I. Background, Preliminary Work:
This section is partitioned into three parts: 1) a brief outline of the first principles simulation techniques that has
been derived by the Goddard group as a part of the NSF-Grand Challenge Application Group(NSF4ASC-9217368 and
NSF-SGER-DBI-9708929) outlines the background and preliminary experimental and theoretical work for the control
self assembly of macromolecules or artificial proteins followed by the proposed work for this part and 3) background
and preliminary theoretical and experimental work on nanoscale polymer filaments and proposed work on control
design of nanofilaments of desired dimensions, tensile strength and mechanical properties.
I.1 First Principles Simulation Methods (Results from Prior NSF support (NSF4ASC-9217368 and NSF-SGER-DBI9708929)) PI - William A. Goddard III
This section describes briefly some first principles simulation methods developed in Goddard group
which provide new tools essential to the proposed project. As a part of the NSF-Grand Challenge
Application Group we have developed various methods and computational tools useful in describing and
modifying the structures of complex bio macromolecules and materials. The publications from this project
have been referred to in the text and it is a part of the reference section. These tools include:
I.1.1 Quantum Mechanical Methods
PS-GVB/Solvate (Tannor et.al 1994) (Pseudo Spectral Generalized Valence Bond) uses ab intio quantum
mechanical methods for predicting accurate structures and molecular properties including solvation effects [
at the Poisson Boltzmann level)]. This allows prediction of structures while including solvation effects. This
method is useful for accurate calculation of energies of small peptides with solvation and would be used for
predicting peptide energies with several mutations.
I.1.2 Fast Monte Carlo Methods
The Continuous Configurational Boltzmann Biased (CCBB) Monte Carlo method (Sadanobu and
Goddard 1997) is a highly efficient variant of the direct evaluation of the partition function, free energy, and
other configurational dependent physical properties for long polymer chains. This method (CCBB) combines
continuous configurational biased sampling with Boltzmann factor biased enrichment. To illustrate the
efficiency and to validate the bias correction for weighting the torsion and chain enrichments, we have applied
this model to isolated single chains using a united atom force field. For a 50 monomer polymer chain CCBB
with 400 chains leads to an accuracy of 0.1% in the free energy whereas simple sampling direct Monte Carlo
requires about 109 chains for this accuracy. This leads to cost savings by a factor of about 350 000. CCBB is
easily extended to multichain systems, to the condensed state, to more realistic force fields, and to evaluating
the mixing free energy for amorphous polymer blends. The CCBB method has been extended to study the
tertiary folding of proteins. The method uses double biasing to obtain an ensemble of compact folded
structures. Algorithmic filtering is needed through the ensemble for native like structures for further study.
We are currently developing such methods for deriving the loop structure of the outer membrane protein A
under the NIH/NIHCD project. The CCBB method has been tested in various proteins up to 75 residues(Debe
and Goddard 1998). This method is a remarkable advancement that can be used for structure based rationale
studies where the native structure is not known. This method would be used in the proposed study to calculate
thermodynamic properties of polyglutamine aggregates.
1.
2.
3.
I.1.3. Molecular Dynamics(MD) Methods
We have developed the Massively Parallel Simulation (MPSim) program(Lim et. al 1997) to allow
simulations for systems with up to a million atoms or smaller systems for long time scales ( up to
microseconds). MPSim was developed for massively parallel computers and includes such important
algorithm developments as :
The Cell Multipole Method (Ding et. al 1992a) CMM, dramatically reduces the cost of long-range Coulomb
and van der Waals interactions while retaining high accuracy. The cost scales linearly with size, allowing
atomic level simulations for million atom systems(ref).
The Reduced Cell Multipole Method(Ding et. al 1992b) RCMM, handles the special difficulties with longrange Coulomb interactions for crystals by combining a reduced unit cell plus CMM for interaction of the
unit cell with its adjacent cells. The cost scales linearly with size retaining high accuracy, allowing simulation
of crystals with a million atoms per unit cell.
The Newton -Euler Inverse Mass Operator (NEIMO) method (Jain et. al 1993; Mathiowetz et. al 1995;
Vaidehi et. al 1996) for internal co-ordinate dynamics which treats some regions of the protein as rigid , while
4.
5.
6.
7.
others are treated with only torsional degrees of freedom. NEIMO allows use of large time steps which
enables long time dynamics. The cost of NEIMO is linear in the number of degrees of freedom , staying
much less than the other costs for million atom systems.
Heirarchical NEIMO(H-NEIMO) is a rigorous coarse grain MD method (Vaidehi and Goddard 1999) based
on atomic level forcefield. The dynamics can be performed at several levels of heirarchy ranging from all
torsion dynamics to coarse grain, domain dynamics. With fine-grain all-torsion NEIMO dynamics, we
normally treat double bonds, terminal single bonds, and rings (benzene) as rigid bodies. In H-NEIMO
dynamics we allow various levels of coarseness, keeping rigid during the dynamics various segments or parts
of a domain. This allows larger time steps and thus H-NEIMO dynamics allows one to simulate long time
scale domain motions in a protein in a shorter simulation timescale by focussing on those degrees of freedom
that bring about the large domain motions especially in enzymes.
Poisson-Boltzmann (PB) description (Tannor et al 1994) included in the dynamics to include solvent effects,
water or membrane, which are critical for simulation of bio-macromolecules. We have also recently included
a faster description of the solvent effects using the Surface Generalized Born(SGB) method(Ghosh et al
1998). We have also shown that the SGB method is as accurate as the PB method in describing the
electrostatics of the solvent.
Various thermodynamic ensembles like the constant temperature, constant volume, constant pressure and
constant energy (like the (T,V,N), (T,P,N) ensemble dynamics have been incorporated in the MPSim program.
This allows us to simulate the dynamics under experimental conditions.
Algorithms and methods suitable for massively parallel high performance (SGI-Origin-2000, IBM-SP2, Cray
T3D/T3E, HP/ Convex-Exemplar, and Intel Paragon).
I.1.4 Non-equilibrium Molecular Dynamics(NEMD) and Transport Properties
Recently we have made considerable progress in the theory of NEMD(Qi et al 1999). We have developed
an approach to predict viscosity by including external shear rates directly into the Hamiltonian equations of
motion. The NEMD technique enables us to determine the transport coefficients from the response of the system
to finite applied fields. In NEMD viscosity is calculated the same way the experimentalist would measure it. The
shear viscosity coefficient, , is obtained directly by evaluating the momentum flux in a system subject to a known
applied shear rate. Also NEMD can evaluate the pressure and viscometric function in addition to the shear
viscosity, enabling us to study the fluid microstructure in the nonequilibrium steady state.
Results from Prior NSF support for Tirrell
NSF DMR-9510031 - David A. Tirrell; $935,000; 9/1/95-2/28/00
Genetically Controlled Syntheses of New Polymeric Materials
The objectives of this work are: i) development of methods for the preparation of architecturally well-defined
artificial proteins containing reactive olefinic, dienyl, acetylenic and pyrrolyl functional groups, and ii) analysis of
the behavior of the resulting proteins. Our general synthetic strategy involves preparation of unnatural amino acids
carrying the functional groups of interest, followed by evaluation of the translational activity of the amino acid
analogues in bacterial cells. Analogues showing good translational activity are then incorporated into artificial
proteins encoded by artificial genes expressed in bacterial culture. Work in the current funding period has focused
on synthetic methods for olefins (alkenes) and acetylenes (alkynes); in each case, substantial successes have been
achieved. The following fifteen papers describing our NSF-supported research have been published or accepted
since the start of funding on September 1, 1995:
1.
S. Kothakota, T.L. Mason, M.J. Fournier, D.A. Tirrell, “Biosynthesis of a Periodic Protein Containing 3Thienylalanine: A Step Toward Genetically Engineered Conducting Polymers,” J. Am. Chem. Soc. 117, 536
(1995).
2.
Kothakota, M.J. Dougherty, M.J. Fournier, T.L. Mason, E. Yoshikawa and D.A. Tirrell, “Templated
Biological Synthesis of Polymers of Abiological Monomers,” Makromol. Symp. 98, 573 (1995).
3.
T.J. Deming, M.J. Fournier, T.L. Mason, D.A. Tirrell, “Structural Modification of a Periodic Polypeptide
through Biosynthetic Replacement of Proline with Azetidine-2-carboxylic Acid,” Macromolecules 29, 1442
(1996).
2
4.
M.T. Krejchi, E.D.T. Atkins, M.J. Fournier, T.L. Mason, D.A. Tirrell, “Observation of a Silk I-like Crystal
Structure in a Genetically Engineered Periodic Polypeptide,” J.M.S.-Pure Appl. Chem. A33, 1389 (1996).
5.
A. Panitch, K. Matsuki, E.J. Cantor, S.J. Cooper, E.D.T. Atkins, M.J. Fournier, T.L. Mason and D.A.
Tirrell, “Poly(L-alanylglycine): Multigram-Scale Biosynthesis, Crystallization, and Structural Analysis of
Chain-Folded Lamellae,” Macromolecules 30, 42 (1997).
6.
M.T. Krejchi, S.J. Cooper, Y. Deguchi, E.D.T. Atkins, M.J. Fournier, T.L. Mason and D.A. Tirrell, “Crystal
Structures of Chain-Folded Antiparallel ß-sheet Assemblies from Sequence-Designed Periodic
Polypeptides,” Macromolecules 30, 5012 (1997).
7.
T.J. Deming, M.J. Fournier, T.L. Mason, D.A. Tirrell, “Biosynthetic Incorporation and Chemical
Modification of Alkene Functionality in Genetically Engineered Polymers,” J. Macromol. Sci.-Pure Appl.
Chem. A34, 2143 (1997).
8.
E.J. Cantor, E.D.T. Atkins, S.J. Cooper, M.J. Fournier, T.L. Mason and D.A. Tirrell, “Effects of Amino
Acid Side-Chain Volume on Chain Packing in Genetically Engineered Periodic Polypeptides,” J. Biochem.
122, 217 (1997).
9.
H. Chen, S.L. Hsu, D.A. Tirrell, H.D. Stidham, “A pH Dependent Coil-to-Sheet Transition in a Periodic
Artificial Protein Adsorbed at the Air-Water Interface,” Langmuir 13, 4775 (1997).
10.
J. G. Tirrell, D. A. Tirrell, M. J. Fournier, T. L. Mason, "Artificial Proteins: De novo Design, Synthesis and
Solid State Properties," in Protein-Based Materials D. Kaplan, K. McGrath, Eds. (Birkhauser, Boston, MA,
1997) pp. 61.
11.
E.A. Ponomarenko, D.A. Tirrell, W.J. MacKnight, “Water-Insoluble Complexes of Poly(L-Lysine) with
Mixed Alkyl Sulfates: Composition-Controlled Solid State Structures,” Macromolecules 31, 1584 (1998).
12.
J.C.M. van Hest and D.A. Tirrell, “Efficient Introduction of Alkene Functionality into Proteins in vivo,”
FEBS Lett. 428, 68 (1998).
13.
J.E. Beecher and D.A. Tirrell, “Synthesis of Protected Derivatives of 3-Pyrrolylalanine,” Tetrahedron Lett.,
39, 3927 (1998).
14.
W.J. MacKnight, E.A. Ponomarenko and D.A. Tirrell, “Self-Assembled Polyelectrolyte-Surfactant
Complexes in Nonaqueous Solvents and in the Solid State,”Acc. Chem. Res., 31, 781 (1998)
15.
A. Panitch, T. Yamaoka, M.J. Fournier, T.L. Mason and D.A. Tirrell, “Design and Biosynthesis of ElastinLike Artificial Extracellular Matrix Proteins Containing Periodically Spaced Fibronectin CS5 Domains,”
Macromolecules, 32(5), 1701 (1999).
I.2.1. Encoded Self-Assembly of Macromolecular Materials: Background and Preliminary Experimental work.
Reversible gels. Recent work from Tirrell’s laboratory has shown that rod-coil-rod triblock copolymers containing leucinezipper endblocks flanking a soluble polyelectrolyte domain undergo reversible gelation in response to small changes in pH
or temperature. The mechanism of gelation in these systems is
unique, in that it relies on controlled dimerization (or higher-order
aggregation) of the leucine-zipper domains under conditions where
the polyelectrolyte “spacer” remains soluble. The leucine zipper is a
common structural motif found in many eukaryotic transcription
factors such as GCN4 (Landshultz et al 1988). The defining
characteristic of the leucine-zipper motif is a heptad periodicity,
typically designated -(abcdefg)-, as shown in Fig. 1, in which the a
and d positions are occupied by hydrophobic groups (often leucine),
and e and g are acidic or basic
Figure 1 Helical wheel of the dimer
3
(i.e., titratable) residues. Such periodic chains readily adopt helical conformations that place a and d on a single
hydrophobic face of the helix, where they are flanked by titratable residues e and g(see Fig. 1). The primary driving force
for protein dimerization is provided by formation of the hydrophobic interface of the dimer, but electrostatic forces arising
from e-g interactions can modulate dimer stability in response to changes in pH and stabilize heterodimeric aggregates in
preference to homodimers (Lumb and Kim 1995). The triblock design preserves the protein dimerization function of the
zipper domain, but turns that function toward entirely new objectives; protein-protein recognition now leads not to DNA
binding, but rather to the formation of switchable hydrogels. The mild conditions under which gelation is reversible (nearneutral pH and near-ambient temperature) make these
triblock copolymer systems attractive candidates for use
in molecular and cellular encapsulation and in
controlled reagent delivery (Figure 2). This preliminary
result is intriguing insofar as it illustrates the strong
connection between molecular structure and materials
properties. Thus manipulations of the molecular
structure would affect the nanoscale properties that can
be tested with hydrogels.
Figure 2. Gelation of triblock artificial proteins
At the same time, it highlights the need for precise,
quantitative descriptions of these connections, such that
real materials design can be accomplished. GCN4-p1 is a
leucine zipper (shown in Fig.4A) peptide containing 33
residues in which four leucine-leucine interactions
contribute to the thermal stability of the coiled coil
architecture. Trifluoroleucine, an unnatural amino acid
that has been incorporated into proteins with high
efficiency, was substituted at all the leucine positions in
GCN4-p1. The choice of trifluoroleucine was based on
two factors. First it has been well documented that
fluorinated amino acids are nearly isosteric to their
natural analogs, and are equally (or more) inert chemically (Rennert et
al 1963). The second and the more crucial factor in choosing
A
trifluoroleucine is that it is accepted by the endogenous leucyl-tRNA
synthetase (LeuRS) during biosynthesis of recombinant proteins
(Kothakota et al 1995). When leucine auxotrophic cells are shifted to a
medium without leucine after induction of protein synthesis, the host
machinery will charge trifluoroleucine in a highly efficient manner. In
fact, it has been observed that over 95% of leucine positions in a
recombinant protein can be substituted by trifluoroleucine (Nakajima
& Tirrell unpublished results). Similar to wild type GCN4-p1, the
Wavelength (nm)
fluorinated peptide is essentially 100% helical as determined by
circular dichroism (Fig.3A). Substitution of leucyl methyls by
trifluoromethyl groups resulted in a significant gain in helix thermal
stability; the thermal melting transition temperature of the fluorinated
peptide was elevated from 49C to 62C(see Fig.3B). The thermal
melting profiles of the peptides were studied using circular dichroism.
The ellipticity at 222 nm was monitored as a function of temperature
and converted to the fraction of peptide unfolded (Fig. 3B). The
difference in thermal behavior is most evident at temperatures higher
than 30C. Tfl-GCN4-p1 denatures to a lesser degree than leu-GCN4B
p1 at the same temperature, indicating a more stable coiled-coil
aggregate.
Temperature (C)
Figure 3. A CD spectra of leu-gcn4-pl(o) and tfl-gcn4-pl(square) at
0oC and 40M. The similar spectra indicates that the peptides are helical. B. Thermal unfolding profiles for native (o) and
tfl-gcn4-pl(square).
100
[q] (103 deg cm2 dmol-1)
80
60
40
20
0
-20
-40
194
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206
210
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218
222
226
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234
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258
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0.9
Fraction Unfolded
0.8
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0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
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4
The fluorinated peptides are also more resistant to guanidine and urea denaturation. Thus incorporation of non-natural
aminoacids may provide proteins of enhanced stability.
I.2.2 Preliminary Results from Theory
The collaboration between theory and experiment is ongoing in this area of nanomaterials between Goddard’s
group and Tirrell’s group. Here we describe some of the preliminary results obtained on the theoretical simulations that
explain the strength of the fluorinated zippers that have been synthesized by Tirrell’s group as compared to the native
triblock leucine zipper based polymers.
Quantum Mechanical and Molecular Dynamics Results for Fluorinated Leucine Zippers.
We have an ongoing collaboration on the design of temperature resistant fluorinated leucine based zippers
described in the previous section. In this section a brief description of the simulation results on molecular level control of
the stickiness of the native gcn4 leucine dimer (fig 4A) and its fluorinated derivatives(Fig4B) is presented. Constant
temperature Nose-Hoover dynamics was performed with a fast and accurate description of the solvent using the SGB
method using the MPSim code on an SGI-Origin 2000 parallel machine for the native gcn4 leucine zipper. The starting
structure for the dynamics was taken from the protein data bank for gcn4 leucine zipper. MD simulations at 300K with
DREIDII (Mayo et al 1984) and charges from CHARMM(Mackerell et al 1995) for the protein show a stable structure with
a net CRMS of <3.5 A, which is the experimental R factor. The binding energy (enthalpy) for this native dimer was
calculated to be -40.0Kcals/mole which is in good agreement with the experimental measurement of the enthalpy of
formation of the dimer which is -35.0 Kcals/mole.
Fluorinated Zippers:
The charges on the fluorine of fluorinated leucine(Leu(F)) zippers are critical in determining the binding energy
of the dimers. These charges have been derived by performing a quantum mechanical(QM) calculation, using Jaguar
(Jaguar 3.5, Schrodinger Inc., Portland, Oregon) on the tripeptide, Gly-Leu(F)-Gly using LMP2 method and 6-31G** basis
set. Using the QM charges on Leu(F) we built the fluorinated dimer starting from the native dimer structures. Since
substitution of one of the methyl groups on the side chain of leucine leads to a chiral center at the -carbon there are several
possible combinations of chiral centers in the dimer. There are four leucine pairs that are selectively substituted with F.
Considering that all the -carbons on a monomer helix is of the same chirality we first studied the four possible dimers as
shown below and in Fig. (5). The case where the carbon attached to the fluorines in the side chain of fluoroleucine is closer
as shown in Fig 6A ( the distance being 4.7A) is called "close". When the flourines are as shown in Fig.6B where the
distance between the carbons attached to the fluorines is 7.1A is called the "far". The other case where one of the carbon
attached to the fluorines is at 7.1A is called the "mixed" case. There are obviously two possibilities here. It is
experimentally tedious to synthesize these optically pure dimers but in such a situation the theoretical simulations are very
helpful in understanding the experimental results.
1.
R R
2. L L
3. R L
4. L R
R R
L L
R L
L R
R R
L L
R L
L R
R R
L L
R L
L R
"far"
"close"
"mixed1"
"mixed2"
Figure 5 The four possible conformers for which simulations were done presently.
Figure 6. The native gcn4-pl dimer is shown in A and the
fluorinated dimer ( F atoms are red) is shown in B. A close up
view of the distances between the F atoms are shown in Fig.7
Using a one point energy of the minimized structures we
determined that the rotamer built using the native structure as the
template is of the lowest energy among the other possible
rotamers. We performed MD simulations on all four fluorinated
dimers for 200ps using MPSim with SGB solvation. The energies
for the flourinated dimers show a separation into two cases.
While the energy of the "close" dimer is high due to the
unfavorable electrostatic repulsions between the fluorines the rest
three dimers "far", and the two "mixed" cases are similar as
shown in Fig. 8. Thus theory can clearly differentiate between the
various optical isomers of the fluorinated dimers using would aid
the design and control of experiments. This is a preliminary
5
demonstration of the strong collaboration that is in the heart of
this exploratory project.
Figure 7. The three possible conformers of fluorinated leucine
zippers . The fourth conformer showed in Fig.4 is the similar to
the “Mixed” case shown above but the fluorines switched
between the two helices. The larger distance shown in the
picture is about 7A and the shorter one is about 4A.
Figure 8 Potential energy(Kcals/mole) variation for various
dimers with time(ps).
II.2.3 Proposed Work: We propose herein a coordinated program of molecular architecture, solution properties, and
gelation behavior of bioengineered block copolypeptides. These designed polypeptides would then be synthesized and
tested for the "stickiness" property using the hydrogel system.
Effect of Fluorine substitution at the molecular design level: Molecular modeling can provide immediate information for
two current approaches in gel engineering. First is the incorporation of even more hydrophobic residues such as
hexafluoroleucine at the d position(Fig. 1). The increase in side chain size may cause even more hydrophobic driving force
for chain association in solution. However, as mentioned earlier, introduction of the cluster of fluorines in the hydrophobic
core might induce unfavorable electrostatic energy (i.e. repulsion). Molecular dynamics can be used to quantify the two
competitive energy contributions and predict the effects of such substitutions (or any other substitution at any position
within the peptide). Hence we would continue to study the effect of fluorination on the stability of the fluorinated zippers as
a function of the degree of fluorination of the side chains. In the simulations we are building optically pure isomers and
would compute the dimerization energy in terms of fundamental forces.
Prediction of the Effect of Length of the helical regions on the stability: The second approach that is underway in
our groups is to lengthen the zipper domain so that it contains more leucine-leucine contacts at the hydrophobic
surface of the coil-coil. Lengthening the zipper domain requires meticulous recombinant DNA manipulation to
produce the protein in vivo since the wild-type GCN4 is already at the length limit of solid-phase peptide
synthesis. The structure of longer zippers is not known. Recent methods in the Goddard lab have yielded folded
structures of short polypetides to the experimental helical propensities(Bertsch et 1l 1998, Peng et al 1999). Hence
we would use the NEIMO MD methods with solvation to study the effect of length on the helical structure of the
zippers. These molecular dynamics results should provide insight into the amount of additional stability that might
be achieved before experiments are performed on extending the helical segments.
Experiments: The preliminary experiments were not done with optical purity at the chiral center of the fluorinated leucyl
methyl carbons. We propose to build optically pure fluorinated gcn4-pl dimers and measure its stability using Circular
6
Dichroism (CD) variation with temperature. From the amino acid analysis and mass spectroscopy data, we will be able to
determine the amount of trifluoroleucine incorporation in the protein . Circular dichroism and DSC will be used to study the
change in energy associated with the different levels of substitution.
Effects of Fluorination on the properties of hydrogels- Mesoscale Dynamics NEMD: Having studied the effect
of fluorine substitution and the effect of the length of the helical regions at the molecular level, the next step is the
mesoscopic simulations of the hydrogel. We would construct hydrogels at various levels of fluorine substitution
and optimal helical length. It is not known whether fluorination has other effects on the hydrogels in addition to
enhancing their thermal stability. To understand how the different levels of fluorination would affect the
properties of the hydrogel, such as the gel-sol transition we would perform the following simulations. The
hydrogels would be built as an infinite periodic system. We would then perform NEMD simulations on the
periodic system and apply a shear at a constant rate. The shear viscosity coefficient(Qi et al 1999) will be
calculated using the momentum flux caused by the shear rate. Now the sharp change in the viscosity coefficient
with temperature would depend on the level of fluorine substitution. Thus we would perform NEMD simulations
at a given shear rate at various temperatures and the temperature at which the viscosity changes sharply would be
plotted vs the level of fluorine substitution.
Experiments: The experimental testing bed for these design predictions are the reversible hydrogels. First, proteins
with different levels( range would be chosen from the optimum level suggested from the design by Vaidehi and
Goddard) of fluorine incorporation will be synthesized by varying the level of trifluoroleucine in the expression
medium. After purification, amino acid analysis and mass spectrometry will be used to determine the level of
incorporation of fluorinated leucines. The pH responses of these hydrogels might also be altered since the
increased binding strength between the helical domains might require a higher level deprotonation (i.e. higher pH)
at the e and g positions to facilitate the transition from gel to solution. In fact, by varying the level of fluorination,
one might be able to obtain a collection of hydrogels with different pH dependent behaviors. The results from the
simulations of the previous section would be used for the design of experiments for various level of fluorination.
The optimum level of fluorination ( from theory) for which the transition temperatures are higher would be chosen
to study the effect of pH on the sol-gel transitions. This could be the first time one can synthesize a series of pH
sensitive proteins using the same amino acid sequences. On the other hand, the free energy gain from entropy as a
consequence of fluorination might be too small compared to the electrostatic repulsion between residues e and g
that the sol-gel transition behavior might not differ significantly from the 100% leucine containing proteins. This
competition in energy can be studied by monitoring the viscosity change in gels as a function of pH. Rolling ball
viscometry has been the method used to observe gelation phenomena in our group. If necessary, commercial
rheometers can be used to study the rheological change in proteins upon fluorination.
I.3 Palette of non-natural aminoacid analogs
Using genetic engineering, Tirrell's laboratory has been able to use protein preparation system of living
organisms to make well-defined protein-based materials. A drawback of the use of biosynthetically prepared
protein is that the limited amount of amino acids that can be incorporated. Incorporation of non-natural amino
acids will partially solve this problem. Interesting functionalities as halides or unsaturated residues can be
introduced into protein polymer. This kind of incorporation can be achieved by: (1) chemical synthesis of peptides;
(2) in vitro synthesis wherein tRNA is chemically acylated with amino acid analog and the protein translated in a
cell-free system; (3) in vivo synthesis wherein the natural protein synthesis apparatus of bacteria utilizes a
structurally related analog in place of the natural amino acid. The in vivo is by far the most advantageous method
due to high yield of amounts of incorporations. Using molecular modeling we want to understand how the
specificity of aminoacid t-RNA synthetase (aaRS) is achieved and elucidate how the binding of most amino acid
residues in the aaRS active site is probably avoided by the size, shape, and hydrophobic nature of the aaRS amino
acid binding pocket.
I.3.1 Preliminary Work: Goddard group has started some initial work on this project with the goal to create
aminoacid analogs that could be incorporated by the corresponding aaRS. We choose the phenylalanyl-tRNA
Synthetase(PheRS) that has the crystal structure and it is the only synthetase in class II that charges the amino acid
with 2’OH group of the 3’-terminal ribose of the RNA. The PheRS is a tetramer with 12,000 atoms and a full
minimization with SGB solvation followed by 100ps of MD was performed using the MPSim program with
DREIDII forcefield and CHARMM charges . The domains showed motions but the overall structure is within
4.5Ao RMS in coordinates. The ligands phenylalanine and fluorophenylalanine were docked(see fig. 9) into the
binding pockets using guides from the description of the crystal structure with the ligand ( not released). Using
conjugate gradient minimization of the ligand and the cavity in PheRS in which the ligand is bound we have
7
calculated the binding energies of –35.6076Kcals/mole and –31.0171Kcals/mole for Phe and fluoroPhe
respectively. At this level of calculation one can say that both these ligands bind with similar affinity.
Figure 9. VanderWaal's surface of docked and substrate-free binding pocket in phenylalanine t-RNA synthetase.
I.3.2 Proposed work: Theory: We would understand the nature of interactions in the binding site of the existing aa and
their analogs for PheRS and MetRS. Fig. 10 shows the list of analogs that have been tested experimentally for be tried to
PheRS, MetRs and IsoLeuRS. Some of these analogs are known to get incorporated and some others are not. The crystal
structures are available in the protein data bank f for GlyRS, HisRS, AspRS, GluRS, SerRS, TyrRS, and GlnRS. We would
hence understand the binding pocket of these aaRS with their corresponding aa docked. Further we would design similar (
to that of MetRS or PheRS) analogs in consultation with Tirrell for the feasibility of the synthesis for these aaRS. Thus we
would design a palette of non-natural aa analogs to be testes experiementally.
III.1.1 Nanoscale Bionano-filaments- Background
Simulation capabilities for nanotubes and nanowires at the Goddard group: We have performed extensive molecular
mechanics and molecular dynamics studies on the energy, structure, mechanical and vibrational properties of single wall
carbon nanotubes (SWNT)(Gao et al 1998a and b; Che et al 1999; Mcclurg et al 1997; Kiang et all 1996a and b) with
different radius and chirality (armchair (n,n), chiral (2n,n), and zigzag (n,0)). In this study an accurate interaction potential
derived from quantum mechanics was used. The simulation studies were primarily carried out using the MPSim(Lim et al
1997) Program. We studied the mechanical properties and stabilities of the carbon nanotubes as a function of radius and
various forms (circular and collapsed). In the bulk phase we determine the specific packing, density, lattice parameters.
Molecular Dynamics and Molecular Mechanics studies led to a triangular packing as the most stable form for all three
forms. More importantly, we determined the Young's modulus along the tube axis for triangular-packed SWNTs using the
second derivatives of the potential energy. They are Y = 640.30 GPa, Y = 648.43 GPa, and Y = 673.49 GPa, respectively.
Normalized to carbon sheet these values are within a few percent of the graphite bulk value. Using classical thin plane
approximation and variation of strain energy as a function of curvature, we calculated the bending modulus of the SWNTs.
The calculated bending moduli are k (n,n) = 963.44(GPa), k (n,0) = 911.64(GPa), and k(2n,n) = 935.48(GPa). We also
calculated the interlayer spacing between the opposite sides of the tubes and found d(n,n) = 3.38(Å), d(2n,n) = 3.39(Å), and
d(n,0) = 3.41(Å). In order to assess the tensile and compressive strength of the SWNTs, a series of compressive and tensile
loading experiments was performed by varying the c lattice parameter, along tube axis. With respect to the optimum c
lattice parameter, co, the strain can be defined as
 = c/co
The tubes are softer under compression due to buckling effect. Four different views along the tube axis and
perpendicular to the tube axis are presented in Fig.10. Based on those energy strain curves, "elastic constants",
are calculated as the second the derivative of energy with respect to applied strain.
III. 1.2 Simulation of lipid bilayers for membrane proteins :
8
Simulation Description: As a part of the ARO-MURI project on optimization of the structure of olfactory
receptors, we carried out a series of MD simulations (Floriano et al 1999) at constant temperature and pressure
(NPT MD) for the 1,2-dilauroyl-DL-phosphatidyl ethanolamine acetic acid (DPE), Disodium betaglycerophosphate hydrate (GP0) and L-alpha-glycerol phosphorylcholine (GPC) crystal structures (Elder et al.,
1977; Lis & Starynowicz, 1985; Abrahamsson & Pascher, 1966). The atomic coordinates were taken from the
Cambridge Structural Database (CSD). Fig. 11 shows the chemical diagrams for DPE, GP0 and GPC. The systems
were chosen in order to evaluate the performance of the force field and atomic charges progressively. Therefore
we simulated a phosphate group (GP0), a polar head group (phosphorylcholine) commonly present in membrane
lipid bilayers (GPC), and the same polar head group combined to alkane chains in the lipid bilayer DPE. The
simulations started with the CSD coordinates. The
atomic coordinates and cell parameters were
minimized using periodic boundary conditions
(PBC). The minimized crystals were submitted to
100 ps of NPT MD at 300K and 1atm, with PBC.
All cell parameters were optimized during
simulations. The dynamics time step was 0.001
ps, the temperature relaxation time was 0.01 ps,
and the Rahman-Parrinello mass term was 0.1.
The non-bonded interactions were calculated
using spline cutoffs (Rcut = 9 Å for GP0 and GPC;
Rcut = 9 and 15 Å for DPE).
Figure 10 Four views of the singlewall carbon
nanotube along and perpendicular tube axis.
We used the Dreiding (Mayo et al 1990)
force field combined with a set of charges derived
from Charge Equilibration method (Rappe and
Goddard 1991). The results were compared to
experimental cell parameters and densities (Elder
et al., 1977; Lis & Starynowicz, 1985;
Abrahamsson & Pascher, 1966), and to previous
theoretical studies (Willians & Stouch, 1993;
Tobias et al., 1997) available in the literature.
Results
As seen in Table 1, the present simulations reproduce the experimental cell parameters and densities of the
DPE, GP0 and GPC crystals with more than 90% accuracy. The errors in density are 4% or less. The cell
parameters are very well reproduced, especially if we consider that both minimization and NPT MD calculations
had no restrictions on their values. The potential and charge scheme used by Tobias et al. (Tobias et al., 1997) also
give results in very good agreement with the experimental parameters for GPC, although it is somewhat worse
than ours in reproducing the density. On the other hand, those authors suggested based on their simulations results
of alkanes, that the alkane chains need more complicated potential forms than those used by them (data not
shown). Our results, on the contrary, are consistent for all three levels of complexity represented by the phosphate
(GP0), phosphorylcholine (GPC) and dilauroylphosphatidylethanolamine (DPE) molecules.
NPT simulations are useful tools for evaluating force field quality. Nevertheless, the present simulations are a
good indication that the combination Dreiding force field/Qeq polar groups is suitable for describing membrane
lipid bilayers. This combination is being currently use to simulate the purple membrane from Halobacterium
halobium, which is composed by lipid bilayers of diphosphatidyl glycerophosphate, in MD simulations of
transmembrane proteins. Figure 12 shows a schematic view of the simulated membrane.
Table 1. Percentage of standard deviation from experimental values for calculated cell parameters (a, b, c, alpha,
beta, gamma) and density (Dens) of DPE, GP0 and GPC crystals at 300K and 1 atm
9
a (Å)
b (Å)
c (Å)
alpha
beta
gamma
Dens
(degrees)
(degrees)
(degrees)
(g/cc)
Minimization
DPEa
1
-7
4
0
0
0
4
a
-1
-1
0
0
-1
0
1
GPCa
-1
-1
0
0
-1
0
1
3
-1
-1
NA
0
NA
NR
3
-7
6
6
-4
3
0
-2
-2
1
-1
-3
-1
-3
-1
3
0
3
0
3
1
0
0
0
0
0
-4
-4
-1
-7
0
-3
0
9
GP0
b
GPC
NPT MD
DPEa
GP0
a
GPCa
GPC S II
c
GPC
CHARMM22c
a
present work
b
c
Williams & Stouch, 1993.
DPE
O
Tobias et al., 1997.
H
NA=not applicable (angles were kept fixed).
NR=not reported.
O
H
O
O
O
P
N
O
O
O
H
GP0
O
O
P
O
OH
O
HO
GPC
CH3
O
3HC
O
OH
P
N
O
O
OH
CH3
Figure 12. Schematic view of a simulated
membrane composed by bilayers of diphosphatidyl
glycerophosphate (DPG). The unit cell has 25 lipid
Figure 11. Structures of the three lipids presently
simulated.
bilayers corresponding to 15500 atoms.
10
In the two sections described above, Goddard group has demonstrated the capabilities to simulate the
strain in a nanotube and simulations of the lipid bilayer membranes for assembling membrane proteins. Both of
these components are vital for the proposed work described below.
III.1.3 Nanotubes for Bio-Sensors: Recent developments in the fabrication of meso- and nanoscale structures
have included the self-assembly of carbon and other materials into nanotubes and quantum wires (1) and the
coalescence of lipid surfactants from solution into sub-micron tubules (2). Formation of bilayer nanotubes from
membrane capsules is a common occurrence in cell biology. For example, when a cell or vesicle sticks to a
foreign surface at a point and is then pulled away, an optically invisible bilayer tube (diameter < 100 nm) usually
connects the capsule to the surface even after displacements of many diameters (4). Two physical conditions are
requisite for nanotube formation from bilayer vesicles: (i) The bilayer must be bonded to a spot on a rigid surface
and (ii) there must be a reservoir of excess bilayer surface beyond that sufficient to enclose the vesicle volume as a
sphere. These two conditions are easily attained and manipulated externally. First, vesicles after preparation are
slightly dehydrated by increasing the osmotic strength of the aqueous suspension. After aspiration into a
micropipet, the excess surface of the vesicle is drawn into a projection inside the pipet (Fig. 13 A), which provides
the reservoir of bilayer for nanotube production. By appropriate control of bilayer composition, strong
attachments can be made when the vesicle is touched to a surface. When the pipet holding the vesicle is
withdrawn, a nanotube is formed (Fig. 13 A and B).
Nanotubes and networks prepared in this way are intrinsically unstable, and cannot survive removal from
the aqueous processing medium. In the preliminary studies of Evans and Tirrell (3), photoinitiated radical crosslinking of polyethylene glycol 1000 dimethacrylate (PEGDMA) confined to the interior of the lipid assembly was
chosen as a prototype chemistry for stabilization of the membrane template. Cross-linked multimethacrylates form
elastic networks that are widely used in applications where strength and shape stability are required (7). PEGDMA
is soluble both in water and in organic solvents, but after cross-linking, the polymerized material forms a resilient
and insoluble polyethylene glycol (PEG) gel. The PEG structure is attractive in the present context for several
reasons: (i) The capability of swelling in aqueous and organic environments enables post-polymerization
processing of patterns and networks in a wide variety of solvents, (ii) PEG does not denature enzymes or other
proteins of potential interest in biosensor or biomaterials technology [PEG-modified enzymes are used currently in
clinical applications (8)], and (iii) PEG surfaces are relatively inert with respect to biological cells, suggesting that
cell viability and function should not be altered in cellular devices based on patterned PEGDMA networks.
Potential Uses for Lipid nanotubes: The nanotube techniques provide a basis for fabrication of functional
nanoscale conduit patterns and networks of various kinds. It has already been demonstrated that lipid tubules can
be used as precursors for formation of submicrometer silica tubes (10) and as templates for deposition of metals
(2) and metal oxides (11). The methods developed in these earlier approaches should be directly applicable to the
patterns made as described here, and should produce robust structures with useful mechanical and electronic
properties. Potentially more versatile, the porous core of a polymerized nanotube provides unrestricted opportunity
for elaboration of network properties. Photocrosslinking of water-soluble conjugated polymers can lead to
networks of organic conductors and impregnation of the core with metal salts can be used to metallize the structure
after reduction. A similar approach for deposition of metals in carbon nanotubes has been reported by others (12).
The cross-linked core of nanotubes can serve to immobilize enzymes or other proteins capable of modulating
electron transfer or biochemical recognition processes, and it is conceivable that nanotube arrays might be used to
link together patterns of biological cells immobilized on substrates (13) to create integrated "cellular" biosensors
and devices. We plan ultimately to explore each of these directions, but the focus of the proposed work is on
fabrication and mechanical behavior of flexible, nanometer-scale polymeric filaments. Because of their small size
(and the associated property of rapid response to signals, such filaments show substantial promise as sensors and
actuators in robotic and other devices.
III.2 PROPOSED WORK
III.2.1. Control of Mechanical Properties
Molecular Modeling of the nanotubes-Variation of the length and concentration of the PEGDMA: Engineering of
the mechanical properties of nanometer-scale polymeric gels should be possible through variation in the structure
and concentration of the polymerizable component entrapped in the lumen of the nanotube used as template.
Goddard group has developed (Section III.1.1 and III.1.2) the forcefields and simulation methods required to
simulate a lipid nanotube filled with various concentrations polyethylene glycol dimethacrylate(PEGDMA). We
also have previous experience in computing the strain in a nanotube caused by bending forces or the ductility of
the
11
nanotube. As shown in Fig. (14) we would
build and optimize the structure of the bilayer
nanotube and compute its ductility as a
function of the length of the PEGDMA. We
would use DREIDII forcefield with charges
from charge equilibration method for the polar
head of the lipid (Floriano et al 1999) and for
the polymer. It will also be important to
examine the role of filament diameter in
A.
B.
determining mechanical properties. Because
mechanical properties in these systems will
Fig. 13. Video microscope images of a lipid bilayer vesicle
depend on the extent of covalent network
(~ 20 µm diameter) held by micropipet suction and tethered
formation within the nanotube template during to a solid microsphere (~ 4 µm diameter) by an invisible
nanotube of bilayer (~ 40 nm diameter) pulled from the
photopolymerization, the increase in surfacevesicle surface. (A) A bright field image shows only the
to-volume ratio in very small structures may
become critical in determining properties such vesicle and microsphere. (B) Epi-illumination is used to
excite fluorescence from labeled lipids doped in the bilayer,
as modulus if connectivity is reduced (or
revealing the 40-nm diameter tube of bilayer emanating
enhanced) by surface effects. These are ideal
from the vesicle.
situations for the study using molecular level
simulations since the molecular level control
alters the properties of the nanostructure.
Measurement of Physical Properties:
Based on bulk measurements, the Young’s modulus E of the polymeric filaments could be
estimated to be between 0.1 and 1 MPa. Since the filament diameters will range from 10 nm to 1 micron,
we can estimate the spring constant k for a typical sample of length L=10 microns using the formula
k=EA/L, where A is the cross sectional area. We thus expect the filaments to have longitudinal spring
constants ranging from 1 pN/micron to 10 5 pN/micron (=100 pN/nm), depending on the diameter. Goddard
group would compute the effect of long range forces on the longitudinal spring constants since it is these
long range forces on the lipid and also the valence forces on the PEG molecules that govern the strain on
the walls of the nanotube. We would construct bilayer nanotubes ranging from 10nm to 100 nm in
diameter. The PEGDMA would be built along the walls of the nanotube as shown in Fig.(?). Both the
valence forces and the nonbond forces in PEGDMA along with the nonbond forces along the head of the
lipid and similar forces along the inner walls of the tube would account for the tensile strength along the
walls of the nanotube. Young's modulus and bending modulii along the tube axis and perpendicular to the
tube axis would be computed as described in the publication Gao et 1l 1999. These quantities would be
computed as a function of
 the length of the PEGDMA molecules
 cross-linking in the PEGDMA molecules
 concentration of the PEGDMA filling the nanotube starting from 10% .
 the tube diameter
Experiments : Preliminary experiments, PEGDMA 1000 at a concentration of 10% (w/v) was used by
Tirrell’s group to demonstrate the effectiveness of polymerization as a method of preparing nanometerscale filaments with some degree of toughness in the aqueous processing environment. In these
experiments the integrity and elasticity of the nanostructured polymeric gels could be readily demonstrated
by deforming the filament with a micropipet. The gels were observed to deform under tension, and then to
relax back to their initial state upon release. Early successes in experiments with methacrylate-functional
polyethyleneglycols (PEGs) prompt us to focus initially on this class of reactive polymers and oligomers.
Two kinds of structural variants are readily available, and will be explored: i). linear PEG dimethacrylates
of a variety of chain lengths, and ii). branched and star-shaped PEGs with up to approximately 100 arms
per chain. The results from the simulation study would offer the experiments insights into the role of
12
PEGDMA and the optimum concentration of PEGDMA for a desired strength of the nanotube. While the
latter class of PEGs is not available in methacrylated form, endgroup functionalization is straightforward
and preparation of multifunctional PEG methacrylates should present no substantial problems. Comparison
of the properties of gels produced from such polymers will allow us to establish the role of network
junction functionality in controlling the mechanical behavior of nanoscale gels.
A second variable to be explored in these studies is the concentration (or volume fraction) of the
prepolymer in the nanotube template. In general, one would expect the modulus and toughness of the gel
to increase with the volume fraction of (pre)polymer, but we anticipate that nanotube formation will not be
possible at very large prepolymer concentrations. Because the maximum concentration at which tube
formation occurs may be dependent on molecular architecture, it is not obvious a priori how one should
design the system to achieve maximum modulus and strength. Here the simulation techniques would offer
pathways by which the strength of the nanotube varies with the cocentration of the PEGDMA. We will
establish the minimum prepolymer concentrations that allow formation of continuous networks, as well as
the maximum concentrations at which tube formation can be achieved. Through systematic variation in
concentration we would study the mechanical strength of polymeric nanostructures. Once this variation and
molecular architecture is understood the experiments could be designed for understanding the relations
between fabrication conditions and the concentration of the polymer.
The Goddard group will examine this question in a systematic way, by computing the molecular
level stress in the nanotube as a function of the diameter of the nanotube( varying from microns to tens of
nanometers) and analyze the stress on bond and angle energies and other components of energy like the
electrostatic and van der Waal’s energy. This would throw light on the role of the filament diameter on its
mechanical strength which is difficult for experimentalists to optimize
TimeLine:
Year I : The fluoroleucine in gcn4-pl would be studied design sticky surfaces in various solvents and other
aminoacid substitutions in the heptad periodicity for the leucine zipper would be designed
Also approximate binding energies would be calcualted in propoer solvent for ligands of various t-RNA
synthetases. This would produce a palette of non-natural aminoacids for synthetic strategies.
13
Year II: The mechanical properties of bilayer nanotubes with PEG would be calculated as a function of the
PEG concentration. Tirrell would also perform the mechanical properties measurements on the bilayer
nanotubes.
14