Anomalous Dehydration Behavior of a Short Collagen Model

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Anomalous Dehydration Behavior of a Short Collagen Model Polypeptide, (L-Prolyl-L-ProlylGlycyl)5, in Aqueous Solution Toshiyuki Shikata,* Nao Yoshida, and Kenji Okuyama Department of Macromolecular Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

ABSTRACT High-frequency dielectric relaxation measurements from 1 MHz to 20 GHz were carried out on aqueous solutions of a short collagen model polypeptide, (L-prolyl-L-prolylglycyl)5 (PPG5), at several temperatures ranging from 10 to 40 C. Experiments revealed that PPG5 behaved as a fully dissociated flexible zwitterionic polymer chain over a wide temperature range. However, its hydration number per PPG5 molecule was altered dramatically and stepwise at Tdh = 27 C. This is close to the triple-helix-to-single-coil (TH-SC) transition temperature (Tt ∼ 30 C) for PPG10, clearly showing a TH-SC transition like that of native collagens in aqueous media. Below the Tdh, PPG5 kept about 135 water molecules per molecule, while it retained less than 50 water molecules above Tdh. This anomalous hydration number change, observed at Tdh (∼Tt), should be a TH-SC transition trigger for the longer PPG10. SECTION Biophysical Chemistry

he collagen model polypeptide, (L-prolyl-L-prolylglycyl)5 (PPG5), one of the simplest of its kind, does not display a triple-helix-to-single-coil (TH-SC) transition in aqueous media. It remains in the single-coil state over a wide temperature range because of its short length. We recently found that this polypeptide shows anomalous dehydration behavior at a temperature close to the TH-SC transition temperature, Tt, of PPG10. This dehydration behavior, exhibited by PPG5, is possibly characteristic for PPGn molecules in their single coil state. It leads to a trigger for the TH-SC transition of PPGn molecules with n g 9. Collagen is the most abundant structural protein in animals with an XYG (where G is glycine; X and Y are any amino acid residue) repeating sequence and exists as a triple-helix structure in nature.1,2 The denaturation temperature of collagen leads to the deconstruction of the triple helix into flexible single coils of constituent chains, based on its amino acid sequence.1-3 To understand the thermal behavior of collagen and its structure, a series of model polypeptides were synthesized.4,5 PPGn shows a reversible TH-SC transition. Furthermore, for n g 9, X-ray diffraction of its single crystals reveals a 7/2-helical symmetry of the triple helix, which is consistent with native collagen.5-7 However, PPG5 never forms triple helices in aqueous media, owing to its short length.8 Thus, it is useful for the investigation of hydration/dehydration and the dynamic behavior of collagen in the single-coil state. The existence of hydration layers on proteins, DNAs, and other biological macromolecules and the dynamics of hydrated water molecules have been fully discussed by using many independent techniques such as dielectric relaxation (DR), nuclear magnetic resonance (NMR), time-resolved fluorescence spectroscopies, and computer simulations.9

T

r 2009 American Chemical Society

DR measurements are powerful for investigating the dynamics of systems containing molecules and/or groups bearing electric dipoles. DR techniques are also useful for the determination of hydration numbers in aqueous solutions, as water possesses a large dipole moment.9,10 In our previous study, we showed that PPG5 behaves as a fully dissociated flexible zwitterionic polymer chain in aqueous media at T = 25 C.10 Moreover, the hydration number (mcoil) per PPG5 molecule in the single-coil state was determined to be approximately 130, with the hydration number per amino acid residue (Mmcoil) being around 9.10 Another recent study11 revealed the hydration number per amino acid residue in the triple-helix state (Mmth) to be around 2 (mth ∼ 65) at 25 C for PPG10, which is much less than the Mmcoil discussed above. On the basis of a chemical equilibrium consideration between two states, the triple-helix and single-coil states, the following equation holds: ðPPG10 3 mth H2 OÞ3 þ 3ðmcoil -mth ÞH2 O a 3ðPPG10 3 mcoil H2 OÞ

ð1Þ

Here, (PPG10 3 mthH2O)3 and PPG10 3 mcoilH2O represent a triple helix of PPG10 bearing mth water molecules, a PPG molecule, and a random coil of PPG with mcoil water molecules, respectively. A relatively large discrepancy between mcoil and (>) mth is naturally required for the chemical reaction at temperatures lower than Tt. This is because the evaluated standard entropy change for eq 1 is negative in this Received Date: November 5, 2009 Accepted Date: December 7, 2009 Published on Web Date: December 14, 2009

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DOI: 10.1021/jz900220x |J. Phys. Chem. Lett. 2010, 1, 412–416

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Figure 1. Frequency, ω, and dependencies of the real and imaginary parts (ε0 and ε00 ) of electric permittivity for an aqueous PPG5 solution at c = 21.1 mM over a temperature range of zT = 10-40 C.

Figure 2. Dependence of the concentration-reduced DR strength (ε4c-1) of the relaxation mode j = 4 on T for aqueous solutions of PPG5. This figure also contains T dependencies of the rotational radius (ÆS2æ1/2) evaluated from ε4c-1, ÆS2æτ1/2 evaluated from τ4, and the hydrodynamic radius (RH) of PPG5 in aqueous solution. Solid, dotted, and dashed lines in this figure are guides for the eye.

range.11 However, mcoil is similar to mth when the reaction is above Tt, as the standard entropy change is positive in the range.11 From these, clear evidence is expected for the dramatic change in mcoil (and mth) around Tt for PPGn in solution. Figure 1 shows the DR spectra and the real and imaginary parts of electric permittivity (ε0 and ε00 ) versus angular frequency (ω) for a 21.1 mM aqueous solution of PPG5 over a temperature range of 10-40 C. The solution showed two characteristic major DR processes in the following ranges: ω = 2 108 and 1011 s-1. According to the linear theory,12 all of the obtained DR spectra were decomposed into Debyetype relaxation modes by the equations given below: 4 4 X X εj εj ωτj 00 þ ε and ε ¼ ð2Þ ε0 ¼ ¥ 2 2 1 þ ω τj 1 þ ω2 τ j 2 j ¼1 j ¼1

given as cNA Æðμbs 1 Þ2 æ ε4 ¼ 2εv kB T

Æðμbs 1 Þ2 æ ¼

2ÆR2 æe2 ðqi þqf Þ2 ð3Þ π2

In these equations, Æ(μbs1)2æ, εv, and kBT represent the mean square of the dipole moment of the first normal mode for the bead-and-spring model chain, the electric permittivity of a vacuum, and the product between Boltzmann's constant and absolute temperature, respectively.10,11,13 The first part of eq 3 quantitatively connects the dielectric increment (ε4 in this case) and the square of dipole moments of solute particles, Æ(μbs1)2æ, which are dissolved in a highly polar media, such as water.10,11,14 The value of ÆS2æ1/2 evaluated from the ε4c-1 data shown in Figure 2, assuming qi = qf = 1 via the equations, is also plotted in this figure. A stepwise reduction in ÆS2æ1/2 with increasing T was clearly observed with a small amplitude that should be directly relevant to dehydration phenomena. A study has been performed on solution properties of model polypeptides bearing a repeating unit, Glycyl-Prolyl-Prolyl, which is similar to PPGn and the same length as PPG5.15 According to this work, the ÆS2æ1/2 value of GPP5 was found to be approximately 1.0 nm at 75 C in 20 mM phosphate buffer with 150 mM sodium chloride. Therefore, the obtained ÆS2æ1/2 value in Figure 2 agreed favorably with the values of GPP5. Consequently, the stepwise reduction in the ÆS2æ1/2 observed at 27 C should be a characteristic behavior for PPG5 in aqueous solution. Because PPG5 is constructed by the sequential formation of amide groups, -C(dO)NH- bearing a finite dipole moment, it possesses characteristics of type-A and type-B polymers13 resulting from the sequence of amide groups. The direction of dipole moment of the amide group is not parallel to the backbone of PPG5; therefore, the characteristics of the type-A polymer is not so strong, but that of type-B polymer is relatively distinctive. Moreover, since the component of the dipole moment of the amide group possesses a direction opposite to that of the dipole due to the dissociation of amino and carboxy groups on PPG5 termini, the characteristics of the type-A polymer would slightly decrease the relaxation strength, ε4, of the mode j = 4. At present, we speculate the segment motion of PPG5 including the contribution of amide groups is also observed nearby the mode j = 3 with τ3. As we

where τj and εj are the relaxation time and strength for each mode, respectively, and ε¥ is the ω-independent permittivity. Aqueous PPG5 solutions showed a pronounced DR mode (j = 4) at ω values of 2 108 to 4 108 s-1 (τ4 = 2.5-5 ns, as indicated in Figure 1 with an arrow), depending on T and irrespective of concentration, c. The relaxation strength (ε4) of the mode j = 4 increased in proportion to c, as evidenced in Figure 2.10 A weak DR mode was identified at ω values of 2 109 to 4 109 s-1 (τ3 = 0.25 to 0.5 ns) and was also independent of concentration. DR spectra shown in Figure 1 reflect the dynamics of PPG5 in the single-coil state. The motions of a pair of opposite electric charges play an essential role in the overall DR processes for this type of single coil. These charges are located at the polypeptide termini, owing to ionization of the amino (N-) and carboxy (C-) termini. These are labeled eqi and -eqf, respectively, where e represents an elementary electric charge. Here qi and qf represent the degrees of ionization of the initial N- and final C-termini, respectively, for PPG5 in this case. The dynamics of zwitterionic polymer chains can be approximately described with the standard bead-and-spring model.13 In this case, the DR modes j = 4 and 3 are assigned to the first and third normal modes, respectively, of a beadand-spring model chain for PPG5.10,11 The magnitude of ε4 is approximately connected to the radius of rotation (ÆS2æ1/2) of PPG5 and the distance between the two termini (ÆR2æ=6ÆS2æ assuming Gussian chain approximation) via the equations


and

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have already discussed in the previous study on PPG5,10 a ratio of ε4ε3-1 (∼7) was slightly smaller than the prediction of the bead-spring model;13 ε4ε3-1 = 9. Thus, we partially attributed the characteristics of the (type-A and) type-B polymer to the small ratio of ∼7. The hydrodynamic radius (RH) of PPG5 in aqueous solution, determined as a function of T at c = 21.1 mM, is also plotted in Figure 2. RH is also a measure of size. Although the data were scattered, a stepwise change in RH identified around 27 C also revealed a reduction in the size. The DR time, τ4, of the mode j = 4 includes information on the size of PPG5. The value of τ4 is relevant to ÆS2æτ1/2 via the equation τ4 = 4πηWÆS2æτ3/2(kBT)-1, where ηW represents the viscosity of water.10,11 The ÆS2æτ1/2 evaluated from τ4 is also plotted in Figure 2. The ÆS2æτ1/2 value, which has a T dependence similar to RH, revealed the presence of a stepwise change in the size of PPG5 around 27 C. The observed small differences between ÆS2æ1/2 and ÆS2æτ1/2 was attributed to the accuracy of the relationship proposed by Oncley14a (the first part of eq 3) used for approximation to evaluate ÆS2æ1/2. Although another small contribution of the dielectric characteristics of PPG5 as an A-type polymer to the value of ε4 due to the presence of amide groups is possible, this contribution would depress the ε4 as described above. The fastest DR mode j = 1, identified at ω values from 0.77 1011 to 1.7 1011 s-1 (τ1 = 6-13 ps), was assigned to the relaxation mode of free water molecules. This is because εW0 and εW00 for pure water display a DR process similar to that of ε10 and ε100 in the same ω range as observed in Figure 1. It has been well documented that the depression in relaxation strength for the mode j = 1 in solution, relative to that of pure water, εW, is described by the equation ε1 1 -10 -3 V P c -10 -3 V W cm ¼ εW 1 þ 10 -3 V P c=2

Figure 3. The relationship between hydration number (mcoil) per PPG5 molecule in aqueous solution and T, evaluated from a ratio of DR strength of the mode j = 1 for aqueous PPG5 solutions relative to that of pure water, ε1εW-1. A schematic depiction illustrating changes in ÆS2æ1/2 and mcoil at the dehydration temperature, Tdh, is also inserted. The line in this figure is a guide for the eye.

The DR mode j = 2 was assigned to an exchange process of hydrated water molecules to PPG5. After a residence time of τ2, the water molecules hydrated to suitable sites of PPG5 would be replaced by free water molecules belonging to bulk water phase.14b This is because the normalized relaxation strength, ε2(cm)-1 g 1.4 M-1, was slightly greater than the value of 10-3V wεW = 1.3 M-1.14b Furthermore, τ2 was approximately 3 times as long as τ1 (= τw), which is typically observed in aqueous systems that include the exchange process of hydrated water molecules.9-11 Some people have referred to this relaxation mode as retarded rotation of water molecules in the hydration layers.9 Lastly, an anomalous dehydration behavior, together with a marked size reduction, was newly identified at Tdh = 27 C for PPG5 in aqueous solution. The value of M mcoil was reduced from around 9 down to approximately 3 above Tdh. The relationship Tdh ∼ Tt for PPG10 suggests that anomalous dehydration is a trigger for the TH-SC transition. However, the fact that Tdh < Tt was found for solutions of PPG15 (Tt ∼ 50 C)4 and PPG20 (Tt ∼ 65 C)4 implies that there might exist other factors that control the n dependence of Tt. The Tdh of PPG10 is speculated to be close to its Tt of 30 C. Above the Tdh, the single-coil state of PPG10 can exist bearing a small mcoil not so different from the value of mth in the triple-helix state. This effectively reduces the standard entropy change for the chemical reaction of TH-SC (eq 1) in the higher temperature range as observed in our previous study.11 However, PPG5 does not possess an enough enthalpy gain to construct the triple-helix state due to its too short length. Then, the dehydration behavior was only observed instead of the TH-SC transition triggered by the dehydration. A project to determine the n dependence of Tdh for PPGn in aqueous media is now in progress. In the temperature region below Tt, PPG5 exists in a single coil state with the help of a high hydration number (Mmcoil ∼ 9). On the other hand, the longer PPG10 prefers the triple-helix state as a result of the total free energy being lower than that of the single-coil state in aqueous media. It bears a lower value of Mmth (∼2).11

ð4Þ

where V p and V w are the partial molar volumes of solute particles and water molecules, respectively, and m is the number of hydrated water molecules per solute molecule (mcoil in this study).10,11 The precisely determined εW values as a function of temperature, T, have been published.16 The T dependence of mcoil evaluated from ε1εW-1 data via this relationship is shown in Figure 3. Because a stepwise reduction in mcoil was clearly observed at the dehydration temperature (Tdh ∼ 27 C), we conclude that the value of mcoil for PPG5 in aqueous solution dramatically changed at a temperature close to the Tt of PPG10. The change in mcoil looks similar to that in ÆS2æ1/2, ÆS2æτ1/2, and RH, as expected. A schematic depiction of the change in size and dehydration behavior for PPG5 in aqueous solution at Tdh is illustrated in Figure 3. The essential reason for the compacting in PPG5 should be the enhancement of intramolecular hydrophobic interaction induced by the anomalous dehydration behavior. According to Mrevlishvili,17 since an entropic change between a free and hydrated water molecule is estimated to be ca. 67 J K-1 mol-1, the entropic gain resulted from the dehydration of 85 water molecules from PPG5 observed at Tdh is evaluated to be ca. 5.7 kJ K-1 mol-1 per PPG5. This value seems compensating for the loss of entropy due to shrinkage of PPG5.


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decay rates, Γi, responsible for the contribution of each solute particle, i.20 The value Γi is related to the translational diffusion constant, Di, for a solute particle, i, in the manner Γi = Di|q|.2 Assuming the Stokes-Einstein relationship Di = kBT(6πηWRHi)-1 for aqueous solutions,21 where RHi is the hydrodynamic radius of the solute particle, the value of RHi for each solute particle, i, was calculated from the Γi value determined by the deconvolution procedure. The contribution from contaminating dust in sample solutions, providing RHi values larger than 100 nm, was ignored. The average hydrodynamic radius, RH, of PPG5 was determined as a function of temperature, T. It showed a relatively sharp distribution and was almost monodispersed, irrespective of T.

EXPERIMENTAL DETAILS Monodisperse PPG5 having purity higher than 99% was purchased from Peptide Institute, Inc. (Osaka, Japan) and used without further purification. Highly deionized water was obtained by an Elix-UV3 system (Millipore-Japan, Tokyo, Japan) with a specific residence higher than 15 MΩ cm and was used as a solvent. The concentrations of PPG5 in aqueous sample solutions were 7.36, 13.8, and 21.1 mM (0.94, 1.77 and 2.71% in weight, respectively). DR measurements were conducted over angular frequencies, ω, ranging from 6.28 106 to 1.26 1011 s-1 (1 MHz to 20 GHz) using two systems at several temperatures ranging from T = 10 to 40 C. The low ω range up to 1.88 1010 s-1 was covered by a 4287A RF LCR meter (Agilent Technologies, Santa Clara, CA) equipped with a homemade electrode cell, which possessed a vacant electric capacitance of C0 = 0.23 pF. In this system, ε0 and ε00 were evaluated by the formulas ε0 = CC0-1 and ε00 = (G - Gdc)C0-1ω-1, where C, G, and Gdc are the electric capacitance of the electrode cell filled with a sample, the conductivity of the sample, and the direct current conductivity due to ionic contaminant components, respectively. An 85070E dielectric material probe system (Agilent Technologies), with an 8720ES network analyzer (HewlettPackard, Palo Alto, CA), was used in the ω range from 3.14 108 to 1.26 1011 s-1. In this system, ε0 and ε00 were calculated by software provided by Agilent Technologies. Sample solution temperatures were controlled by circulating thermostatted water. Details of the measurement procedures are described elsewhere.18 The reproducibility of all the data was confirmed. Values of the partial molar volumes (V p) for PPG5 in aqueous solutions at T = 10 to 40 C were determined to be 902-925 cm3 mol-1 (slightly dependent on T). This measurement was performed using density measurements of the sample solutions that were taken with a DMA5000 densitometer (Anton Paar, Graz, Austria). Dynamic light scattering experiments were carried out on an aqueous solution of PPG5 at 21.1 mM over a temperature range of 10-50 C using an ELSZ plus system (Otsuka Electronics Co., Ltd., Osaka, Japan). The light source used was a laser diode (660 nm wavelength), and the scattering angle was 160. Consequently, the magnitude of the scattering vector of the system was |q| = 0.0249 nm-1. The obtained time (t) dependent autocorrelation functions (ÆI(0)I(t)æ = g(2)(t)) of scattered light intensity (I(t)) from sample solutions were analyzed with deconvolution software, which was based on the Levenberg-Marquardt algorithm19 supplied by Otsuka Electronics Co., Ltd. Theoretically, the autocorrelation function of scattered light is described as gð2Þ ðtÞ ¼ Bð1 þ f jgð1Þ ðtÞj2 Þ and X jgð1Þ ðtÞj ¼ gi e -Γi t

AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: shikata@ chem.sci.osaka-u.ac.jp.

ACKNOWLEDGMENT This work was supported by KAKENHI

(Grant-in-Aid for Scientific Research on Priority Area “Soft Matter Physics”) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and also another KAKENHI (Grant-in-Aid for Scientific Research (B) 21350064) from the Japan Society for the Promotion of Science. Otsuka Electronics Co., Ltd. kindly cooperated for dynamic light scattering experiments.

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i

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