Evidence of Coupling between the Motions of Water and Peptides

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Letter

Evidence of Coupling between the Motions of Water and Peptides Silvina Cerveny, Izaskun Combarro-Palacios, and Jan Swenson J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b01864 • Publication Date (Web): 29 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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The Journal of Physical Chemistry Letters

Evidence of Coupling between the Motions of Water and Peptides Silvina Cerveny*1,2, Izaskun Combarro-Palacios1 and Jan Swenson3 1

Centro de Fisica de Materiales (CSIC, UPV/EHU)-Materials Physics Center (MPC), Paseo Manuel de Lardizabal 5, 20018, San Sebastián, Spain 2

3

Donostia International Physics Center, 20018, San Sebastián, Spain

Department of Applied Physics, Chalmers University of Technology, SE-412 96 Göteborg, Sweden.

Corresponding Author Silvina Cerveny, [email protected]

ACS Paragon Plus Environment

1


ABSTRACT. Studies of protein dynamics at low temperatures are generally performed on hydrated powders and not in biologically realistic solutions of water, due to water crystallization. However, here we avoid the problem of crystallization by reducing the size of the biomolecules. We have studied oligomers of the amino acid L-lysine, fully dissolved in water, and our dielectric relaxation data show that the glass transition related dynamics of the oligomers is determined by the water dynamics, in a similar way as previously observed for solvated proteins. This implies that the crucial role of water for protein dynamics can be extended to other types of macromolecular systems, where water is also able to determine their conformational fluctuations. Using the energy landscape picture of macromolecules the thermodynamic criterion for such solvent-slaved macromolecular motions may be that the macromolecules need the entropy contribution from the solvent to overcome the enthalpy barriers between different conformational sub-states.

TOC GRAPHICS

2 Solute

0

Solvent 1-Lys 3-Lys 4-Lys 10-Lys ε-PLL

-2

log (τ)

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

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-4

3

-6 -8 -10 3

4

-1

5

1000/T [K ]

KEYWORDS. Amino acid, slaving, peptide, protein, dynamics


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Proteins are biopolymers made up of long strings of amino acids having biological functionality. In the functional state, proteins are organized in multi-scale structures and this, together with its dynamics, determines their functions1, 2, 3, 4. The dynamics includes motions of the protein itself (such as vibrations, rotations and transitions between sub-states5) as well as motions in the hydration shell and the bulk solvent and all of them are necessary for the functions. Actually, proteins evolve on a rugged energy landscape with conformational substates separated by energy barriers. Fluctuations of the protein structure correspond to transitions from one sub-state to another and water plays a dominant role increasing the entropic contribution and facilitating the passage of the enthalpic barriers. This causes the most important protein motions to follow the same temperature dependences as the water motions5, 6, 7. Thus, it has been experimentally proved that the protein conformational motions are “slaved” by the hydration shell and the bulk solvent5. This implies that the large-scale conformational protein fluctuations exhibit the same non-Arrhenius temperature dependence as the cooperative relaxation in the surrounding solvent6,

8, 9, 10, 11, 12

. However, these protein fluctuations are

typically 103-106 times slower due to that many solvent fluctuations are needed to cause all the steps needed for a large-scale conformational change of the protein. There is a large number of experimental and simulation studies supporting this close coupling6, 13, 14, 15, 16. Another common viewpoint is that water acts as a plasticizer for the protein17 and thereby facilitates its motions. Furthermore, molecular dynamics (MD) simulations have shown that translational motion of hydration water and its hydrogen bonding to the protein surface control the internal dynamics of the biomolecule18,

19

. The importance of translational motions of hydration water for protein

dynamics has also been observed by neutron scattering, where an onset of anharmonic protein dynamics (i.e. the so-called dynamical transition) occurred at the same temperature as the onset


3


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of translational water dynamics20. Related to this finding is also the suggestion that there is a mutual effect on both water and protein dynamics21. This coupling between the solute and the solvent has, as far as we are aware of, not been observed for any other water mixtures than protein solutions. However, in this work we have extended the protein studies to smaller molecules of peptides by investigating the dynamical behaviour of oligomers of the amino acid L-lysine (of chain lengths 1-10 monomers) and εpolylysine (ε-PLL) with a chain length of 32 monomers similar to what He22 et al did to analyse the dynamical transition phenomena. All samples were completely dissolved in water and studied at supercooled temperatures without any problem of crystallization due to their high solubility in water (up to 8 M). Lysine has three hydrophilic moieties (two amino groups and one carboxylate group) as well as a large hydrophobic tail, and this provides different environments for water molecules. In spite of the fact that L-lysine and its oligomers are small and relatively simple molecules it has been shown that they can also explore different conformations23, 24, 25. However, the smallness of these molecules reduces the topological disorder and the number of possible conformational fluctuations of the biomolecule, which, in turn, leads to a much narrower glass transition, than for proteins, as shown in Table 1 and supplementary information (SI). Nevertheless, the results presented here show that the previously observed coupling between protein and solvent dynamics is maintained even if the protein is replaced by short chains of a single amino acid. This implies that solvent slaved motions are not unique for proteins, but present in all types of solute molecules which need the dynamics in the surrounding solvent to reach different conformational sub-states. To study the slow relaxation dynamics of the aqueous solutions of n-lysine (n = 1, 3, 4 and 10) and ε-polylysine (n = 32) we used broadband dielectric spectroscopy. To avoid crystallization at


4

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all temperatures the concentration of water was limited to cw = 40 wt% (in weight). The chosen system has also the advantage of having chain-length dependent conformations (ε-PLL has a secondary structure of β-sheet, 10-lys has an α-helix conformation, whereas the short peptides (1-, 3- and 4-lys) have no defined secondary structure for this concentration (see Infrared results in Figure S1 in SI)), so that its possible influence on the dynamics can be studied. Moreover, the platelet β-sheet of ε-PLL differs from that of proteins in the number of carbon atoms in the main chain. Table 1: General characterization of n-lysine samples at cw = 40 wt%. Note that Tg has not a monotonous molecular weight dependence. cw = 40 wt% Samples

N

Mn [g/mol]

Tg,DSC [K]

∆Tg [K]

pH

N

N/n

1-Lys

1

146.2

194.0

8

10.4

5.4

5.40

3-Lys

3

402.5

190.0

9

--

14.9

4.96

4-Lys

4

530.7

186.0

6

--

19.7

4.90

10-Lys

10

1300

194.6

7

10.0

48.1

4.81

ε −PLL

32

4090.0

219.5

6

10.3

173.9

4.96

Mn is the molecular weight of each oligomer, Tg,DSC represents the calorimetric glass transition temperature and ∆Tg is the width of the transition. N is the number of water molecules per n-lysine molecule and N/n is the number of water molecules per monomer of the n-lysine molecule.

Figure 1 (a-d) shows both components of the complex dielectric permittivity data for the 3lysine water solution at some temperatures. The derivative of the real part of the complex permittivity, which is practically identical to ε´´(ω) but not affected by conductivity26, is shown in (e) and (f). The other oligomers show very similar behaviors to that showed for 3-lysine. We previously analyzed the dynamics of water in solutions of 1-lysine at different water concentrations27 and this work builds on our prior study. For longer chains of lysine, we also find


5


a rich dynamical behavior with the appearance of seven different relaxations. Below the glass transition temperature (Tg) we observe three dynamical processes (1, 2 and 3), whereas at temperatures above Tg, the dielectric data show as much as five dynamical processes (3 to 7), although in this work we mainly focus on the behavior of processes 3 and 4. These relaxation processes are analogous to those observed in protein solutions28, 29. Technical details about the fitting of the processes are provided in SI (figures S2-S6). 4

3-Lys (cw = 40 wt%)

10

(b)

195.7 K

(a)

2

10

190

2

10

182.5 ε´´

Process 3

Process 2

ε´

170

Process 1

0

10

Process 2

155

140

-2

-2

0

10

2

10

10

4

6

10

8

10

f [Hz]

10

10

-2

0

10

2

10

6

10

8

10

10

f [Hz]

10

(c)

6

4

10

8

10

(d)

230 237.5

247.5 K

6

10 4

10

220

212.5

ε´

ε´´

4

10

205

Process 4 2

2

Process 3

10

10

(d) 0

10

-2

10

0

10

2

10

4

6

10

10

8

f [Hz]

10 -2 10

0

2

10

4

10

6

10

10

8

10

f [Hz]

2

3

10

10

195.7 K

(e)

Process 3

190

205 2

10

1

10

Process 2

εD´´

182.5

εD´´

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

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170

0

10

237.5 K

230 220 212.5

(f) Process 4

1

10

155

Process 3

140 0

10

-1

10

-1

10

1

10

3

10

f [Hz]

5

10

-1

10

1

10

3

10

5

10

7

10

f [Hz]

Figure 1. (a, b) Real and Imaginary part of the complex dielectric permittivity focus (ε*(ω) = ε´(ω) – i ε´´(ω)) of the 3-lysine water solution (cw = 40 wt%) at temperatures lower than Tg. Solid lines through the data points represent fits to the experimental data. (c, d) Same as in (a, b) at temperatures higher than Tg. (e, f). εD´´(ω) calculated from the derivative of ε´(ω).


6

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Fig. 2 shows temperature dependences of relaxation times τ for 3-lysine (a), 10-lysine (b) and

ε-poly(L-lysine) (ε-PLL) (c) (cw = 40 wt%). In our previous work27, we analysed the molecular origin of each process by studying samples of 1-lysine at several water concentrations. Processes 1 to 3 are caused by dipole reorientations of water molecules, whereas process 4 is related to reorientational motions of 1-lysine molecules27. The dielectric response for the longer lysine chains studied here reveals the same relaxation processes and we attribute them to the same molecular origin, although process 4 in 1-lysine is of more intermolecular origin than in the case of the oligomers. Assignments of processes 3 and 4 to water and peptide dynamics, respectively, can be also established by determine how the dielectric strength (∆ε) changes with water content, as shown in figures S7 and S8 in SI. These figures show that ∆ε3 increases with increasing water content, whereas ∆ε4 increases with increasing peptide content, in agreement with our assignments. Moreover, process 3 slows down when water is replaced by D2O, also confirming that process 3 is due to a relaxation of water molecules. Although we do not focus here on the dynamics of the solutions below Tg, but rather on the properties above Tg, we should note that processes 1 to 3 have Arrhenius temperature dependences below Tg and their activation energies are shown in SI (Table S1). In addition, the presence of more than one water related process below Tg (well below the physiological regime) is not commonly observed in other aqueous solutions30 than protein solutions7, 31. Process 5 is also present in 1-lysine, but for longer chains we need two more processes (6 and 7) to fit the dielectric relaxation data at higher temperatures. However, these processes have large dielectric amplitudes (∆ε > 104) and therefore it is difficult to rationalize them as standard relaxation processes. The temperature at which the structural (α) relaxation time extrapolates to 100 s defines a temperature usually called the dielectric glass transition temperature, Tg,100 s. For 3-lysine, τ4


7


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reaches 100 s at T = 192 K, whereas τ5 reaches 100 s at T = 200 K. Considering the width of the glass transition (9 K, see Table 1), these values are both in good agreement with the calorimetric Tg (191 K) and both processes can therefore be related to the glass transition phenomenon. However, we have to be aware that from the crossing frequency of the real and imaginary parts in the ε* (permittivity), M* (modulus) and σ* (conductivity) representations, processes 5 to 7 are coupled to the process of ionic conductivity32 (see figure S9 in SI). Thus, an identification of process 5 to a glass transition related relaxation process is not straightforward. Processes 6 and 7 were also detected in protein solutions and attributed to electrode polarization33, 34. Also in our case, the peak in σ´´ (see figure S9 in SI) is a signature of that electrode polarization is the origin of these processes35. The relaxation times of processes 3 and 4 follow the very same non-Arrhenius temperature dependence (see figure 2), and the relaxation strengths of these processes decrease with increasing temperature. In the case of polymers, these characteristics are strong indications for that such relaxations are caused by cooperative motions, as typical for the cooperative and glass transition related structural (α) relaxation36. However, in our case the cooperative character of process 4 comes from the cooperativity of the water relaxation (process 3). Therefore process 4 should be denoted as collective rather than cooperative, although it is responsible for the observed glass transition. Table 2 shows all the fit parameters of the Vogel-Fulcher-Tammann (VFT) equations used to describe the temperature dependences of the relaxation times of the processes 3 and 4 for the different oligomers. For both processes (3 and 4), the fragility associated parameter (D) is the same, supporting that process 4 is slaved by process 3. In figures 3(a) and 3(b) the chain-lengths dependences of the relaxation times of processes 3 and 4, respectively, are shown. The water relaxation is almost unaffected by the chain-length,


8

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except for the ε-PLL solution at temperatures lower than 235 K, where this relaxation process becomes considerably slower than for the shorter oligomers. In fact, the deeply supercooled water in the ε-PLL solution shows a relaxation behaviour close to what has been predicted to occur for bulk water, if crystallization could had been avoided37, 38

39

. This observation further

suggests that the water molecules in the solution of ε-PLL form larger and more bulk-like clusters than in the solutions of the shorter oligomers, probably due to that the comparably large

β-sheets of ε-PLL cannot be mixed with the water molecules on as short length scale as the smaller n-lysine molecules. Since process 4 is slaved by process 3 it can be seen in figure 3(b) that also process 4 of the ε-PLL solution slows down much more rapidly at low temperatures, compared to the same process in the solutions of the shorter oligomers. In this figure it is also interesting to note that process 4 is slower for the monomer than for n = 3-4. This indicates that the origin of process 4 is not the same for the monomer as for the oligomers. The slowness of the monomer suggests that this process involves the motion of more than single monomers, i.e. that it is of intermolecular origin in the case of the monomer system. The influence of intermolecular interactions on process 4 should decrease with increasing chain-length, but the intermolecular interactions are furthermore expected to change with the molecular weight due to that the different chain-lengths of n-lysine adopt different conformations in water. Hence, this can be the structural reason for the deviation from the normal molecular weight dependence. Further support for the anomalous molecular weight dependence and the assignment of process 4 to the structural (α) relaxation of the oligomers comes from the calorimetric Tg values presented in Table 1. It is there seen that Tg decreases with increasing chain-length up to 4 monomer units, in consistency with the corresponding dielectric relaxation process. Thus, it is evident that the


9


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aqueous solutions of n-lysine do not follow the expected molecular weight dependence observed for other aqueous solutions as n-ethylene glycol oligomers40. A large difference between the here studied n-lysine-water solutions and previous protein solution studies is that n-lysine-water mixtures exhibit a narrow glass transition, (see Table 1,

∆Tg) in contrast to proteins, which display a glass transition that extends over a large temperature range (more than 70 K has been reported for myoglobin11 and other proteins28, 41). The reason for this difference is that for proteins the glass transition is due to the “freezing in” of a wide range of different types of relaxations (conformational fluctuations, electrostatic interactions, rotations of small side groups, collective motions of the full chain, etc.) occurring on very different time scales at a given temperature11,

42

, whereas for the n-lysine-water systems we expect a

substantially reduced number of intra-molecular motions to be present because of the smaller size of the oligomers and their lower degree of structural complexity as compared to proteins. In fact, as mentioned above, only one relaxation (process 4) is clearly due to molecular motions of n-lysine. The important role of water for this relaxation process, as further discussed below, is also supported by the fact that it is undetectable for low hydrated lysine powders (cw = 5 wt%)27. This implies that it is the water molecules that give rise to these n-lysine motions that freeze in at Tg. The same role of water (or solvent) has also been observed by Shinyashiki et al for hydrated bovine serum albumin29 and by Jansson et al for myoglobin11.


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Figure 2. Temperature dependences of the relaxation times of 3-lysine, 10-lysine and ε-PLL (cw = 40 wt%) respectively. Vertical coloured areas represent the width of the glass transition. Data obtained from isothermal measurements are shown in full points; open points were obtained from TSDC experiments and crosses from the derivative analysis. The straight lines below Tg represent fits to the experimental data by the Arrhenius equation (not discussed in this work, see activation energies in SI), whereas above Tg full lines represent VFT-fits to the experimental data. Upper panel in each figure represents the heat flow measured by DSC of 3-lysine, 10-lysine and ε-PLL (cw = 40 wt%) respectively. Average errors in the determinations of τ1, τ3 and τ4 are 10%, 5% and 4%, respectively. For τ2 the average error is about the same as the size of the symbols.


11


log (τ)

4 (a)

process 3

0 -4

1-Lys 3-Lys 4-Lys 10-Lys ε-PLL

-8 -12

3

4

log (τ)

0 (b)

5 6 -1 1000/T [K ]

7

process 4 1-Lys 3-Lys 4-Lys 10-Lys ε-PLL

-5

-10

0

3

4 5 -1 1000/T [K ]

6

(c)

-2

log (τ4)

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

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-4

1-Lys (1.07) 3-Lys (1.01) 4-Lys (1.00) 10-Lys (1.04) ε-PLL (1.07)

-6 -8

-10

-10

-8

-6

log (τ3)

-4

-2

Figure 3. (a and b) Temperature dependences of the relaxation time for processes 3 and 4, respectively. Solid lines through the data symbols represent VFT fits above Tg and Arrhenius fits below Tg (see text). (c) Relaxation times of process 4 as a function of the relaxation times for process 3. An almost perfect linear dependence, with a slope of one, is found for all the oligomers.


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Table 2: VFT parameters (τα = τ0 exp (DT0 / (T-T0)) for processes 3 and 4 above Tg, where τ0 is a pre-exponential factor, D is a parameter related to fragility and To is the temperature at which the relaxation time extrapolates to infinity.

Process 3 Sample

Process 4

D

To (K)

log (τo)

D

To(K)

log (τo )

1-Lys

7.9

152.4

-13.3

7.9

155.9

-12.1

3-Lys

7.9

149.0

-13.2

7.9

149.0

-12.1

4-Lys

7.9

148.5

-13.3

7.9

149.6

-12.5

10-Lys

12.1

136.5

-14.2

12.1

138.8

-12.5

ε-PLL

3.4

191.2

-12.6

3.4

195.6

-9.8

In view of our results it is now interesting to elucidate the relationship between the processes 3 and 4 in the same way as previously has been done for protein and solvent motions5, 7. As mentioned above, the important role of water for protein dynamics (and related biological activities) has been linked to the observation that structural protein fluctuations are determined or “slaved” by motions in the surrounding solvent5, 6, 7, 8, 9, 10, 11. This slaving means that the protein and solvent relaxations exhibit the same temperature dependence, but are occurring on different time scales. The slaving model is tested in figure 3(c), where it, indeed, can be seen that processes 3 and 4 exhibit almost identical temperature dependences. Thus, figure 3(c) provides evidence for that the observed structural relaxation of n-lysine is slaved by the α-like water relaxation. This behaviour has previously only been observed for proteins with a surrounding solvent7, 9, and not for other types of aqueous solutions43, 44. The present finding thereby shows that the solvent plays the same important role for the dynamics of oligomers of amino acids, as for proteins, and that the biological functions of amino acids are also crucially dependent on the


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surrounding water. For both types of systems the solvent-slaved relaxations involve both conformational intra-chain fluctuations as well as collective inter-chain motions. Furthermore, since the small oligomers (3- and 4-lysine) lack the three-dimensional structure observed in folded proteins, it is evident that the slaving behaviour is not directly dependent on the topology of the system (but maybe indirectly by a change of the internal enthalpy barriers between different conformational sub-states). In conclusion, for the solutions of n-lysine and water studied here it is clear that the glass transition related conformational fluctuations of the oligomers exhibit the same temperature dependence as the α-like water relaxation, although the time scale of these fluctuations is considerably slower, as also observed for protein solutions. This is the case even if the timescale of the water dynamics (see figure 3(a)) is affected by the structure of the solute molecules and the interactions between the two components. The similar role of water for the dynamics of peptides and proteins founded here may have important implications for both the general understanding of the role of water in biological systems, as well as of how proteins work. In fact, our findings suggest that the observed slaving behaviour is not limited to biological systems, but can be present also in non-biological systems if their conformational motions can only occur due to motions in the surrounding solvent. Using the concept of energy landscape and a thermodynamic terminology the criteria for solvent-slaved conformational motions can be expressed as a need for a substantial entropic contribution from the solvent to overcome the enthalpy barriers between different sub-states in the solute molecules5. 45


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Experimental Methods Sample preparation Oligomers of L-lysine (n-Lys, n = 1, 3, 4 and 10) from Sigma Aldrich Chemical Co. (n = 1, 3, and 4) and Byomatic (n = 10) were used to prepare solutions to a concentration of cw = 40 wt%. ε-Poly-L-lysine 25% water solution from JNC Corporation (n = 32) was also analysed to a concentration of cw = 40 wt% (in weight). ε-(poly-L-lysine) (ε-PLL) has a secondary structure of

β-sheet whereas 4- and 10-Lysine have a random coil conformation for this concentration (see Figure S10 in SI). Differential scanning calorimetry measurements were performed on a DSC Q-2000 from TA Instruments, using cooling and heating rates of 10 K/min. No sign of crystallization was found on cooling or heating and a single narrow glass transition temperature (Tg) was observed for all the samples (see Figure S11 in SI). Table 2 shows the general characteristics of the solutions and Tg values. Dielectric experiments To analyze the dynamics we used broadband dielectric spectroscopy (BDS), which is a powerful technique suitable to evaluate the dielectric properties of biological systems and also to assess the molecular dynamics on various time and length scales. These measurements were performed at temperatures much lower than the physiological temperatures because conductivity and polarization effects are reduced at lower temperatures and this makes it easier to observe the different relaxation processes of the solvent and the biomolecule. To measure the complex dielectric permittivity, ε´´(ω) = ε (ω)- i ε´´(ω), we combined different dielectric techniques to obtain a wide spectral range (0.1 Hz to 20 GHz). For the frequency range from 10-1 Hz to 106 Hz, we used a Novocontrol Alpha- Analyser, whereas for the frequency


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range from 106 Hz to 109 Hz, an Agilent rf impedance analyser 4192B was used. The sample thickness was for all measurements 0.1 mm, and the sample diameter was 30 mm and 10 mm for the low and high frequency measurements, respectively. In the frequency range of 0.2–20 GHz a dielectric probe kit Agilent 85070E (bandwidth 200 MHz to 20 GHz) with an open ended coaxial probe connected to a vector analyser 8361 (VNA) was used to measure the dielectric permittivity from 323 to 283 K of 1-lysine46 and ε-PLL solutions. Thermo stimulated depolarization current (TSDC) was used to determine relaxation times at longer times (longer than 100 s). TSDC is a dielectric technique in the temperature domain47. TSDC measurements were carried out using a Keithley 6517 in combination with a Novocontrol sample cell for TSDC measurements. Experimental conditions were polarization temperature, Tp = 183 K, and heating rates (q) of 0.5, 1, 3, 5 and 7 K/min. To analyse the complex permittivity (ε*), simultaneous fitting of both real (ε´) and imaginary (ε´) components were performed by the use of symmetric Cole-Cole functions48. In the supplementary information section, the fitting procedure is explained in detail.

ASSOCIATED CONTENT Infrared response of all the samples; fitting procedures,; calorimetric response of all the samples, relaxation strength for different water contents (1-Lys and ε-PLL) and molecular structure of the oligomers (PDF).


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ACKNOWLEDGMENT S.C acknowledges funding by Elkartek Program, the Basque Government (IT-654-13) and the Spanish Ministry “Ministerio de Economia y Competitividad" (project MAT2015-63704P (MINECO/FEDER, UE)). J.S. thanks the Swedish Research Council for financial support. REFERENCES 1. Chaplin, M. Opinion - Do We Underestimate the Importance of Water in Cell Biology? Nat. Rev. Mol. Cell Biol. 2006, 7, 861-866. 2. Frauenfelder, H.; Fenimore, P. W.; Chen, G.; McMahon, B. H. Protein Folding Is Slaved to Solvent Motions. Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 15469-15472. 3. Johnson, M. E.; Malardier-Jugroot, C.; Murarka, R. K.; Head-Gordon, T. Hydration Water Dynamics near Biological Interfaces. Journal of Physical Chemistry B 2009, 113, 40824092. 4. Ball, P. Water as a Biomolecule. ChemPhysChem 2008, 9, 2677-2685. 5. Fenimore, P. W.; Frauenfelder, H.; McMahon, B. H.; Young, R. D. Bulk-Solvent and Hydration-Shell Fluctuations, Similar to Alpha- and Beta-Fluctuations in Glasses, Control Protein Motions and Functions. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, 14408-14413. 6. Frauenfelder, H.; Chen, G.; Berendzen, J.; Fenimore, P. W.; Jansson, H.; McMahon, B. H.; Stroe, I. R.; Swenson, J.; Young, R. D. A Unified Model of Protein Dynamics. Proceedings of the National Academy of Sciences of the United States of America 2009, 106, 5129-5134. 7. Jansson, H.; Bergman, R.; Swenson, J. Role of Solvent for the Dynamics and the Glass Transition of Proteins. J. Phys. Chem. B 2011, 115, 4099-4109. 8. Beece, D.; Eisenstein, L.; Frauenfelder, H.; Good, D.; Marden, M. C.; Reinisch, L.; Reynolds, A. H.; Sorensen, L. B.; Yue, K. T. Solvent Viscosity and Protein Dynamics. Biochemistry 1980, 19, 5147-5157. 9. Swenson, J.; Jansson, H.; Bergman, R. Relaxation Processes in Supercooled Confined Water and Implications for Protein Dynamics. Phys. Rev. Lett. 2006, 96, 247802. 10. Finkelstein, I. J.; Massari, A. M.; Fayer, M. D. Viscosity-Dependent Protein Dynamics. Biophysical Journal 2007, 92, 3652-3662. 11. Jansson, H.; Swenson, J. The Protein Glass Transition as Measured by Dielectric Spectroscopy and Differential Scanning Calorimetry. BBA-Proteins Proteomics 2010, 1804, 2026. 12. Schiro, G.; Cupane, A.; Vitrano, E.; Bruni, F. Dielectric Relaxations in Confined Hydrated Myoglobin. Journal of Physical Chemistry B 2009, 113, 9606-9613. 13. Karplus, M.; Kuriyan, J. Molecular Dynamics and Protein Function. Proceedings of the National Academy of Sciences of the United States of America 2005, 102, 6679-6685. 14. Doster, W.; Cusack, S.; Petry, W. Dynamical Transition of Myoglobin Revealed by Inelastic Neutron Scattering. Nature 1989, 337, 754-756.


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Page 18 of 22

15. Lewandowski, J. R.; Halse, M. E.; Blackledge, M.; Emsley, L. Direct Observation of Hierarchical Protein Dynamics. Science 2015, 348, 578-581. 16. Demuth, D.; Haase, N.; Malzacher, D.; Vogel, M. Effects of Solvent Concentration and Composition on Protein Dynamics: 13c Mas Nmr Studies of Elastin in Glycerol–Water Mixtures. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2015, 1854, 9951000. 17. Doster, W. The Dynamical Transition of Proteins, Concepts and Misconceptions. Eur Biophys J 2008, 37, 591-602. 18. Tarek, M.; Tobias, D. J. Role of Protein-Water Hydrogen Bond Dynamics in the Protein Dynamical Transition. Physical Review Letters 2002, 88, 138101. 19. Tournier, A. L.; Xu, J.; Smith, J. C. Translational Hydration Water Dynamics Drives the Protein Glass Transition. Biophysical Journal 2003, 85, 1871-1875. 20. Levantino, M.; Schiro, G.; Lemke, H. T.; Cottone, G.; Glownia, J. M.; Zhu, D. L.; Chollet, M.; Ihee, H.; Cupane, A.; Cammarata, M. Ultrafast Myoglobin Structural Dynamics Observed with an X-Ray Free-Electron Laser. Nat. Commun. 2015, 6, 041713. 21. Khodadadi, S.; Roh, J. H.; Kisliuk, A.; Mamontov, E.; Tyagi, M.; Woodson, S. A.; Briber, R. M.; Sokolov, A. P. Dynamics of Biological Macromolecules: Not a Simple Slaving by Hydration Water. Biophysical Journal 2010, 98, 1321-1326. 22. He, Y. F.; Ku, P. I.; Knab, J. R.; Chen, J. Y.; Markelz, A. G. Protein Dynamical Transition Does Not Require Protein Structure. Physical Review Letters 2008, 101, 178103. 23. Leng, Y.; Zhang, M.; Song, C.; Chen, M.; Lin, Z. A Semi-Empirical and Ab Initio Combined Approach for the Full Conformational Searches of Gaseous Lysine and Lysine–H2o Complex. Journal of Molecular Structure: THEOCHEM 2008, 858, 52-65. 24. Boeckx, B.; Maes, G. Experimental and Theoretical Observation of Different Intramolecular H-Bonds in Lysine Conformations. The Journal of Physical Chemistry B 2012, 116, 12441-12449. 25. Bazsó, G.; Najbauer, E. E.; Magyarfalvi, G.; Tarczay, G. Near-Infrared Laser Induced Conformational Change of Alanine in Low-Temperature Matrixes and the Tunneling Lifetime of Its Conformer Vi. The Journal of Physical Chemistry A 2013, 117, 1952-1962. 26. Wübbenhorst, M.; van Turnhout, J. Analysis of Complex Dielectric Spectra. I. OneDimensional Derivative Techniques and Three-Dimensional Modelling. Journal of NonCrystalline Solids 2002, 305, 40-49. 27. Cerveny, S.; Swenson, J. Dynamics of Supercooled Water in a Biological Model System of the Amino Acid L-Lysine. Physical chemistry chemical physics 2014, 16, 22382-90. 28. Panagopoulou, A.; Kyritsis, A.; Shinyashiki, N.; Pissis, P. Protein and Water Dynamics in Bovine Serum Albumin-Water Mixtures over Wide Ranges of Composition. J. Phys. Chem. B 2012, 116, 4593-4602. 29. Shinyashiki, N.; Yamamoto, W.; Yokoyama, A.; Yoshinari, T.; Yagihara, S.; Kita, R.; Ngai, K. L.; Capaccioli, S. Glass Transitions in Aqueous Solutions of Protein (Bovine Serum Albumin). J. Phys. Chem. B 2009, 113, 14448-14456. 30. Swenson, J.; Cerveny, S. Dynamics of Deeply Supercooled Interfacial Water. J. Phys.: Condens. Matter 2015, 27, 033102. 31. Khodadadi, S.; Sokolov, A. P. Protein Dynamics: From Rattling in a Cage to Structural Relaxation. Soft Matter 2015, 11, 4984-4998. 32. Floudas, G. Dielectric Spectroscopy. In Polymer Science: A Comprehensive Reference, Möller, K. M., Ed.; Elsevier: Amsterdam, 2012, pp 825-845.


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Page 19 of 22

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


33. Ishai, P. B.; S. Talary, M.; Caduff, A.; Levy, E.; Feldman, Y. Electrode Polarization in Dielectric Measurements: A Review. Measurement Science and Technology 2013, 24, 102001. 34. Kundu, S. K.; Choe, S.; Sasaki, K.; Kita, R.; Shinyashiki, N.; Yagihara, S. Relaxation Dynamics of Liposomes in an Aqueous Solution. Physical Chemistry Chemical Physics 2015, 17, 18449-18455. 35. Samet, M.; Levchenko, V.; Boiteux, G.; Seytre, G.; Kallel, A.; Serghei, A. Electrode Polarization Vs. Maxwell-Wagner-Sillars Interfacial Polarization in Dielectric Spectra of Materials: Characteristic Frequencies and Scaling Laws. The Journal of Chemical Physics 2015, 142, 194703. 36. Ngai, K. L. Relaxation and Diffusion in Complex Systems (Partially Ordered Systems); Springer 2011. p 650. 37. Swenson, J.; Teixeira, J. The Glass Transition and Relaxation Behavior of Bulk Water and a Possible Relation to Confined Water. J. Chem. Phys. 2010, 132, 14508. 38. Elamin, K.; Jansson, H.; Kittaka, S.; Swenson, J. Different Behavior of Water in Confined Solutions of High and Low Solute Concentrations. Phys. Chem. Chem. Phys. 2013, 15, 18437-18444. 39. Cerveny, S.; Mallamace, F.; Swenson, J.; Vogel, M.; Xu, L. Confined Water as Model of Supercooled Water. Chemical Reviews 2016, 116, 7608-7625. 40. Capaccioli, S.; Ngai, K. L.; Shinyashiki, N. The Johari−Goldstein Β-Relaxation of Water. The Journal of Physical Chemistry B 2007, 111, 8197-8209. 41. Khodadadi, S.; Malkovskiy, A.; Kisliuk, A.; Sokolov, A. P. A Broad Glass Transition in Hydrated Proteins. BBA-Proteins Proteomics 2010, 1804, 15-19. 42. Doster, W. The Protein-Solvent Glass Transition. BBA-Proteins Proteomics 2010, 1804, 3-14. 43. Cerveny, S.; Alegria, A.; Colmenero, J. Universal Features of Water Dynamics in Solutions of Hydrophilic Polymers, Biopolymers, and Small Glass-Forming Materials. Physical review. E, Statistical, nonlinear, and soft matter physics 2008, 77, 031803. 44. Cerveny, S.; Colmenero, J.; Alegria, A. Dynamics of Confined Water in Different Environments. European Physical Journal-Special Topics 2007, 141, 49-52. 45. Shabbir, A.; Javakhishvili, I.; Cerveny, S.; Hvilsted, S.; Skov, A. L.; Hassager, O.; Alvarez, N. J. Linear Viscoelastic and Dielectric Relaxation Response of Unentangled UpyBased Supramolecular Networks. Macromolecules 2016, 49, 3899-3910. 46. Rodriguez-Arteche, I.; Cerveny, S.; Alegria, A.; Colmenero, J. Dielectric Spectroscopy in the Ghz Region on Fully Hydrated Zwitterionic Amino Acids. Physical Chemistry Chemical Physics 2012, 14, 11352-11362. 47. Arrese-Igor, S.; Alegría, A.; Colmenero, J. Polymer Chain Dynamics: Evidence of Nonexponential Mode Relaxation Using Thermally Stimulated Depolarization Current Techniques. Physical Review Letters 2014, 113, 078302. 48. Cole, K. S.; Cole, R. H. Dispersion and Absorption in Dielectrics - Direct Current Characteristics. Journal of Chemical Physics 1942, 10, 98-105.


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209x147mm (300 x 300 DPI)


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Figure 2 209x296mm (300 x 300 DPI)



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Figure 3 209x296mm (300 x 300 DPI)


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Evidence of Coupling between the Motions of Water and Peptides

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