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Osmolyte Effects on the Growth of Amyloid Fibrils Aswathy Narayanan Muttathukattil, and Govardhan Reddy J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b09215 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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The Journal of Physical Chemistry

Osmolyte Effects on the Growth of Amyloid Fibrils Aswathy N. Muttathukattil and Govardhan Reddy⇤ Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore, Karnataka, India 560012 E-mail: [email protected] Phone: +91-80-22933533. Fax: +91-80-23601310

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ACS Paragon Plus Environment


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Abstract Understanding the role of naturally occurring protective osmolytes such as Trimethylamine N-oxide (TMAO) on the growth of amyloid fibrils implicated in neurodegenerative diseases is important to prevent fibril growth. The effect of TMAO on the growth of amyloid fibrils formed by the Sup35 prion peptide NNQQNY is studied using molecular dynamics simulations. The free energy surface for the growth of the protofibril shows three major basins corresponding to the free state where the peptide is in solution, the docked state where the peptide in solution interacts with the surface of the protofibril, and the locked state where the peptide is tightly bound into the protofibril becoming a part of the fibril. The free energy surface in the presence of TMAO shows that TMAO stabilizes the locked state of the peptide compared to the free state, indicating that TMAO aids in fibril growth. On dissecting the interaction of TMAO with individual amino acids in the peptide shows that TMAO interacts both directly and indirectly with the amino acids depending on the nature of the side chains. The methyl groups in TMAO interact strongly with the hydrophobic aromatic ring in the Tyr residue. In the locked state, the surface area of Tyr available for interaction with TMAO decreases, as a result the Tyr residue in the peptide flips out from the locked position increasing the fluctuations of the peptide locked in the protofibril. Such strong direct interactions of amino acids with TMAO destabilize the folded or aggregated states in proteins. The overall increased stability of the peptide locked in the protofibril by TMAO is due to the entropic or indirect interactions with the backbone, Asn and Gln residues, which form a major component of the NNQQNY peptide.

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Introduction Proteins generally fold into distinct three dimensional structures to perform their functions. The folded structure of a protein is sensitive to the amino acid sequence, and the physical and chemical environment where the protein resides and functions. The physio-chemical factors 1–4 that can alter the structure of the protein include temperature, pressure, pH, ionic strength, and co-solvents. The deviation from optimum protein folding conditions can lead to misfolding and aggregation of proteins, which is implicated in a number of neurodegenerative diseases like Alzheimer’s and Parkinson’s. 5,6 These diseases are associated with the formation of fiber like structured aggregates known as amyloid fibrils. Various physio-chemical factors influence the structure of the protein, and hence will contribute to the thermodynamics and kinetics of protein aggregation. The effect of osmolytes on protein aggregation is important because these are generally small organic molecules and have the potential to act as drugs for diseases related to amyloid formation. Trimethylamine N-oxide (TMAO) is one of the naturally occurring osmolytes, which stabilizes folded states of proteins against denaturant stresses. The structure of the protein in the folded and aggregated state is different. 7 So the proteins have to at least partially unfold to form aggregates and it is important to understand the role of protective osmolytes such as TMAO in protein aggregation. Experiments have investigated the role of osmolytes on the aggregation of both folded and intrinsically disordered proteins. 8–21 The general conclusions from experiments are that the protective osmolytes inhibit or delay the aggregation of folded proteins, as they have to partially unfold to form aggregates, 8–13 whereas the aggregation rates of intrinsically disordered proteins are generally enhanced. 13–19 Amyloid fibrils rich in -sheet content are formed by proteins with diverse amino acid sequences and folded structures, 22–24 which implies that the mechanism of protein aggregation could be universal. 25 The mechanism of amyloid formation involves cascade of events, among them nucleation and growth are the two important steps. 26–33 A nucleus of the structured protein aggregate emerges from the oligomers formed by the association of aggregation 3



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prone protein conformations, 25,34 and this nucleus subsequently grows to form amyloid fibrils. Fibril growth occurs via two steps and is known as dock-lock mechanism. 35–40 In the docking step, the free monomer in solution interacts with the surface of the fibrils and docks onto the fibril if sufficient number of contacts are formed with the fibril. This is a reversible step, the docked monomer may detach from the fibril and go back into the solution or it may lead to the locking step, where the docked peptide undergoes structural rearrangement to get locked into the fibril to become part of the fibril. 36–40 The dock-lock process repeats resulting in the formation of elongated protein fibers known as protofibrils, which interact and intertwine with other protofibrils to form mature amyloid fibrils. 6 The dock-lock process is studied computationally using molecular dynamics simulations for various peptides using different levels of atomic resolution to understand the fibril elongation process. 39–45 The role of protective osmolytes on protein 46–51 and RNA folding 52 is well studied using computations, but their role in protein aggregation is relatively less studied 40,53–55 although experiments predict rich behavior depending on whether the protein is intrinsically disordered or folded. 8–21 In this work we study the effect of TMAO on the growth of an amyloid fibril formed by the NNQQNY peptide from the Sup35 Prion protein 22 using molecular dynamics simulations. We find that TMAO stabilizes the locked state of the peptide in the protofibril compared to the free monomer state. The dissection of the interaction of TMAO with individual amino acids present in the peptide reveal that TMAO can interact both directly and indirectly depending on the type of the amino acid. The side chain of the Tyr residue present in the peptide interacts strongly and directly with TMAO. This strong interaction increases the fluctuations of the locked peptide in the protofibril in the presence of TMAO. However, the enhanced stability of the locked peptide compared to the free monomer is due to the indirect interaction of TMAO with the Asn and Gln residues which make up a majority of the peptide. In the case of Asn and Gln residues, the number of hydrogen bonds the

C = O group in the side chain forms with water decreased due to the strong interaction

of TMAO with water. To compensate for this loss in the number of hydrogen bonds, the 4


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C = O group prefers to form hydrogen bonds with other peptides, increasing the stability of the locked state.

Methods To study the effect of the osmolyte, TMAO, on the growth of amyloid fibrils, a fibril formed by a six residue peptide, NNQQNY, from the Sup35 prion protein is chosen. From the crystal structure, 22,56 we created a protofibril made of 6 peptides (Figure 1A). The protein data bank (PDB) ID for the crystal structure is 1YJO. The six peptides in the protofibril are labeled from P1 to P6. A defect is created in the protofibril by moving the peptide P1 ⇡ 10 Å into the solution from the peptide P2 in the protofibril (Figure 1A). The figure is rendered using visual molecular dynamics program. 57 This system is solvated with TIP3P model water 58 and TMAO in a 70 Å cubic simulation box. This simulation set up is created to study the thermodynamics of the peptide P1 binding into the defect created in the protofibril in the presence of TMAO. Two systems with TMAO concentration, [TMAO] of 0 M and 4 M are studied. Molecular dynamics simulations are performed using NAMD 59 in the N P T ensemble. The temperature, T , and pressure, P , are maintained at 300 K and 1 atm, respectively using Nose-Hoover Langevin piston with a barostat oscillation time of 200 fs. 60,61 CHARMM22 force field 62 with the CMAP corrections 63 is used for the peptide. The force field parameters reported by Kast et al. are used for the TMAO molecule. 64 To check the dependence of results on TMAO model, we performed additional simulations with the Osmotic model 49 of TMAO and compared with the Kast model. van der Waals interactions are truncated at 12 Å with a force switch smoothing function from 10 Å, while long-range electrostatic interactions are calculated using particle-mesh Ewald method with a grid size of 1 Å and real space cut-off 12 Å . 65 Periodic boundary conditions are applied in all the directions and a time step of 2 fs is used to integrate the equations of motion. All covalent bonds involving hydrogen atom are

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kept rigid using the RATTLE algorithm. 66 The initial configurations are minimized using conjugate gradient algorithm. After minimization, the system is equilibrated for 5 ns by applying harmonic constraints to the heavy atoms of the peptide units. After equilibration, well-tempered metadynamics 67,68 simulations are performed to obtain the free energy of peptide P1 binding into the protofibril. The simulations are well-tempered using a bias temperature of 2000 K. Harmonic constraint of 10 kcal/mol/Å2 is applied on all the C↵ atoms in the peptides P2-P6 in order to keep the protofibril stable. Collective variables (CV) used to project the free energy of P1 binding in to the protofibril are center of mass distance, dcm , between the peptides P1 and P2, and root mean square deviation (RMSD) of the peptides P2 and P1 with respect to their structure in the protofibril without the defect (Figure 1A). All the atoms of peptides P1 and P2 are used to calculate RMSD. Gaussians of height 0.01 kcal/mol are added after every 1000 steps of the metadynamics simulation. Width of the Gaussians for different CVs are determined by calculating their fluctuations in a trial MD simulation of 5 ns. Width of the Gaussians for the CV RMSD is 0.25 Å, and for dcm is 2 Å in 0 M, and 1 Å in 4 M TMAO solution. In order to confine the space of exploration of the P1 peptide, artificial boundary potentials are applied on the CVs with a wall force constant of 10 kcal/mol/Å2 if RMSD

9 Å, and dcm

15 Å. The

convergence of the simulation is tested by monitoring the changes in free energy profiles for the last 100 ns of the simulation (Figure S1 in SI), and the free energy reported is computed using the NAMD 59 software package. To dissect the influence of TMAO on the free energy of dimer formation of the amino acid residues Asn, Gln and Tyr present in the peptide, we have performed adaptive biasing force 69,70 (ABF) simulations using NAMD 59 and CHARMM22 force field 62 with the CMAP corrections. 63 Systems consisting of dimers of acetamide, propanamide, p-cresol and hexaglycine are made, and solvated with TIP3P water and TMAO in a 40 Å cubic simulation box. Acetamide, propanamide, and p-cresol mimic the side chains of Asn, Gln and Tyr, respectively, and hexaglycine the backbone of the peptide. The initial positions for 6


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the dimers are obtained from the side-chains of the two peptides forming -strands in the protofibril. For each system, simulation boxes with [TMAO] of 0 M, 4 M and 6 M are made. The movement of each residue is restricted to only along fibril axis by applying harmonic constraint of 25 kcal/mol/Å2 to the coordinates of the other two axis for the heavy atoms in the residue. Center of mass distance between the two residues, dcm is used as the CV to project the free energy of dimer formation. The ABF calculation for each side chain is run for a total time of ⇡ 250 ns to compute the PMF. These free energy calculations are repeated with the Osmotic model 49 of TMAO to check for model dependence. Hydrogen Bond Definition: A hydrogen bond exists between two atoms if the distance between the donor atom (O or N) and the acceptor atom (O or N) is less than 3.5 Å, and the angle between the donor, hydrogen and acceptor atoms is greater than 135 degrees.

Results and Discussion The effect of TMAO on the growth of an amyloid fibril formed by the NNQQNY peptide from the Sup35 Prion protein is studied using molecular dynamics simulations. The free energy surface for the binding of a peptide in TMAO solution into a defect created in the protofibril leading to the growth of the fibril (Figure 1A) is estimated from metadynamics simulations. 68 Two systems with TMAO concentration, [TMAO] of 0 M and 4 M are studied. The simulations for each [TMAO] are run for a total time of ⇡ 1.0 µs (Figure S2). In the first set, the simulations are started with an initial conformation in which the peptide P1 is completely unbound from the protofibril (Figure 1A). These simulations are run for ⇡ 0.8 µs to get the free energy surface for peptide binding into the protofibril. During these simulations the P1 peptide successfully bound and unbound from the defect in the protofibril (Figure S2). To ensure proper sampling of the bound state, a second set of simulations are run for ⇡ 0.25 µs starting from an initial conformation in which the peptide P1 is bound in the protofibril and are continued until the peptide is completely unbound. The free energy 7



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surface reported (Figure 1B and 1C) is obtained at the end of the second set of simulations. The free energy surface projected on to CVs, RMSD and dcm , contains three major basins (Figure 1B and 1C). The first basin is the free peptide in solution (dcm > 10 Å), the second basin is the docked stage (dcm ⇡ 6.5 Å) where the binding peptide interacts with the protofibril, and the third stage is the locked state (dcm ⇡ 4.8 Å) where the peptide binds into the defect in the protofibril confirming the dock-lock mechanism proposed for the growth of amyloid fibrils. 36,38–40 The generic mechanism for the growth of amyloid fibrils by smaller peptides is well described by the reaction K DF

K LD

Free monomer state (F) ) * Docked state (D) ) * Locked state (L). The free energy difference between the locked and free state,

(1)

GLF (= GL GF ) computed

using the difference in the minima of the locked and monomer basins for [TMAO] = 0 M and 4 M are 1.3 kcal/mol and -4.1 kcal/mol, respectively. The

GLF decreased in the presence

of TMAO indicating that it stabilizes the locked state of the monomer compared to the free state. However, the free energy basin corresponding to the locked state of the monomer moved to higher RMSD values for [TMAO] = 4 M (Figure 1C). This is clearly observed when the free energy surface is projected onto a single CV RMSD (Figure 1D) by using the equation

G(T, P, RM SD) =

kB T ln

Z

exp[ G(T, P, RM SD, dcm )/kB T ] ddcm .

(2)

The free energy calculations support the generic dock-lock growth mechanism represented by Equation 1 and show that TMAO shifts the equilibrium of the reaction towards the lock state of the peptide by stabilizing the locked state compared to the free and dock states. The effect of cosolvents on the equilibrium constants of reactions involving macromolecules can be described by the Wyman-Tanford equation. 71–73 This equation relates the influence of cosolvent concentration on equilibrium constants of reactions to the preferential interaction 8


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of the cosolvent with the macromolecule. The rate of change of equilibrium constants K DF and K LD in Equation 1 with respect to the cosolvent, TMAO, activity a3 is given by

✓

✓

@ln K DF @ln a3 @ln K LD @ln a3

◆ ◆

=

D 23

F 23

=

DF 23

(3)

=

L 23

D 23

=

LD 23 .

(4)

T,P

T,P

In the above equation, subscripts 1, 2 and 3 in the equation stand for water, peptide and TMAO, respectively.

L 23 ,

D 23 ,

and

F 23

are the coefficients of preferential interaction of

TMAO with the fibril in solution and the peptide in the locked, docked and free states, respectively. The preferential interaction coefficient,

23 (r)

=

*

nlocal (r) 3

23 ,

nlocal (r) 1

is given by equation, 74–76

⇣ nbulk ⌘ 3

nbulk 1

+

(5)

,

here n denotes the number of molecules, and r denotes the radius of the local domain of the protein separating it from the bulk. The quantity inside the angular brackets h i is time averaged. To compute nlocal (r) we used the definition that a TMAO molecule is in 3 the local domain of the peptide if the nitrogen atom of TMAO is within a distance r from any atom of the peptide.

23 (r)

is calculated for the free, docked and locked states from

metadynamics simulation trajectories (Figure S2). Negative values of

23

implies preferential

exclusion of TMAO relative to water from the vicinity of the fibril and peptide system. 77 Simulation studies 78 on the effect of TMAO on hydrophobic polymer collapse suggested that protective osmolytes are not necessarily excluded from the surface of the protein, which means

23

can be positive. For the osmolyte to be protective, what is more important is the

relative depletion of the osmolyte from the vicinity of the polymer or protein as it changes its conformation, i.e.

23

should be positive as the polymer collapses from a coil like extended

state to a globule.

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The large r,

F 23 , L 23

>

D 23 , D 23

and >

L 23 F 23

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plotted as a function of the local domain radius, r, shows that at

(Figure 2). As a result, @ ln K DF /@ln a3

T,P

and @ ln K LD /@ln a3

T,P

in Equation 4 are positive indicating that TMAO shifts the equilibrium in the amyloid growth reaction (Equation 1) towards the right leading to enhanced growth of the fibril. The order of the values of the preferential interaction coefficients,

L 23

>

D 23

>

F 23 ,

shows that TMAO

molecules are preferentially excluded more in the free state compared to the locked state. This suggests that the relative differences in the preferential exclusion of TMAO from the vicinity of the fibril in solution and the peptide shifts the equilibrium from the free state to the docked state, which is further shifted to the locked state leading to the enhanced stability and growth of the fibrils in the presence of TMAO. The shift in the free energy basin corresponding to the locked state to higher RMSD values in the presence of [TMAO] is due to the increase in the fluctuations of the Tyr sidechain of the locked P1 peptide in the presence of TMAO (Figure 3A). The Tyr RMSD compared to the other residues in the P1 peptide is also higher, and this is not because Tyr is a terminal residue since the RMSD of the N-terminal Asn is smaller compared to Tyr (Figure 3B). The increased fluctuations of the Tyr residue in the presence of TMAO is due to the strong direct interaction of TMAO with Tyr. The radial distribution function, g(r), between the acidic hydrogen (H⇣ ) in Tyr with the negatively charged oxygen (OT ) in TMAO (Figure 4A), and g(r) between the hydrophobic aromatic carbons (C , C , C✏ , C⇣ ) in Tyr with the methyl carbons (CT ) in TMAO (Figure 4B) shows the preferential interaction of TMAO with Tyr. The spatial density maps of water and TMAO within 5 Å of the peptide shows that the residues Asn and Gln predominantly interact directly with water, whereas Tyr interacts directly with TMAO (Figure 4C). The spatial maps are computed using volmap plugin in Visual Molecular Dynamics package. 57 The space around the peptide is divided into a grid of size of 0.1 Å3 . The grid points take values 0 or 1 depending on whether it contains an atom. Each atom of water or TMAO is treated as a sphere and a grid point is occupied if it lies inside a sphere representing an atom. The value at a grid point is averaged 10


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over all the simulation frames to compute the fractional occupancy of TMAO or water at a site. In the spatial density maps the fractional occupation of TMAO near the side chain vicinities of the residues Asn and Gln in the NNQQNY peptide is / 0.3 whereas for water it is / 0.8 showing that water prefers to solvate these residues. Whereas for the Tyr side chain (near the aromatic ring) the fractional occupancy of TMAO is / 0.56 and water ⇡ 0 demonstrating TMAO relatively prefers to solvate Tyr side chain. The spatial density map of TMAO within 5 Å of a neutral non-Zwitterionic (neutral) form of TMAO clearly shows that CT of TMAO interacts with the plane of the aromatic ring in Tyr, and OT in TMAO interacts with the acidic protons in Tyr (Figure 4D). In addition to the Kast TMAO model 64 there exits other TMAO models 49,79,80 in literature. We also computed the g(r) between Tyr and the Osmotic model of TMAO 49 parameterized to reproduce the osmotic pressure in TMAO-water mixtures accurately. Even the osmotic TMAO model shows a strong direct interaction between TMAO and Tyr (Figure S3). The osmotic TMAO model exhibits a much stronger interaction between H⇣ in Tyr with the negatively charged OT in TMAO than the Kast model, as the charges in the osmotic model are scaled by a factor of 1.2 compared to Kast model. 49 However, the Kast model exhibits relatively stronger interaction than the Osmotic model between the methyl carbon CT of TMAO and the hydrophobic aromatic ring in Tyr. The spatial density maps of water and Osmotic model of TMAO within 5 Å of the NNQQNY peptide show that the residues Asn and Gln predominantly interact directly with water, whereas Tyr interacts directly with TMAO in agreement with the Kast model of TMAO (Figure S3C and S3D). To further understand the origin of stability of the locked peptide in TMAO solution at the individual residue level we studied the interactions of residues present in the NNQQNY peptide with TMAO. We performed a different set of simulations to dissect the role of TMAO’s interaction with the amino acids Asn, Gln and Tyr. In these simulations we computed the free energy of dimer formation of the side chains of amino acids Asn, Gln and Tyr in the presence of TMAO using adaptive biasing force (ABF) 69 simulation technique. The 11



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molecules acetamide, propanamide and p-cresol are the side chains of the amino acids Asn, Gln and Tyr, respectively, and hexaglycine (Gly6) is the backbone of the peptide NNQQNY. Formation of dimers by these molecules in presence of TMAO can reveal how TMAO modulates protein-protein interactions responsible for the growth of amyloid fibrils. The potential of mean force (PMF) of dimer formation is calculated as a function of the center of mass distance between the monomer molecules, dcm . The PMF obtained at the end of 250 ns of ABF simulation is reported (Figure 5). The error bars (standard deviation) are smaller than the symbols and are computed using the PMF data from 150 ns to 250 ns by accumulating the data every 10 ns. The minima in the PMF corresponds to the dimer, and distance between monomers in the dimer is ⇡ 4.8 Å. In the cases of acetamide (Asn side chain), propanamide (Gln side chain) and Gly6 the free energy of dimer formation decreased in the presence of [TMAO] indicating that TMAO promotes dimer formation in these amino acids, and hence aggregation of peptides consisting of these residues (Figure 5A, 5B and 5C). In the case of p-cresol (Tyr side-chain) the free energy of dimer formation increased with [TMAO], which means that TMAO inhibits the dimer formation in Tyr residues (Figure 5D). This is consistent with our earlier observation that TMAO in solution interacts strongly with the Tyr residue in the P1 peptide locked in the protofibril, and this strong interaction leads to flipping out of the Tyr residue from the locked position resulting in higher RMSD (Figure 3 and 4). This also contributes to the shifting of the locked state basin of the peptide in the free energy surface to higher RMSD values on addition of TMAO (Figure 1C). The destabilization of the p-cresol dimer by TMAO due to direct interaction is also consistent with the recent finding 81 that TMAO in concentrations greater than 1 M destabilizes compact globular states of hydrophobic polymers. Since TMAO overall stabilizes the locked state of the NNQQNY peptide in the fibril (Figure 1B and 1C), this implies that the decrease in

GLF is due to the 3 Asn and 2

Gln residues where dimerization is favored in the presence of TMAO compensating for the loss in stability due to the strong direct interaction of Tyr with TMAO (Figure 4). We also 12


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computed the PMFs for the dimer formation using the osmotic model 49 of TMAO to check for the model dependence (Figure S4). We do observe some differences in the PMF profiles as expected with different models. 82 No significant stabilization of the dimer is found for the Asn side chain and Gly6 compared to the monomers (Figure S4A and S4C). The dimer of Gln side chain is stabilized and the Tyr side chain is destabilized (Figure S4B and S4D) in agreement with the Kast model data (Figure 5B and 5D). The interaction of TMAO with the amino acid residues may be direct or indirect, which leads to the changes in their dimerization free energy 48,49,77 (Figure 5 and S4). TMAO is found to decrease the free energy of dimer formation in the case of acetamide, propanamide and Gly. The indirect interactions hypothesized to play a critical role in protein stability by TMAO are excluded volume effects 48,83–85 and changes in water structure. 46,86–99 The relative interaction of the peptide with TMAO compared to water is quantified using the preferential interaction coefficient,

23 ,

given by the Equation 5 and the partition coefficient, 100 Kp

defined as Kp (r) = nlocal (r)/nlocal (r) ⇤ nbulk /nbulk . 3 1 1 3 For the neutral Tyr residue, both

23

and Kp indicate that TMAO interacts directly

with the residue (Figure 6) in agreement with the spatial density map (Figure 4). The

23

for Tyr is positive at large r indicating that TMAO preferentially solvates Tyr over water (Figure 6A). Even Kp value in the local domain at r ⇡ 4

5 Å is greater than 1 showing

that the number of TMAO molecules are in excess compared to the bulk indicating a strong direct interaction. This strong interaction of TMAO with the Tyr naturally destabilizes the dimer state of Tyr compared to the monomer states as observed in the RMSD and PMF calculations (Figure 3 and 5D). For the neutral Asn residue, both indirect interaction of TMAO. The

23

23

and Kp indicate

value at large r is negative, and the Kp value is

always less than 1, indicating water preferentially solvates this residue over TMAO. So the relatively small stabilization of the dimers of the side chains of Asn residue (Figure 5A) could be due to the indirect interaction of TMAO with this residue. We have also computed

23

values for the neutral Asn, Gln and Tyr residues using the Kirkwood-Buff integral analysis 101 13



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(Figure S7 and S8) as computed by Ganguly et al. 102 and the signs of the

23

values are

consistent (see SI for more information). In the case of neutral Gln residue, the range r ⇡ 4

23

value is positive and even Kp values in the

5 Å is slightly greater than 1, showing that TMAO directly interacts with

the Gln residue slightly stronger than water. However, the PMF data shows that TMAO promotes dimer formation among the Gln side chains (Figure 5B) unlike neutral Tyr residue (Figure 5D). In the case of the side chain of Asn, which is the lower homologue of Gln, it shows preferential exclusion of TMAO (Figure 6). The side chain amide nitrogen present in both Asn and Gln interacts equally strongly with TMAO oxygen (Figure S5A). So, the additional hydrophobic methylene group present in Gln must be responsible for the preferential interaction with TMAO. TMAO has an hydrophobic umbrella due to the three methyl groups that can interact with the hydrophobic patches of amino acids. Compared to Gln, Asn has a shorter hydrophobic patch so its interaction with TMAO is less favorable compared to Gln. The radial distribution function, g(r), also shows that C of Gln prefers to interact with TMAO while Asn equally prefers to interact with both TMAO and water (Figure S5B and S5C). This leads to changes in the sign of

23

for Asn and Gln. When the

dimer of Gln forms, there is no loss in the number of TMAO molecules interacting with Gln (Figure S6A). As a result the direct interaction of TMAO with Gln does not destabilize the dimer formation. But in the case of Tyr the direct interaction of TMAO destabilizes the dimer formation as there is exclusion of TMAO molecules from the plane of the aromatic ring (Figure S6B). The Gln dimer in the presence of TMAO should probably be stabilized by the strong interaction of TMAO with water, which in turn controls the hydrogen bonding within the protein or the peptide promoting compact structures or aggregation. In addition to the mechanism 48,49,88 of preferential exclusion of TMAO from the protein surface in stabilizing the folded states of proteins, the other mechanism hypothesized to play a role in stabilizing compact structures of proteins is the strong interaction of TMAO with water. A recent study 103 shows that subtle changes in the osmolyte-water interactions can 14


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either enhance or prevent hydrophobic association of molecules. The strong TMAO-water interaction can reduce the hydrogen-bond forming ability of water with the amide oxygen in the protein weakening the water-protein hydrogen bonds. As a result the unfolded state of the protein becomes less stable and also the intramolecular hydrogen bonds in the protein become stronger thereby stabilizing the folded state of the protein. 85,92,94,98 To test this hypothesis we computed the total number of hydrogen bonds acetamide, propanamide, Gly6 and p-cresol form with water and TMAO in various [TMAO] (Table S2 in SI). In the case of acetamide, propanamide, and Gly6, where TMAO promotes dimer formation, the total number of hydrogen bonds formed by these molecules decreased with the increase in [TMAO] (Table S2). Water can form hydrogen bonds with both the

C = O and

N H2 groups present in the amide functional group, whereas TMAO can form hydrogen bonds only with the the

N H2 group. The number of hydrogen bonds formed by water with

N H2 group in acetamide, propanamide, and

NH

group in Gly6 decreased with

the increase in [TMAO], but this decrease is compensated by the increase in the number of hydrogen bonds formed with TMAO (Table S2). However the number of hydrogen bonds formed by water with the

C = O group decreased with the increase in [TMAO], and this

decrease in the number of bonds cannot be compensated by TMAO. The overall decrease in the number of hydrogen bonds is due to the decrease in the number of bonds formed between water and the

C = O group (Table S2). To compensate for this loss, the protein

backbone and amino acids with the amide functional group in their side chains prefer to form intramolecular hydrogen bonds leading to the stability of protein folded or aggregated states. In the case of p-cresol, where TMAO destabilizes the dimer formation by direct interaction with the

OH group, the number of hydrogen bonds between the

OH group in p-cresol

and water also decreased with the increase in [TMAO] (Table S2). The hydrogen bonds formed by TMAO with the

OH group in p-cresol do not completely compensate for the

loss in hydrogen bonds (Table S2). The destabilization of Tyr dimers could be due to the strong interaction of TMAO with the side chain of Tyr (Figure 4). 15



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Page 16 of 38

To dissect the role of electrostatic and van der Waals interactions in determining the presence of TMAO or water in the vicinity of NNQQNY peptide we have computed the interaction energies of both TMAO and water in the first solvation shell (FSS) of the amino acid side chains present in the peptide and compared them to their bulk values. The distribution of the binding energies of TMAO and water in the FSS of the side chains of the peptide and in the bulk are shown in the Figure 7. The region within 3.5 Å of any atom of the side chain of a residue is defined as the FSS. The binding energies show that for both TMAO and water it is the van der Waals energies that are more favorable in the FSS for all the side chains compared to the bulk. To compare objectively whether the side chains prefer TMAO or water in the FSS based on the energy analysis we note that each TMAO in FSS can replace 4-5 water molecules as the radius of TMAO is approximately twice as that of water. The water accessible surface area of TMAO is equal to the surface area of a sphere 104 of radius 5.4 Å and the radius of water is 2.8 Å which is approximately the position of the first peak in the oxygen-oxygen radial distribution function of water. 105 The volume of TMAO is ⇡ 8 times of that of water and we assume that each TMAO replaces 4 water molecules from the FSS. With this assumption, from the binding energies of TMAO and water in the FSS of Tyr (panel-F in Figure 7), we show that it is energetically preferable for TMAO to be present in the FSS of Tyr ( E =

0.43

(4 ⇤

0.05) =

0.43 + 0.2 =

0.23

kcal/mol), and this is consistent with the spatial density maps and the TMAO preferential interaction coefficient (Figure 4 and 6). The binding energy data of TMAO and water in the vicinity of Asn1, Asn2 and Asn5 (panel-A, B and E in Figure 7), show that it is energetically favorable for water to be in the FSS compared to TMAO in agreement with the

23

and Kp

data (Figure 6). The binding energy data for Gln3 and Gln4 (panel-C and D in Figure 7) shows that it is energetically preferable to place TMAO in FSS of compared to water. For example, TMAO in the vicinity of Gln4 is stabilized by -0.19 kcal/mol compared to water, which is in agreement with

23

data for the Gln residue (Figure 6). The energy analysis is

also consistent with the picture presented using the spatial density maps and preferential 16


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interaction coefficients.

Conclusions In summary, we have studied the effect of TMAO on the growth of an amyloid fibril formed by the peptide NNQQNY from the Sup35 Prion protein using molecular dynamics simulations. We find that TMAO stabilizes the locked state of the peptide in the protofibril compared to the free monomer state, where the peptide is in solution. In the presence of TMAO, the basin corresponding to the locked state in the free energy surface projected onto the collective variables RMSD and dcm is stabilized compared to the free state (Figure 1B and 1C). The locked state basin in the free energy surface moved to higher RMSD values due to the direct strong interaction of TMAO with the plane of the aromatic hydrocarbon ring present in the side chain of Tyr residue. In the locked state, the surface of the aromatic ring available to interact with TMAO decreases. As a result, the Tyr residue in the peptide flips out from it locked position to interact with TMAO and this results in higher RMSD for the locked peptide on addition of TMAO. Analysis of the interactions of TMAO with the residue Asn reveals that TMAO has indirect interactions with Asn and it is preferentially excluded from the protein surface compared to water (Figure 4 and 6). The exclusion of TMAO from the Asn residue surface is minimized in the aggregated state. As a result TMAO stabilizes the dimerization of Asn residues (Figure 5A), which promotes the stabilization of the peptide in the locked state. The Gln residue, which is a higher homologue of Asn with an additional -CH2 group interacts favorably with TMAO. However, the dimer of Gln is not destabilized unlike the dimer of the residue Tyr, as the number of TMAO molecules interacting with Gln do not decrease upon the dimer formation (Figure S6). The increased stability of Gln dimers in the presence of TMAO might be due to the strong interaction of TMAO with water. The stabilization of compact conformations (folded or aggregated) by TMAO in peptide chains

17



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primarily composed of amino acids with amide functional group in the side chain (Asn and Gln) can also arise due to the reduced number of hydrogen bonds between the -C=O group in the amide and water. The premise is that TMAO interacts strongly with water, as a result the number of hydrogen bonds between water and the -C=O group decreases, and the side chains prefer to form intra molecular hydrogen bonds or hydrogen bonds with other peptide groups resulting in the stabilization of compact or aggregated protein conformations.

Acknowledgement G.R. acknowledges start up grant from Indian Institute of Science (IISc), Bangalore, and funding from Nano mission, Department of Science and Technology, India. Aswathy acknowledges research fellowship from Council of Scientific and Industrial Research, India. The computations are performed using the Cray XC40 cluster at IISc, PARAM Yuva-II and BRAF resources at C-DAC.

Supporting Information Available Figures S1-S8; Tables S1-S2. This material is available free of charge via the Internet at http://pubs.acs.org/.

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(77) Canchi, D. R.; Garcia, A. E. Cosolvent Effects on Protein Stability. Annu. Rev. Phys. Chem. 2013, 64, 273–293. (78) Mondal, J.; Stirnemann, G.; Berne, B. J. When Does Trimethylamine N-Oxide Fold a Polymer Chain and Urea Unfold It? J. Phys. Chem. B 2013, 117, 8723–8732. (79) Schneck, E.; Horinek, D.; Netz, R. R. Insight into the Molecular Mechanisms of Protein Stabilizing Osmolytes from Global Force-Field Variations. J. Phys. Chem. B 2013, 117, 8310–8321. (80) Larini, L.; Shea, J.-E. Double Resolution Model for Studying TMAO/Water Effective Interactions. J. Phys. Chem. B 2013, 117, 13268–13277. (81) RodriÌĄguez-Ropero, F.; RoÌĹtzscher, P.; van der Vegt, N. F. Comparison of Different TMAO Force Fields and Their Impact on the Folding Equilibrium of a Hydrophobic Polymer. J. Phys. Chem. B 2016, 120, 8757–8767. (82) Chand, A.; Chettiyankandy, P.; Pattanayak, S. K.; Chowdhuri, S. Effects of Trimethylamine-N-oxide (TMAO) on Aqueous N-methylacetamide Solution: A Comparison of Different Force Fields of TMAO. J. Mol. Liq. 2016, (83) Hilaire, M. R.; Abaskharon, R. M.; Gai, F. Biomolecular Crowding Arising from Small Molecules, Molecular Constraints, Surface Packing, and Nano-Confinement. J. Phys. Chem. Lett. 2015, 6, 2546–2553. (84) Sagle, L. B.; Cimatu, K.; Litosh, V. A.; Liu, Y.; Flores, S. C.; Chen, X.; Yu, B.; Cremer, P. S. Methyl Groups of Trimethylamine N-Oxide Orient Away from Hydrophobic Interfaces. J. Am. Chem. Soc. 2011, 133, 18707–18712. (85) Ma, J.; Pazos, I. M.; Gai, F. Microscopic Insights Into the Protein-Stabilizing Effect Of Trimethylamine N-Oxide (TMAO). Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 8476–8481. 27



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(86) Bruzdziak, P.; Panuszko, A.; Stangret, J. Influence of Osmolytes on Protein and Water Structure: A Step To Understanding the Mechanism of Protein Stabilization. J. Phys. Chem. B 2013, 117, 11502–11508. (87) Freda, M.; Onori, G.; Santucci, A. Hydrophobic Hydration and Hydrophobic Interaction in Aqueous Solutions of Tert-Butyl Alcohol and Trimethylamine-N-Oxide: A Correlation with The Effect of These Two Solutes On the Micellization Process. Phys. Chem. Chem. Phys. 2002, 4, 4979–4984. (88) Hu, C. Y.; Lynch, G. C.; Kokubo, H.; Pettitt, B. M. Trimethylamine N-Oxide Influence On the Backbone of Proteins: An Oligoglycine Model. Proteins 2010, 78, 695–704. (89) Hunger, J.; Tielrooij, K.-J.; Buchner, R.; Bonn, M.; Bakker, H. J. Complex Formation in Aqueous Trimethylamine-N-oxide (TMAO) Solutions. J. Phys. Chem. B 2012, 116, 4783–4795. (90) Meersman, F.; Bowron, D.; Soper, A. K.; Koch, M. H. J. Counteraction of Urea by Trimethylamine N-Oxide Is Due to Direct Interaction. Biophys. J. 2009, 97, 2559– 2566. (91) Panuszko, A.; Bruzdziak, P.; Zielkiewicz, J.; Wyrzykowski, D.; Stangret, J. Effects of Urea and Trimethylamine-N-oxide on the Properties of Water and the Secondary Structure of Hen Egg White Lysozyme. J. Phys. Chem. B 2009, 113, 14797–14809. (92) Pazos, I. M.; Gai, F. Solute’s Perspective on How Trimethylamine Oxide, Urea, and Guanidine Hydrochloride Affect Water’s Hydrogen Bonding Ability. J. Phys. Chem. B 2012, 116, 12473–12478. (93) Stanley, C.; Rau, D. C. Assessing the Interaction of Urea and Protein-Stabilizing Osmolytes with the Nonpolar Surface of Hydroxypropylcellulose. Biochemistry 2008, 47, 6711–6718.

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(94) Reddy, P. M.; Taha, M.; Venkatesu, P.; Kumar, A.; Lee, M.-J. Destruction of Hydrogen Bonds of Poly(N-Isopropylacrylamide) Aqueous Solution by Trimethylamine N-Oxide. J. Chem. Phys. 2012, 136, 234904. (95) Sarma, R.; Paul, S. Exploring the Molecular Mechanism of Trimethylamine-N-oxide’s Ability to Counteract the Protein Denaturing Effects of Urea. J. Phys. Chem. B 2013, 117, 5691–5704. (96) Sharp, K. A.; Madan, B.; Manas, E.; Vanderkooi, J. M. Water Structure Changes Induced by Hydrophobic and Polar Solutes Revealed by Simulations and Infrared Spectroscopy. J. Chem. Phys. 2001, 114, 1791–1796. (97) Usui, K.; Hunger, J.; Sulpizi, M.; Ohto, T.; Bonn, M.; Nagata, Y. Ab Initio Liquid Water Dynamics in Aqueous TMAO Solution. J. Phys. Chem. B 2015, 119, 10597– 10606. (98) Wei, H.; Fan, Y.; Gao, Y. Q. Effects of Urea, Tetramethyl Urea, and Trimethylamine N-Oxide on Aqueous Solution Structure and Solvation of Protein Backbones: A Molecular Dynamics Simulation Study. J. Phys. Chem. B 2010, 114, 557–568. (99) Zou, Q.; Bennion, B. J.; Daggett, V.; Murphy, K. P. The Molecular Mechanism of Stabilization of Proteins by TMAO and Its Ability to Counteract the Effects of Urea. J. Am. Chem. Soc. 2002, 124, 1192–1202. (100) Courtenay, E.; Capp, M.; Anderson, C.; Record, M. Vapor Pressure Osmometry Studies of Osmolyte-Protein Interactions: Implications for the Action of Osmoprotectants In Vivo and for the Interpretation of “Osmotic Stress” Experiments in Vitro. Biochemistry 2000, 39, 4455–4471. (101) Kirkwood, J.; Buff, F. The Statistical Mechanical Theory of Solutions.1. J. Chem. Phys. 1951, 19, 774–777.

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(102) Ganguly, P.; Hajari, T.; Shea, J.-E.; van der Vegt, N. F. A. Mutual Exclusion of Urea and Trimethylamine N-Oxide from Amino Acids in Mixed Solvent Environment. J. Phys. Chem. Lett. 2015, 6, 581–585. (103) Ganguly, P.; van der Vegt, N. F.; Shea, J.-E. Hydrophobic Association in Mixed Urea– TMAO Solutions. J. Phys. Chem. Lett. 2016, 7, 3052–3059. (104) Street, T. O.; Bolen, D. W.; Rose, G. D. A Molecular Mechanism for Osmolyte-induced Protein Stability. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 13997–14002. (105) Sorenson, J. M.; Hura, G.; Glaeser, R. M.; Head-Gordon, T. What Can X-ray Scattering Tell Us about the Radial Distribution Functions of Water? J. Chem. Phys. 2000, 113, 9149–9161.

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(A)

+

P4

P3

(B)

P1

Y

X

P5 P6

P2 P3

(C) F

1.4 0M

P4

P6

P2 P1

[TMAO]

P5

Z

F

1.4 4M 15 1.2

1 0.8

5

D

dcm (nm)

10

10

1 5

0.8 0.6

0.6 L

(D)

0.2

0.4

0.6

RMSD (nm)

0

0.8

8

(E) 0M 4M

6 4 2 0

L

D

0.2

0.4

F 0.6

RMSD (nm)

0.8

G(dcm) (kcal/mol)

dcm (nm)

1.2

G(RMSD) (kcal/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


L

0.2

D

0.4

0.6

RMSD (nm)

0.8

0

4 3 2 1 0M 4M

0

0.6 0.8 1.0 1.2 1.4 dcm (nm)

Figure 1: (A) Simulation setup for the NNQQNY peptide protofibril growth. A protofibril made of 6 peptides, labeled P1-P6, is constructed using the crystal structure (PDB ID: 1YJO). The protofibril with axis along the z-axis consists of 2 sets of -strands shown in red and green (figure on right). To study the fibril growth, an initial conformation, where the peptide labeled P1 is moved away from the protofibril into the solution creating a defect in the crystal, which is shown as a step using dashed lines (figure on left). In this state, dcm between the peptides P1 and P2 is ⇡ 10.5 Å. Metadynamics simulations are used to compute the free energy of binding of the peptide P1 into the defect created in the protofibril. The CVs used to project the free energy of binding are dcm and RMSD with reference to the structure where the peptide P1 is bound in the protofibril (figure on right). Free energy surface for a NNQQNY monomer in solution interacting with a protofibril (B) [TMAO] = 0 M, and (C) [TMAO] = 4 M. Free energy projected onto a single collective variable (D) RMSD and (E) dcm . The three energy basins related to the free monomer state [F], docked state [D] and locked state [L] are shown. In panels (D) and (E) the free energy profiles are shifted so that the locked state basins are aligned. As [TMAO] increases from 0 M to 4 M, the locked state of the monomer becomes globally stable. The energy basin related to the locked state shifts to higher RMSD values because of the strong interaction of the Tyr residue in the monomer with TMAO. RMSD as a CV can resolve the three states in the fibril growth as shown in (D) for [TMAO] = 0 M. 31 ACS Paragon Plus Environment


0

Γ23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 38

F

-2

Γ23

-4

Γ23

-6

Γ23

D L

-8 -10 -12 -14 1

2

3

4

5

6

7

8

r (Å) Figure 2: TMAO preferential interaction coefficient, 23 , for the peptide and the fibril system in the free (F), docked (D) and locked (L) states as a function of the local domain radius, r, for [TMAO]=4 M. TMAO is relatively less excluded from the vicinity of the peptides in the locked state compared to the docked and free states making it a more stable state in the presence of TMAO.

32


Page 33 of 38

(A) 3.5

RMSD (Å)

3.0 2.5

[TMAO] = 0 M [TMAO] = 4 M

2.0 1.5 1.0 0.5 0

2

(B)

4

6

time (ns)

8

10

8

10

3.0

RMSD (Å)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[TMAO] = 4 M 2.0 1.0 0.0 0

2

4

6

time (ns)

Figure 3: (A) RMSD as a function of time for the Tyr residue in the P1 peptide locked in the protofibril for different [TMAO]. RMSD increases with the increase in [TMAO] indicating a strong interaction between Tyr and TMAO. (B) RMSD as a function of time for each individual residue present in the locked P1 peptide in [TMAO] = 4 M solution. RMSD of Asn1, Asn2, Gln3, Gln4, Asn5 and Tyr6 are shown in cyan, green, red, blue, purple and yellow colored lines, respectively. RMSD of Tyr is higher compared to other residues.

33



(B) 1.4

(A) OT Hζ

1.2

-

1.0

2

g(r)

g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

0.8 0.6

CT - Cγ

0.4

CT - Cδ1

CT - Cζ CT - Cδ2

0.2

CT - Cε1

CT - Cε2

0.0

0 2

(C)

Page 34 of 38

4

6

2

8

r (Å)

3

(D)

4

5

6

7

8

9

r (Å)

Figure 4: Tyr shows strong interaction with TMAO, which is responsible for the increased RMSD of the locked peptide P1 in the protofibril. The data in this figure is obtained for [TMAO] = 4M. (A) The radial distribution function, g(r), between TMAO oxygen (OT ) and Tyr acidic hydrogen (H⇣ ). (B) The g(r) between TMAO methyl carbon atoms (CT ) and the various aromatic carbon atoms present in the Tyr side chain. (C) The spatial density map of water (blue color) and TMAO (orange color) within 5 Å of the NNQQNNY peptide. The figure is rendered for the condition that the fractional occupancy for water or TMAO at a position should be greater than 0.3. The map shows that the aromatic ring in Tyr residue directly interacts with TMAO, whereas the Asn and Gln residues interact directly with water relatively more than TMAO. (D) The spatial density map of TMAO within 5 Å of the neutral Tyr. The hydrophobic aromatic carbons present in the Tyr interact directly with the methyl carbons (green color) present in TMAO. The fractional occupancy of TMAO above the Tyr aromatic ring is much greater than water. The acidic hydrogens present in the side chain and in the peptide group can interact with TMAO oxygen (red color). The fractional occupancy of water near the acidic hydrogens is much greater than TMAO. Water is not shown in the figure to emphasize that TMAO also can interact with the acidic hydrogens although water has a predominant occupancy there. 34


Page 35 of 38

(A)

(B)

1.0

Asn

0.5

PMF (kcal/mol)

PMF (kcal/mol)

1.0 0.0

-0.5

6

(C)

2

8

dcm (Å)

10

12

PMF (kcal/mol)

0 -2

6

8

dcm (Å)

10

6

1.0

8

dcm (Å)

12

0.0 [TMAO] = 0M [TMAO] = 4M [TMAO] = 6M

-2.0

12

10

Tyr

-1.0

[TMAO] = 0M [TMAO] = 4M [TMAO] = 6M

4


4 (D)

Gly6

-4

0.0

-2.0

-1.5 4

Gln

-1.0


-1.0

PMF (kcal/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4

6

8

dcm (Å)

10

12

Figure 5: The free energy of dimer formation as a function of the distance between the monomers along the fibril axis. The decrease in free energy with the increase in [TMAO] for acetamide (Asn side chain), propanamide (Gln side chain) and Gly6 at r ⇡ 5 Å indicates stabilization of dimers by TMAO. In the case of p-cresol (Tyr side chain) the free energy increased with [TMAO] indicating that Tyr dimer is destabilized by TMAO. The error bars (standard deviation) are smaller than the symbols and are computed using the PMF data from 150 ns to 250 ns by accumulating the data every 10 ns.

35



1

(A)

Γ23

0 Asn Gln Tyr

-1 1

2

3

4

5

r (Å)

6

7

8

(B) 1.0

Kp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 38

0.5

Asn Gln Tyr

0.0 1

2

3

4

5

r (Å)

6

7

8

Figure 6: (A) The preferential interaction of TMAO, 23 , with neutral amino acids as a function of the local domain radius, r, around the amino acid. (B) The partition coefficient, Kp , of neutral amino acids plotted as a function of r.

36


Water

TMAO 6 4 2 0

6 4 2 0 -40

-30

-20

Gln3 -3

2 0

10x10

4 2 0

-3

Gln4

TMAO in bulk TMAO in FSS

2 0

10x10

4 2 0

-3

8

Asn5


10x10 Probability density

Probability density

-3

6 4 2 0

2 0

-3

Tyr6


2 0

-3

6 4 2 0 -50 -40 -30 -20 -10 Electrostatic energy (kcal/mol)

10x10

6 4 2 0

-3


4 2 0 -4

-20

10x10

-3

Gln3

6 4 2 0

-10

Water in bulk Water in FSS

-4

6 4 2 0

10x10

-3

Gln4


8

-3

6 4 2 0

-3

Asn5


8 6 4 2 0 -40

-30

-20

6 4 2 0

8 6 4 2 0 -10 -5 0 5 VDW energy (kcal/mol)

-3

8 6 4 2 0 -4

10x10

-3

Tyr6



6 4 2 0 -4

10x10

6 4 2 0 -40 -30 -20 -10 Electrostatic energy (kcal/mol)

0 4 8 12 VDW energy (kcal/mol)

8

-10

8

0 4 8 12 VDW energy (kcal/mol) Water in bulk Water in FSS

-3

Electrostatic energy (kcal/mol) 10x10


8

-4

10x10


8

-40 -30 -20 -10 Electrostatic energy (kcal/mol)

8

10x10

-30

8

-10 -5 0 5 VDW energy (kcal/mol)

Probability density

(F)

4




6

10x10

4

-3

6


6

-50 -40 -30 -20 -10 Electrostatic energy (kcal/mol) 8x10


Asn2

8


6

(E)

10x10

4

-50 -40 -30 -20 -10 Electrostatic energy (kcal/mol)

8x10


8

Electrostatic energy (kcal/mol)

6

Probability density

Probability density

-3

-3

-40

8


(D)

0


6

8x10

10x10

4

-10



6

Probability density

Probability density

8x10

2


-3


8

Electrostatic energy (kcal/mol)

(C)

4

Probability density

-50

-3

10x10

Probability density

Probability density

10x10

6

5

Probability density


Asn2


Probability density

-10 -5 0 VDW energy (kcal/mol)


Asn1

8

Probability density

8

-3

Probability density

0

10x10

Probability density

2


Probability density

4

-3

Probability density

10x10

6

(B) -3 8x10 Probability density


Probability density

Asn1

-3

Probability density

Probability density

8x10

Probability density

(A)

Probability density

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Probability density

Page 37 of 38

-3


8 6 4 2 0 -4

0 4 8 12 VDW energy (kcal/mol)

Figure 7: Binding energies of TMAO and water within the bulk and the first solvation shell (FSS) regions of the side chains of Asn1, Asn2, Gln3, Gln4, Asn5 and Tyr6. The total binding of energy of TMAO or water is E tot = E vdw + E el , where E vdw ( E el ) is the difference of the average van der Waals (electrostatic) binding energies in the FSS and bulk regions. 37



Monomer

+

Protofibril

Free Monomer (F)

Docked State (D)

8 G(RMSD) (kcal/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Locked State (L)

[TMAO] = 0M [TMAO] = 4M

6 4 2

L

D

0 0.2

0.4

F 0.6

RMSD (nm)

0.8

TOC figure

38


Page 38 of 38

Osmolyte Effects on the Growth of Amyloid Fibrils - The Journal of

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