E L Glutamine GLN / Q L
Lysine LYS / K L Serine SER / S N
Table 2.2: Sequence mapping between 20-letter amino acid and adapted CG four letter code.
The FF also needs to be modied to cover the new changes in the dihedral angles, bond distances
(which are no longer constrained) and non-local interactions between beads. The new FF is given by
the equations 2.11, 2.12 and 2.13 below:
H
adap
=

b
1
2
k
b
(bb
0
)
2
+

1
2
k

(
0
)
2
+

_
V
weak
() +V
sti f f
()

(2.11)
+

i, ji+3
4
H
S
1
_
_

r
i j

0
_
12
S
2
_

r
i j

0
_
6
_
V
weak
() =
h
6

k=0
_
A
k
cos
k
()
_
(2.12)
V
sti f f
() = B
0
1
2

h
B
1
(
0
)
2
(2.13)
where now the bond distances are explicitly described by the spring potential, as CM
3
D package used
for the CG simulations employs a reversible multiple time-step integrator, the stiffness is given by
k
b
= 33
h
and b
0
= 3.84

A consistent with the measured all-atomC

distance distributions. The

bond angles have the same treatment as in the original HG model, with the parameters given by k

=
20
h
and
0
= 105

extracted from the atomistic simulations. The parameters A

k
, B
0
, B
1
and
0
set
the dihedral angles between four subsequent C- atoms that show either periodic behaviour with two
minima (weak) or harmonic with one minima (stiff) depending on the four beads sequence, as the
11
Figure 2.5: Shifting the Lennard-Jones potential has the effect of shortening the range of the potential.
The position of the nearest neighbors and the second nearest neighbors between strands in the silk part
are given by the dashed vertical lines. In the traditional L-J potential the second nearest neighbours
still feel the interaction.
new avor (proline) plays an important role on the stiffness of the dihedral angle. The L-J potential
is shifted and scaled in order to coincide with the previous model optimized for the slik-part [14],
where the scaled parameter sets the range of the interaction and
0
shifts the potential to match
the size of the bead, as can be seen in the g. 2.5. The strength of the non-bonded interaction k
LJ
=
4
h
was previously optimized for the silk part by M. Schor [14] by comparing the potentials of mean
force (PMF) from steered MD (SMD) simulations of the atomistic and the coarse-grained model. The
PMFs from SMD were calculated following the method of Park and Schulten [63, 64].
Simulations with CM
3
D were carried out for different sequence sizes. We started with the 30
residues structure built in MolMol and kept the positions of the C atoms. From this basic structure
we built sequences of 45, 60, 75, 90, 120, 150 and 200 beads by sticking together the pieces of short
peptides. We used VMD to manipulate the PDB les of the different sizes of collagen-like sequence.
Then the sequences were put in a cubic box (as the CG simulation employs implicit solvent, the shape
of the box is not relevant)with periodic boundary conditions in all x, y and z directions. The time step
used is 2fs.
We started relaxing the protein at low temperature. The rst simulation was carried out with
velocities given by a Boltzmann distribution, at 30K for 1ns in a NVT ensemble with Nos e-Hoover
chain with 4 units and time step of 1.6ps. Then we performed further 1ns simulations based on
previous velocities and positions and raising gradually the temperature up to 60K, 100K, 200K and,
nally, 300K. Then we equilibrated the system for 1ns in 300K and started a 50ns simulation to
sample the averages. The 1ns simulations were carried out in a local machine and took no more than
30 minutes at most for the 200 residues sequence. For the 50ns long we simulated on LISA cluster
12
with 1 processor 2 Intel Xeon 3.4GHz and took at most 20 hours for the longest sequence (200 beads).
2.5 Order Parameters
The conguration space is very high-dimensional and a visualization of its direct quantities is
meaningless. To overcome this problem, we need to project the phase space in one-dimensional
representations. They can monitor the (un)folding transitions, characterize native and unfolded states
and some of them can directly be compared to experimental measurements.
Since a protein chain is not a regular object and because it is subject to dynamic structural equi-
librium that involves motion, it is necessary to consider a statistical measure of a chain size. Then the
end-to-end distance is a key description for the statistical behavior of the chain.
A commonly used order parameter is the Root Mean Square Deviation (RMSD) from a refer-
ence structure, usually obtained experimentally by X-ray or NMR. RMSD was calculated minimizing
under rotations and translations and is dened as:
RMSD =
_
1
M
N

i=1
m
i
|r
i
r
re f
i
|
2
_1
2
(2.14)
A second order moment about the mean chain position is the radius of gyration. It describes the
overall spread of the molecule and it is dened as the root mean square distance of the collection of
atoms from their common centre of gravity:
R
g
=
_
1
M
N

i=1
m
i
|r
i
r
cm
|
2
_1
2
(2.15)
where r
cm
denotes the position of center of mass of the protein. This measure gives a valuable way to
compare our CG method with the experimental data available for the collagen-like system.
Root Mean Square Fluctuation (RMSF) is a measure of the deviation between the position of
particle i and some reference position.
RMSF =
1
T
T

t
j
=1
_
r
i
(t
j
) r
i
_
2
(2.16)
where T is the time over which one wants to average, and r
i
is the reference position of particle i.
Typically this reference position will be the time-averaged position of the same particle i, ie. r
i
. Note
that, instead of averaging over the particles (as in RMSD), RMSF averages over the simulation time,
giving a value for each particle i, usually the C atoms.
13
3 Results
The results of the MD simulations described in the chapter 2 are presented here. We analyse the
bond distances, bend and dihedral angles and order parameters obtained from the Atomistic simu-
lations and use them to t the CG adapted model for the collagen-like block copolymer. Thus, we
present the improved CG model and compare the results with the previous atomistic simulations.
Lastly, we summarize the results obtained from the adapted model analysing the order parameters.
3.1 Atomistic Simulations
Atomistic simulations of the short peptides provided enough information about bonds, bends and
dihedral distributions, while the 30 residues collagen-like simulation revealed several details about
the dynamics of the protein. Bond distance distributions (see g. 3.1 (left)), strongly peaked around
3.84 0.12

A representing the distance between C- atoms of subsequent amino acids, justify the
use of a stiff harmonic potential for the bonds. Also based on the distributions calculated from the
atomistic simulations, the rather narrow exibility of the bend angles (see g. 3.1 (right)) justies the
same treatment as in the original HG model. The dihedral angles between four subsequent C- atoms
show periodic behaviour with two minima (exible) or harmonic with one minima (stiff) leading to
an expansion of the model in such a way that these details can be taken into account.
Figure 3.1: Bond distances (left) and bend angles (right) distributions obtained from an all-atom
simulation of the short peptide A1.
14
Analyzing the dihedral distributions shown in the g. 3.2, we can see three examples of rather
different potentials that were tted either with cosine expansion or harmonic potentials depending on
the position of proline in the dihedral angle.
Figure 3.2: Negative Logarithm of the dihedrals distibutions of the sequences LNPL (left), PNLP
(center) and LLNL (right) obtained froman all-atomsimulation of the short peptides in the minimalist
four-letters description. We can easily see how the distribution changes according to the relative
position of the Proline amino acid in the sequence.
After obtaining all the required parameters for the CG model, we present the results for
the 30 residues peptide which was simulated for 60ns to have a reference system and com-
pare with the new minimalist model. This sequence was simulated under the same procedure
adopted for the small pieces of the collagen-like sequence. We chose an intermediate sequence
|GNEGQPGQPGQNGQPGEPGSNGPQGSQGNP|to sample as many different amino acids as possible and cal-
culated the order parameters (see g. 3.3) for the RMSD, R
g
and RMSF.
Figure 3.3: RMSD (left), R
g
(center) and RMSF (right) calculated for a 60ns simulation of the 30
residues sequence. It can be observed that RMSD reaches a plateau after 45ns and also R
g
does not
change its value, but instead remains at during the whole simulation. The xaxis in the RMSF
graphic represents the C atoms and it can be seen that none of the atoms is more likely to nd a
more stable position related to the others.
15
3.2 Development of the adapted CG model
We tted the dihedrals potentials according to the distributions generated by the A1-A12 short
peptides 10ns simulations. An effetctive potential can be extracted by taking the negative logarithm
of the average distribution V() = lnP(). We found in the atomistic simulations distributions
rather different from the adapted HG model for the silk part, but which could all be tted either
in a cosine expansion (weak) or with a parabolic function (stiff): where the parameters A
k
, B and
C assume a wide range of values due to the specif characteristic of each dihedral distribution to
reproduce the measured dihedral angles of the short peptide, and at the same time not be inconsistent
with the average dihedral angles in the collagen-like sequence. We summarize in the table 3.1 the
characteristics of all possible dihedral angles between four beads in the collagen-like sequence, with
their correspondent tting function.
We consider proline as a neutral for the purposes of non-bonded interactions. Alanine is treated as
hydrophobic (S
1
=S
2
= 1) with a attractive potential. Glycine and serine are treated as neutral (repul-
sive) beads and interact with the hydrophobic and hydrophilic beads under the same potential (S
1
=
1; S
2
= 0). The lysine, aspargine, glutamine and glutamic acid residues are treated as hydrophilic
(repulsive) (S
1
= 1/3; S
2
=1) and also when they interact with hydrophobic beads.
To summarize, based on all-atom simulations of a short peptide in solution we have optimized
the CG model to reproduce the structural properties of the collagen-like sequence. Our model is
given by eqs. 2.11, 2.12 and 2.13. In the gures below we compare the atomistic simulation and the
tting functions with a 30 residues CG simulation of a piece of the collagen-like for the dihedrals
distributions. We can see that the harmonic function for the LNPL (g. 3.4 (left)) sequence has a
good agreement with the atomistic model, as expected, due to the strong connement of the potential.
Indeed the weak potential represented by the PNLP 3.4 (g. (center)) with the proline on the anks
also has a nice behaviour compared to the potential. The cosine tting of the LLNL 3.4 (g. (right))
sequence seems to have some steric hindrance, or maybe the phase space was not sampled enough,
but this tting did not interfer on the nal behavior of the collagen-like, as will be shown in the nal
results. However, as will be shown later, the results for the radius of gyration are already in a good
agreement with the theory, showing that even a rough tting of some dihedral distributions do not
interfer on the mesoscopic result, as the proline governs the dynamics with its stiff potential. A list of
all the dihedral distributions with the tting parameters is presented in appendix A.
16
Figure 3.4: Comparison between the Atomistic short peptides and 30 residues CG simulations for
the negative logarithm of the dihedrals distibutions of the sequences LNPL (left), PNLP (center) and
LLNL (right). It is observed that the tting for PNLP and LNPL sequences are good enough but the
agreement for the LLNL potential seems to show some histeric hindrance, or maybe the phase space
was not sampled enough.
3.3 Analysis of the collagen-like block
The analysis of all the dihedral angles in the atomistic simulations showed a strong sequence-
dependence of the distributions, leading us to adapt the original Head-Gordon model to achieve a
more accurate description of our collagen-like proteins. From our simulations, we concluded that
the high concentration of proline randomly spread in the sequence plays an important role on the
stiffness of the dihedral angles between four consecutive C and therefore proline must be taken
into account as a separated avour. In this way, we characterized the dihedrals distributions using
four avours: hydrophilic (L), hydrophobic (B), neutral (N) and proline (P). The relation between the
amino acids present in the collagen-like and their four letters minimalist codes are given in Table 2.2.
After taking the negative logarithm of the dihedral distributions, we tted them either with cosine
expansion (
6
k=0
_
A
k
cos
k
()

) or parabolic function (B
0
1
2
B
1
(
0
)
2
) according to the position of
the proline. The results of the observations of the dihedrals stiffness can be summarized in the table
below, whegre the distributions were divided in groups according to their main characteristcs. The
rst group, where the proline is not present, shows a exible and smooth logarithmic distribution
of the dihedral angles. The second group has a proline at the third position, and it is observed that it
makes the dihedral angles stiff and the logarithmic distribution is therefore very stiff with one minima.
The third group has a proline at the second position and, despite of its stiffness, it still can be tted
by a cosine expansion. The fourth and last group has proline in one or both anks, and it makes the
dihedral angles very exible and the distribution is, therefore, smooth.
Analyzing the table 3.1 and the gs. 3.4 above, it is possible to infer some conclusions about the
role that Proline plays in the dihedrals distributions:
1. Proline makes the dihedral angles stiffer when it is on the second or third position from the rst
17
Sequence Characteristics Fitting Function
NNLN, LLNL, NLNB, BNLN, NLLN exible and smooth Cosine
NNPN, LNPL, NNPL, NBPN very stiff with one minima Parabola
BPNL, LPNL, LPNN exible Cosine
NLNP, LLNP, PNLP, PBNL, LNLP very exible Cosine
Table 3.1: List of dihedral sequences and their tting functions in the minimalist four letters model.
It can be observed that the sequences are labeled in four categories, according to the exact position of
proline in the chain.
-carbon, as can be easlily seen in g. 3.4 (left).
2. Without Proline (see g. 3.4 (right)), the distribution has a periodic behaviour that can easily
be tted with a cosine expansion.
3. When Proline is positioned on the ankes (see g. 3.4 (center)), the periodic distribution of
dihedral angles is in addition very smooth, being tted easily with a weak potential in cosine
expansion.
The radius of gyration was calculated for 30 in the adapted HG model (see g. 3.5 (left)). We
can see from the two snapshots presented in the g. 3.5 (right) that the collagen changes its con-
formation in a very exible way, going from an almost completely extended situation to collapsed
conformations, conrming the experimental observations [11].
Figure 3.5: Radius of Gyration for the 30 residues sequence calculated from the adapted HG model
(left) and snapshots (right) of the 30 residues minimalist sequence at 300K showing the extended and
collapsed (bottom left) possible conformations achieved by the collagen-like in the 50ns simulations.
The red beads represent proline, the green are hydrophilic and blue are neutral.
18
3.4 Dependence of the R
g
with the number of residues
We then calculated the radius of gyration from the output of a CG simulation runned in CM
3
D
for many collagen sizes and plotted in g. 3.6 a logarithm scale curve for R
g
as a function of the
number of residues. There is a very good agreement with the experimental value for 400 residues
within the statistical error, which serves to validate the adapted HG model for the collagen-part block-
copolymer. Calculating the slope of the curve, we can see the dependence of the radius of gyration
with the number of molecules to be R
g
= 1.391(N)
0.528
in good agreement with the Florys exponent
(0.583) [62] in a good solvent (which means that the particles affectively repel each other), where N
denotes the number of residues.
Figure 3.6: Logarithm dependence between the radius of gyration (in nm) and the number of residues
on the collagen-like sequence. The simulation values are plotted with the experimental result for 400
residues sequence and tted with a linear function.
19
4 Conclusions and Future Perspectives
Finally, we can conclude that the adapted HG model developed for the collagen-like protein
can predict the experimentally observed order parameter value. Therefore, as stated in the begin,
it is conrmed that the high concentrations of proline and the charged/hydrophilic residues in the
sequence play an important role on avoiding the structure to folds into any specic state, but instead
retains its randomness. The radius of gyration has a very good agreement with experiments, behaving
in a logarithmic dependence with the number of residues and providing a good value for the Flory
expoenent.
In the future, this adapted collagen-like CG model will be combined with the adaped CG model
previously developed for the silk-part to be applied for the whole collagen-silk-collagen block copoly-
mer. Thus, it will enable us to study the effect of the collagen-like on silk-like block folding and
self-assembling.
20
Acknowledgements
Many people contributed to the accomplishment of this work. I pay here special attention to some
of them, not neccerily the most important, but the ones who were essential, in precise moments, to
consolidate this achievement.
First of all I thank God, for having given me the ability to learn and understand, always supplying
me with vitality to face the challenges and keeping up achieving my goals, never allowing me to
surrender, but instead keeping me humble.
I thank my supervisor Peter Bolhuis, for giving me the opportunity to start this Msc. project at the
University of Amsterdam. I also thank Marieke Schor, who co-supervised me during the project, and
made my life much easier with your expertise on protein folding and coarse-grained simulation. I also
thank the Molsim group, Bernd, Francesco, Anna, Grisell, Murat, Wolfgang, Rosanne and Zerihum,
my friend, with whom I had a great time in the course of this project. I also acknowledge Sara cluster
for the computer power provided for the simulations.
I also thank my friends in Amsterdam Pedro, Dimas, Max, Igor, Raquel, Vinicius, Girry, Anthony,
Adrien, and many other students, thanks you all for the great time we had here in Amsterdam. To
my friends in Lyon Roberto, Diego, Rodrigo, Franck, Dorian, Jakub, Alex, Aion, Jana, that made that
short stay in France one of the best periods in my life.
I would like to thank the coordinators of the AtoSim Programme for accepting my application to
this course and Erasmus Mundus for the scholarship.
Finally, to all of them who contributed directly or indirectly to the accomplishment of this project.
Thank you very much!
21
APPENDIX A -- Dihedral Angles
Here we present all the dihedral angles distributions analized in the collagen-like sequence as
well as the tting parameters. It can be observed that some of them have almost the same behavior
and can be tabulated in four categories dened by the position of the proline. These groups are shown
in the table 3.1. All the sequences are listed in the gures subsequent, with the tting parameters at
the captions.
Figure A.1: The tting coefcients are NNLN: A
0
=-3.34, A
1
=1.51, A
2
=2.19, A
3
=-1.22, A
4
=-3.34,
A
5
=-0.69; LLNL: A
0
=-3.31, A
1
=-1.99, A
2
=-0.64, A
3
=2.17, A
4
=-0.14, A
5
=-0.69; NLLN: A
0
=-4.25,
A
1
=0.69, A
2
=2.27, A
3
=-0.29, A
4
=-1.32, A
5
=0.36.
Figure A.2: The tting coefcients are BNNL: A
0
=-3.98, A
1
=0.63, A
2
=-0.42, A
3
=-1.98, A
4
=0.70,
A
5
=0.89; NLNB: A
0
=-1.61, A
1
=-0.33, A
2
=-4.59, A
3
=2.08, A
4
=1.73, A
5
=-0.81; PBNL: A
0
=-2.68,
A
1
=0.08, A
2
=1.72, A
3
=0.55, A
4
=0.33, A
5
=-0.11.
22
Figure A.3: The tting coefcients are LNPL - NNPL: B
0
=-5.81, B
1
=0.051,
0
=-50.24; NNPN:
B
0
=-6.73, B
1
=0.23,
0
=-109.86; NBPN: B
0
=-6.54, B
1
=0.28,
0
=-115.27.
Figure A.4: The tting coefcients are BPNL: A
0
=-2.34, A
1
=-2.73, A
2
=-1.08, A
3
=10.75, A
4
=-12.81,
A
5
=5.07; LPNL - LPNN: A
0
=-2.33, A
1
=-0.23, A
2
=-2.84, A
3
=-0.02; LNLP: A
0
=-3.73, A
1
=-2.24,
A
2
=1.02, A
3
=1.02, A
4
=-1.41, A
5
=2.03.
Figure A.5: The tting coefcients are NLNP: A
0
=-4.59, A
1
=-1.02, A
2
=2.63, A
3
=-0.18, A
4
=-0.43,
A
5
=0.91; LLNP: A
0
=-1.61, A
1
=-0.33, A
2
=-4.59, A
3
=2.08, A
4
=1.73, A
5
=-0.81; PNLP: A
0
=-3.72,
A
1
=-2.25, A
2
=1.02, A
3
=1.01, A
4
=-1.41, A
5
=2.03.
23
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