52
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of polypeptides [1]. This study indicates that the specific conformation produced by hydrophobic interactions of the polypeptide sequence might be important for the cross-linking process. The aggregation process of polypeptide I was investigated under the three different heating processes described in previous chapters (“Slow heating”,
“Fast heating” and “Heat shock”) using circular dichroism (CD) spectroscopy and the
hydrophobic characteristics were monitored using a fluorescent reagent (ANS).
Li et al., simulated the molecular basis for the extensibility of the (GVGVP)18
elastin model polypeptide by molecular dynamics (MD). MD is a form of computer simulation in which atoms and molecules are allowed to interact for a specified period of time by approximations of physical parameters. This provides a view of the motion of the atoms and molecules [2-4]. The principles of MD have also been described as
“statistical mechanics by numbers” and “Laplace's vision of Newtonian mechanics.”
MD is used to predict biomolecular interactions and provide insights into molecular motion at the atomic scale [7-8]. Therefore, in order to characterize the aggregation process, the aggregation of three molecules of (GVGVP)18 in water was simulated by MD to identify the amino acid residues involved in hydrophobic interactions among the three molecules. The reactivity of single amino acid mixtures containing a combination of Val, Gly, or Pro were examined by amino acid analysis after gamma-ray irradiation
54
to identify the cross-linking points.
Materials and Methods Materials
Elastin protein-based polypeptide I: (GVGVP)251 was supplied from Prof. Urry (Minnesota University) and BRL (Bioelastic Research, Ltd.) through Bioelastic Japan Co. [5]. 8-Anilino-1-naphthalenesulfonic acid magnesium salt (ANS-Mg) ((C6H5NHC10H6SO3)2Mg・H2O) was obtained from Nacalai Tesque, Inc. Sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4) potassium dihydrogen phosphate (KH2PO4), valine, glycine, and proline were obtained from Wako Pure Chemical Industries Ltd.
Circular dichroism spectroscopy of aggregated polypeptide I
The polypeptide structure was analyzed using circular dichroism (CD) spectroscopy after heating to 42ºC using three different heating processes, “Slow heating”, “Fast heating”, and “Heat shock”.
Aqueous solutions of the polypeptide I were prepared at 4ºC and heated to 42ºC Slow heating: 25 mg of the polymer was dissolved in 5 ml of deionized water in a stoppered Pyrex glass test tube and cooled on ice. The polymer solution was slowly
55
heated from 4ºC to 42ºC at a rate of 1.3°C/min while stirring in a water bath (EYELA SB-350) with a magnetic stirrer (EYELA RCN-3D) at a speed of 600 rpm.
Fast heating: 25 mg of the polymer was dissolved in 5 ml of deionized water at room temperature (5 mg/ml) in a stoppered Pyrex glass test tube and cooled to 4ºC. The polymer solution was quickly heated from 4ºC to 42ºC for 10 min. while stirring in a water bath (EYELA SB-350) with a magnetic stirrer (EYELA RCN-3D) at a speed of 600 rpm.
Heat shock: 25 mg of the polymer was dissolved in 1 ml of deionized water at room temperature and cooled to 4ºC. Drops of the solution at 4ºC were added to a 4-fold volume of water at 42ºC in a stoppered Pyrex glass test tube using a 1-ml tuberculin syringe with a 27-G needle (0.4 × 19 mm) (TERUMO) while stirring in a water bath (EYELA SB-350) with a magnetic stirrer (EYELA RCN-3D) at a speed of 600 rpm.
The heated samples were diluted to 0.5 mg/ml in deionized water to bring the absorbance within the measurable range (<1.0) for CD spectroscopy.
CD spectra were measured using a JASCO J-720 spectropolarimeter (JASCO Corporation, Tokyo, Japan) with a standard analysis program. The temperature was controlled using a recirculating water bath (42ºC), and the spectra were recorded over the wavelength range 190–250 nm using a cell with a path length of 0.1 cm at a
56
scanning speed of 50 nm/min. The spectral bandwidth was 1.0 nm, and the integration time was 5 s. Data are represented as molar ellipticities ([θ] deg cm2/d mol).
Gamma irradiation
The heated samples were placed in thermos bottles (TIGER MWE-C350) and irradiated with 60Co gamma-rays at doses of 8.5–30 kGy using the 60Co-gamma irradiation pool at Osaka Prefecture University (dose rate: 10.5 kGy/h). The temperature of the sample was maintained at 42ºC during irradiation. The gamma irradiated samples were subjected to CD spectroscopy in the same manner as described above.
Fluorescence spectroscopy of polypeptidemixed with 8-anilino-1-naphthalenesulfonic
acid magnesium salt
The aggregates of polypeptide Iprepared by each heating process were mixed with 8-anilino-1-naphthalenesulfonic acid magnesium salt (ANS-Mg) in 10 mM PBS. The concentrations of the polypeptide and the ANS-Mg were 5 mg/ml and 0.1 mM, respectively. Fluorescence spectroscopy measurements were performed using a Shimadzu spectrofluorophotometer RF-5000 (Shimadzu Co. Ltd.). The emission spectra at 42ºC were measured with excitation at 370 nm [6].
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Analyzing the aggregation of the (GVGVP)18 with molecular dynamics simulation
Molecular dynamics simulations were performed using an energy refinement (AMBER) force field [9] with the Groningen Machine for Chemical Simulations (GROMACS 3.3.0) software package [10] using the GROMACS 96 force field [11] and the TIP3P water model. Each molecular system simulated was described using an all-atoms representation of both the polypeptide and water. The idealized β-spiral structure proposed by Urry was used as the starting structure for all simulations [2, 12].
The initial structure was immersed in a periodic water box with a truncated octahedral shape. Electrostatic energy was calculated using the particle mesh Ewald method [13].
Cutoff distances for the calculation of the Coulomb and van der Waals interactions were set at 0.9 nm. After energy minimization using a steepest descent method, the system was equilibrated at 315 K and normal pressure for 20 picoseconds (ps) under the LINCS constraints [14] for all bonds. The system was coupled to the external bath by the Berendsen pressure and v-rescale temperature coupling [15]. The final MD calculations were performed under the same conditions used for the 10000 ps calculations. The results were analyzed using the standard software provided by the GROMACS package.
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Amino acid analysis of irradiated single amino acid mixtures
Single amino acid mixtures combining single amino acids such as Val-Val, Pro-Pro, Gly-Gly, Val-Pro, Val-Gly, Pro-Gly, and Val-Pro-Gly were prepared. Each mixture was dissolved in deionized water (concentration:1720 nmol/ml) and irradiated with 60Co gamma-rays at a dose of 30 kGy as described above. After gamma irradiation, these samples were freeze dried and redissolved in 0.1 N HCl (concentration: 200 nmol/ml). Amino acid analyses were performed with a HITACHI Model L-8500 analyzer using MCl buffer and ninhydrin.
Results and Discussion
CD spectroscopy of polypeptide I
Polypeptide I prepared by the three different heating processes (“Slow heating”,
“Fast heating” and “Heat shock”) was characterized by CD spectroscopy (0.5 mg/ml in
deionized water) at 42ºC (above CP). The CD spectra of the samples are shown in Fig.
4-1. The absorbance of the aggregated polypeptides in an aqueous solution was under OD 1.0, and the intensities of bands of the CD spectra were within the measurable range.
As shown in Fig. 4-1, the CD spectrum of the sample prepared by “Heat shock” has a negative band near 225 nm which has been defined as a characteristic band of a type-II
59
β-turn by Urry et al., [16]. This spectrum is distinct from the others.
Fig. 4-1 Circular dichroism (CD) spectra of polypeptide I before irradiation. The concentrations of all the samples are 0.5 mg/ml. The dotted line is the sample prepared by “Slow heating”. The broken line is the sample prepared by “Fast heating”. The solid line is the sample prepared by “Heat shock”.
CD spectroscopic measurements were also obtained for gamma-ray cross-linked samples prepared using the optimal “Heat shock” process with a 27-G needle. The intensity of the negative band near 225 nm was reduced after gamma irradiation as shown in Fig. 4-2. This suggests that the type-II β-turn structure was partially distorted by gamma irradiation.
The type-II β-turn is formed by the hydrogen–bonded ring at the Val-Pro-Gly-Val portion. These Val residues are closer to each other [16-21]. The MD simulation of
60
Fig. 4-2 Circular dichroism (CD) spectra of polypeptide I prepared by “Heat shock”
before and after irradiation. The concentrations of all the samples are 0.5 mg/ml. The solid line is before irradiation. The dotted line is after irradiation.
(VPGVG)18 in water showed that side-chain contact at 42ºC is 1.5 times that of the side-chain contact at 20°C [2]. This is caused by an increase in hydrophobic interactions between aliphatic side-chains. From this evidence, it is suggested that the cross-linking might occur between Val and Val. Therefore, as shown in Fig. 4-1, the formation of more type-II β-turns within the polypeptide I chains by the “Heat shock” process would be effective in forming the organized structure and stimulating cross-linking between the polypeptides to yield stable nanoparticles by gamma irradiation. After gamma irradiation, the formation of the type-II β-turn within the polypeptide I chains was
61
observed in the cross-linked nanoparticles. The extent of formation of the type-II β-turn was less than the amount observed prior to gamma irradiation.
Fluorescence spectrometry for the polypeptide I
The hydrophobic characteristics of the polypeptide following aggregation by various heating processes were investigated using ANS-Mg as a probe. This fluorescence reagent interacts with hydrophobic side-chains of the polypeptides. As
Fig. 4-3 Fluorescence spectra of 0.1 mM of the polypeptide IwithANS-Mg in PBS (excitation at 370 nm). The spectra were measured at 42ºC. The dotted line is the sample prepared by “Slow heating”. The broken line is the sample prepared by “Fast heating”. The solid line is the sample prepared by “Heat shock”. The gray line is ANS alone.
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shown in Fig 4-3, the aggregates prepared by the “Heat shock” process showed greater fluorescence intensity than the “Fast heating” and “Slow heating” processes. This indicate that “Heat shock” might induce a greater extent of hydrophobic side-chain interactions between the polypeptides.
MD simulation for polypeptide I
In order to determine the optimal conditions for the aggregation process and formation of gamma-ray cross-linking between the molecules, a MD simulation of the aggregation of three molecules of (GVGVP)18 was performed. Urry and co-workers have proposed that the VPGVG unit adopts a type-II β-turn (Fig. 4-4 (A)) structure around the Pro-Gly pair. Repetition of this β-turn gives rise to a right-handed helix which is known as a β-spiral (Fig. 4-4(B)) [12, 16-21]. Therefore, the simulation was initiated from the β-spiral structure for three molecules of (GVGVP)18 in water from 0 to 10000 ps at 42ºC. During this time period, 60% of the type-II β-turn was maintained within the three molecules of (GVGVP)18. The torsion angles of Gly23 Ψ and Val24 ϕ in this structure predicted that a type-II β-turn was formed as shown in Fig 4-5. The angles (Gly23 Ψ -16.6° and Val24 ϕ -138°) measured at 10000 ps were generally maintained over the range of 0 to 10000 ps. These angles are favorable for formation of
63
the type-II β-turn [2]. The distances in this structure predicted the formation of a hydrogen bond between Val21_O and Val24_H as shown in Fig. 4-6. The distance between these atoms was 1.94 nm at 10000 ps and is generally maintained from 0 to 10000 ps. This distance is favorable for formation of the type-II β-turn [21]. Moreover, Fig. 4-7 shows the assembly status of the threemolecules of (VPGVG)18 from 0 to 10000 ps. Within the initial 20 ps, the (VPGVG)18 molecules undergo a change in their conformation from the initial -spiral structure and began to aggregate. However, the three molecules of (VPGVG)18 did not become entangled amongst themselves.
64
Fig. 4-4 (A) Model for the type-II β-turn of VPGVG. The white lines indicate the presence of a hydrogen bond. (B) Model for the β-spiral of (GVGVP)18. The red spheres and sticks are oxygen (O), the white spheres and sticks are hydrogen (H), the purple spheres and sticks are nitrogen (N) and the gray spheres and sticks are carbon (C) [12, 16-21].
65
Fig. 4-5 The angles of Gly23 Ψ and Val24 ϕ. Representative dynamics of type-II β-turn formation between Val21-Val24 at 42°C. The black line is Gly23 Ψ and the gray line is Val24 ϕ.
Fig. 4-6 The distance between Val21_O and Val24_H. Representative dynamics of type-II β-turn formation between Val21- Val24 at 42ºC. Val21_O and Val24_H predict the formation of a hydrogen bond.
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Fig.4-7 Conformational behavior of the three molecules of (VPGVG)18 as probed by dynamics simulations in water at 42ºC. The simulations were initiated from a β-spiral structure. Wireframes are Gly, spheres and sticks are Val, and space-fills are Pro.
67
Fig. 4-8 Inter-atomic distances for the carbon (C) atoms of Val-Val (expect for C of C=O) within the three molecules of (VPGVG)18 as probed by dynamics simulations in water at 42ºC. The simulations were initiated from the β-spiral structure. The data-assembled distance of the carbon-carbon bond is less than 1.0 nm.
68
Fig. 4-9 Inter-atomic distances for the carbon (C) atoms of Pro-Pro (expect for C of C=O) within the three molecules of (VPGVG)18 as probed by dynamics simulations in water at 42ºC. The simulations were initiated from the β-spiral structure. The data-assembled distance of the carbon-carbon bond is less than 1.0 nm.
69
Fig. 4-10 Inter-atomic distances for the carbon (C) atoms of Gly-Gly (expect for C of C=O) within the three molecules of (VPGVG)18 as probed by dynamics simulations in water at 42ºC. The simulations were initiated from the β-spiral structure. The data-assembled distance of the carbon-carbon bond is less than 1.0 nm.
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Fig. 4-11 Inter-atomic distances for the carbon (C) atoms of Val-Pro (expect for C of C=O) within the three molecules of (VPGVG)18 as probed by dynamics simulations in water at 42ºC. The simulations were initiated from the β-spiral structure. The data assembled distance of the carbon-carbon bond is less than 1.0 nm.
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Fig. 4-12 Inter-atomic distances for the carbon (C) atoms of Val-Gly (expect for C of C=O) within the three molecules of (VPGVG)18 as probed by dynamics simulations in water at 42ºC. The simulations were initiated from the β-spiral structure. The data assembled distance of the carbon-carbon bond is less than 1.0 nm.
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Fig. 4-13 Inter-atomic distances for the carbon (C) atoms of Pro-Gly (expect for C of C=O) within the three molecules of (VPGVG)18 as probed by dynamics simulations in water at 42ºC. The simulations were initiated from the β-spiral structure. The data assembled distance of the carbon-carbon bond is less than 1.0 nm.
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In the zwitterions, all OH radicals react preferentially at the α-carbon bond of simple amino acids by removal of a hydrogen atom to form a free radical [22-24]. While Gly is a simple amino acid, Val and Pro are not. Therefore all of the carbon inter-atomic distances were calculated (with the exception of C=O) for the Val-Val, Pro-Pro, Gly-Gly, Val-Pro, Val-Gly, and Pro-Gly units within the three molecules of (VPGVG)18
as probed by dynamics simulations in water at 42ºC. The data-assembled distance of the carbon-carbon bond is less than 1.0 nm. This indicates that a pair of carbon atoms in close proximity might give rise to a conjugate. As a result, within the 9000–9999 ps range, the distribution of the distances between the carbon-carbon atoms of Val-Val, Pro-Pro, Gly-Gly, Val-Pro, Val-Gly, and Pro-Gly were closer than in the 0-999 ps range (Fig 4-8-4-13). In the 9000–9999 ps range, the distribution of the distances of the carbon-carbon atoms between Val-Val, Val-Pro, Gly-Val, and Pro-Gly were closer than the others. It is suggested that hydrophobic amino acids such as Val and Pro give rise to intermolecular interactions within the (VPGVG)18 polypeptide.
Reactivity of single amino acids with respect to gamma irradiation
The reactivity of Val, Pro, or Gly with respect to gamma irradiation were examined for mixtures containing Val, Pro, and Gly which might be assembled closer to each
74
other within the (GVGVP)18 molecules according to the MD simulation of the aggregation of the three polypeptides. As shown in Table 4-1, the amounts of single Val and Pro are greatly reduced in each solution containing the combinations of Val-Val, Pro-Pro, Gly-Gly, Val-Pro, Val-Gly, Pro-Gly, and Val-Pro-Gly after irradiation. Val and Pro have high rate constants for the reactions of OH radicals in pH 6.6–6.8 (Table 4-2). Moreover, the comparison of Val and Pro indicates that the reduction of the amount of Val is greater than the reduction in the amount of Pro (Table 4-1). According to these results, it is suggested that Val may be cross-linked by gamma irradiation to a greater extent than Pro.
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Table 4-1. Extent of reduction of amino acids (Val Gly and Pro) by gamma irradiation.
Each amino acid was mixed as follows; I: Val-Val, II: Val-Pro, III: Gly-Gly, IV:
Val-Pro, V: Val-Gly, VI: Pro-Gly and VII: Val-Pro-Gly. The mixtures were subsequently irradiated.
Group Amino acid Amount of
amino acid Before (n mol/ml)
Amount of amino acid
After (n mol/ml)
Reactivity (%)
I: Val-Val Val 190 0 100
II: Pro-Pro Pro 238 0 100
III: Gly-Gly Gly 202 26 87
IV: Val-Pro Val 211 12 94
IV: Val-Pro Pro 231 63 72
V:Val-Gly Val 207 0 100
V:Val-Gly Gly 188 167 11
VI:Pro-Gly Pro 231 0 100
VI:Pro-Gly Gly 207 170 18
VII:Val-Pro-Gly Val 208 19 91
VII:Val-Pro-Gly Pro 228 75 67
VII:Val-Pro-Gly Gly 194 181 7
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Table 4-2. Rate constants for the reactions of OH radicals with the amino acids (pH 6.6–6.8) [25].
Amino acid Rate constant (k)
Val 6.6×108
Pro 6.5×108
Gly 1.7×107
Conclusions
The formation of type-II β-turns within the polypeptide I chains by the “Heat shock” process is effective in forming the organized structure which stimulates the process of cross-linking the polypeptides to yield stable nanoparticles by subsequent gamma irradiation. Moreover, the intermolecular contact between hydrophobic side-chains such as Val may be important for efficient formation of cross-linked structures.
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Chapter 5 Loading and timed release of drugs by using the cross-linked