Characteristic interaction of Ca2+ ions with elastin coacervate: Ion transport study across coacervate layers of α-elastin and elastin model polypeptide, (Val-Pro-Gly-Val-Gly)n

Ion transport characteristics across a macrocoacervate layer membrane composed of aqueous elastin model polypeptides with a specific repeating pentapeptide sequence, H-(Val-Pro-Gly-Val-Gly)_n-Val-OMe (n ≥ 40), were investigated. Transmembrane potential responses for NaCl, MgCl₂, and CaCl₂ concentration-cell systems were measured and examined systematically by comparing with those across a coacervate membrane composed of bovine neck ligamental α-elastin. In the case of the NaCl and MgCl₂ systems, potential responses across these protein liquid membranes were different noticeably from each other depending upon the molecular structure with and without charged peptide side chains, whereas in the CaCl₂ systems the transmembrane potential responses across the noncharged polypentapeptide coacervate membrane were comparable with those across the α-elastin coacervate membrane carrying both the positively and negatively charged amino acid residues as an amphoteric ion-exchange membrane. These results indicated that mechanisms of major Ca²⁺ ion transport are based on the specific and selective interactions with electrically neutral sites of elastin, such as the polypentapeptide backbone chain. © 1996 John Wiley & Sons, Inc.     

Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies

The temperature dependence of the composition of coacervate and equilibrium phases is examined for the polypentapeptide of elastin (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n in water. This provides for the development of a phase diagram. CD data is presented that provides information on associated polypeptide structure changes that, when added to previous CD, nmr, and dielectric relaxation data at lower water composition, allow construction of a phase-structure diagram of the polypentapeptide–water system. The molecular-weight dependence of phase change (coacervation) is included. The volume–composition studies as a function of temperature also provide temperature coefficients of expansion and of composition important in analyzing the mechanism of elasticity.     

Protein elasticity based on conformations of sequential polypeptides: The biological elastic fiber

Fibrous elastin of the biological elastic fiber is a cross-linked condensed state in which there is roughly one-half polypeptide and one-half water. The precursor protein tropoelastin, a chemical fragmentation product -elastin, and a sequential polypeptide (l·Val¹-l·Pro²-Gly³-l·Val⁴-Gly⁵) _n , which is a prominent primary structural feature of tropoelastin, are each soluble in all proportions in water at 20°C. On heating to physiological temperatures, each undergoes aggregation and forms a dense viscoelastic phase, which as the fiber itself, is about 60% water. This reversible heat-elicited condensed phase is called the coacervate. Circular dichroism studies show coacervation to be a process of increasing intramolecular order. Electron microscopy (light, scanning, and transmission) shows coacervation to be a process of increasing order intermolecularly. Thus a rise in temperature between 20 and 40°C results in an increase in order of the polypeptide. Coacervation is an inverse temperature transition, and the condensed state is anisotropic at the molecular level. Thermoelasticity studies in water on bovine ligamentum nuchae fibrous elastin and on -irradiation cross-linked polypentapeptide coacervates show increases in elastomeric force,f, over the same 20–40°C temperature range in which the inverse temperature transition gives rise to the coacervate, and the constancy off/T with temperature, once the transition is effectively completed, suggests a high-entropy component to the elastomeric force. Thus the data argue for an anisotropic-entropic elastomer.Detailed conformational studies on the polypentapeptide result in the development of a -spiral conformation in which there are regularly recurring -turns in loose helical array (a structure that forms on raising the temperature) and in which there are recurring dynamic suspended segments that are the focal point of large, low-energy oscillatory motions called librations. The structure gives rise to a librational entropy mechanism of elasticity wherein the amplitudes of the rocking motions become damped on stretching. This perspective is substantiated by dielectric relaxation studies on the coacervate state and by characterization of synthetic analogs of the polypentapeptide. Dielectric relaxation studies on a concentrated state of about 60% water show the development of a regular structure over the same temperature range as for the development of the coacervate state, and the development of the regular structure with increasing temperature is seen to parallel the development of elastomeric force with increasing temperature. Increasing elastomeric force coincides with increasing regularity of structure! Synthetic analogs of the polypentapeptide, designed to interfere with the librational processes of the suspended segment, impair elastic function, and an analog that makes the -turn more rigid results in increased elastic modulus. This development of a librational entropy mechanism for protein elasticity is a departure from the kinetic theory of rubber elasticity, the random network perspective that has dominated the traditional view of biological elasticity for the past several decades. The new perspective opens the way to insightful consideration of new elastomeric biomaterials with numerous biomedical applications.     

D-Ala5 analog of the elastin polypentapeptide. Physical characterization

The D-Ala5 analog, (L-Val1-L X Pro2-Gly3-L X Val4-D-Ala5) of the polypentapeptide (PPP) of elastin is synthesized and characterized by a series of physical methods. Carbon-13 and proton nuclear magnetic resonance spectroscopies are used to verify purity and, by means of solvent dependence of peptide C-O chemical shift and of temperature dependence of peptide NH chemical shift, to establish by comparison with the PPP of elastin the presence and increased stability of the Type II Pro2-Gly3 beta-turn. The temperature dependence of aggregation in water to form a viscoelastic phase called the coacervate is reported for several concentrations. Comparison of carbon-13 nuclear magnetic resonance spectra obtained under identical conditions for the coacervate states of the PPP of elastin and the D-Ala5 analog shows the effect of replacing the Gly5 residue by a D-Ala5 residue to be one of greatly restricting mobility of the polypeptide chain. Scanning electron micrographs, of the coacervate alone and of the coacervate cross-linked and compounded to a Dacron fabric before and after stress-strain studies, are reported which show the D-Ala5 PPP matrix to rupture during the stresses of drying and of stretching while wet. Thus, the effect of adding a methyl moiety to the Gly5 residue of the PPP of elastin is to decrease markedly the mobility of the polypeptide chain and to destroy elasticity. The results are presented as a test of the proposed librational entropy mechanism of elasticity of the PPP of elastin.     

Proton and carbon magnetic resonance studies of the synthetic polypentapeptide of elastin.

Proton and ¹³C magnetic resonance studies are reported on the synthetic polypentapeptide of elastin, HCO-(Val₍₁₎-Pro₍₂₎-Gly₍₃₎-Val₍₄₎-Gly₍₅₎)_n-Val-OMe, where n ∼- 18. Temperature and solvent dependence of peptide N$\text{H}$ chemical shift and solvent dependence of peptide carbonyl chemical shift were used to delineate these moieties preliminary to identification of secondary structure.Based on these studies it is proposed, for the organic solvents of dimethyl sulfoxide, methanol, and low-temperature trifluoroethanol, that dynamic hydrogen bonds form in order of decreasing frequency of occurrence between the Val₍₁₎$\text{C}$O and the Val₍₄₎ N$\text{H}$ (a β-turn), between the Gly₍₃₎ N$\text{H}$ and the Gly₍₅₎$\text{C}$O (an 11-atom, hydrogen-bonded ring), and a more limited interaction between the Gly₍₃₎$\text{C}$O and the Gly₍₅₎ N$\text{H}$ (a γ-turn).Arguments are presented that relate the conformational features proposed above to the coacervate, which is a filamentous state.     

Temperature-correlated force and structure development in elastomeric polypeptides: the Ile1 analog of the polypentapeptide of elastin

The Ile¹ analog of the polypentapeptide of elastin, (L · Ile¹-L · Pro²-Gly³-L · Val⁴-Gly⁵)_n, abbreviated as Ile¹-PPP, was synthesized with n > 100 to determine the effect of the increased hydrophobicity of the pentamer resulting from Val¹ replacement by Ile¹ on the previously characterized inverse temperature transition of the polypentapeptide of elastin (PPP). Ile¹-PPP, dissolved in water at 4°C, was found to aggregate, forming a viscoelastic coacervate on raising the temperature. The onset of aggregation was 8°C for Ile¹-PPP, as compared to 24°C for PPP. Characterization by CD demonstrated an increase in intramolecular order on raising the temperature from 8°C to 25°C, and demonstrated similar conformations for PPP and Ile¹-PPP before and after their respective transitions. The CD-characterized transition also occurred at a temperature some 15°C lower than that of PPP. By means of 20-Mrad γ-irradiation cross-linking of the Ile¹-PPP coacervate, an elastomeric matrix was formed with an elastic modulus, similar to that of 20-Mrad cross-linked PPP. The temperature dependence of elastomeric force of cross-linked Ile¹-PPP showed an abrupt increase from essentially zero at 8°C to three-quarters of full force at 10°C and essentially full force by 20–25°C. This development of elastomeric force for the more hydrophobic Ile¹-PPP matrix, which parallels the increase in intramolecular order characterized by the CD studies, also occurs at a temperature some 15°C lower than that for the PPP matrix. Thus, in these elastomeric polypeptides, development of elastomeric force is coupled to an inverse temperature transition, the temperature of which depends inversely on the hydrophobicity of the constituent pentamer. It appears that a series of elastomeric polypeptide biomaterials are possible in which the temperature over which elastomeric force develops can be varied.     

Solubilized elastin substrate for continuous fluorimetric assay of kinetics of elastases

Elastolysis is central to progression of emphysema and aortic aneurysms. Characterization of steady-state enzyme kinetics of elastolysis is fettered by the insolubility of mature elastin and the polydispersity of solubilized elastin. We prepared a fluor-tagged, 100-kDa fraction (fEln-100) from commercial α-elastin. It is soluble, less heterogeneous in mass, cross-linked like mature elastin, and likely to retain the capacity of α-elastin to self-assemble. fEln-100 has introduced the ability to compare quantitatively the apparentk_cat and K_m of elastases. For example, metalloelastase (MMP-12) displays higher apparent affinity for fEln-100, while MMP-2 displays faster catalytic turnover.     

Nuclear overhauser enhancement demonstration of the type II β-turn in repeat peptides of tropoelastin

Nuclear Overhauser enhancement (NOE) experiments have been performed with the elastin peptides, namely; HCO-Val₁-Pro₂-Gly₃-Gly₄-OMe, t-Boc-Val₁-Pro₂-Gly₃-Val₄-Gly₅-OMe and t-Boc-Val₆-Ala₁-Pro₂-Gly₃-Val₄-Gly₅-OMe in DMSO-d₆. An NOE of approximately 10% was observed between the αCH of Pro₂ and the NH of Gly₃ involved in the β-turn of all three peptides. This finding shows the close proximity of two aforementioned protons and thus shows the occurrence of Type II β-turn in the repeat elastin peptides. The intermolecular distances are calculated and compared with the distances obtained from other model systems.     

Conformational transitions of solubilized trout elastins

CD measurements on polypeptides obtained from trout elastin by mild acid hydrolysis (α-elastin) or cyanogen bromide cleavage (CB-elastin) are compared with the results of analogous studies on bovine α-elastin. The spectra show that bovine and trout elastin, despite their compositional differences, have a similar β-turn content. This is discussed with reference to the molecular evolution of the protein and the function of β-turns in elastin.     

Differential scanning calorimetry studies of NaCl effect on the inverse temperature transition of some elastin-based polytetra-, polypenta-, and polynonapeptides

Differential scanning calorimetry studies of the effect of NaCl on protein-based polymer self-assembly has been carried out on six elastin-based synthetic sequential polypeptides- i.e., the polypentapeptide (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n and its more hydrophobic analogues (L -Leu¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n and (L -Val¹-L -Pro²-L -Ala³-L -Val⁴-Gly⁵)_n; the polytetrapeptide (L -Val¹-L -Pro²-Gly³-Gly⁴)_n and its more hydrophobic analogue (L -IIe¹-L -Pro²-Gly³-Gly⁴)_n; and the polynonapeptide (a pentatetra hybrid), (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵-L -Val⁶-L -Pro⁷-Gly⁸-Gly⁹)_n. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order of the polypentapeptide, as the temperature is raised from below to above that of the transition, has been repeatedly observed using different physical characterizations. In the present experiments, it is observed that the transition temperatures of the polypeptides studied are linearly dependent on NaCl concentration. The molar effectiveness of NaCl in shifting the transition temperature ΔT_m/[N], is about 14°C/[N], with the dependence on peptide hydrophobicity being fairly small. Interestingly, however, the δΔQ/ [N] does depend on the hydrophobicity of a polypeptide.     

Dielectric relaxation studies on bovine ligamentum nuchae

Dielectric relaxation studies of bovine ligamentum nuchae are reported over the frequency range of 1 MHz to 1 GHz and over the temperature range of 23–48°C. A temperature-dependent relaxation process was observed at low megahertz-frequency with the correlation time of around 40 ns. The result is quite similar to that of a synthetic polypentapeptide (VPGVG) and of α-elastin. The relaxation is proposed to arise in part from the peptide libration within the polypentapeptide of bovine ligamentum nuchae.     

Entropic elastic processes in protein mechanisms. II. Simple (passive) and coupled (active) development of elastic forces

The first part of this review on entropic elastic processes in protein mechanisms (Urry, 1988) demonstrated with the polypentapeptide of elastin (Val¹-Pro²-Gly³-Val⁴-Gly⁵)_n that elastic structure develops as the result of an inverse temperature transition and that entropic elasticity is due to internal chain dynamics in a regular nonrandom structure. This demonstration is contrary to the pervasive perspective of entropic protein elasticity of the past three decades wherein a network of random chains has been considered the necessary structural consequence of the occurrence of dominantly entropic elastomeric force. That this is not the case provides a new opportunity for understanding the occurrence and role of entropic elastic processes in protein mechanisms. Entropic elastic processes are considered in two classes: passive and active. The development of elastomeric force on deformation is class I (passive) and the development of elastomeric force as the result of a chemical process shifting the temperature of a transition is class II (active). Examples of class I are elastin, the elastic filament of muscle, elastic force changes in enzyme catalysis resulting from binding processes and resulting in the straining of a scissile bond, and in the turning on and off of channels due to changes in transmembrane potential. Demonstration of the consequences of elastomeric force developing as the result of an inverse temperature transition are seen in elastin, where elastic recoil is lost on oxidation, i.e., on decreasing the hydrophobicity of the chain and shifting the temperature for the development of elastomeric force to temperatures greater than physiological. This is relevant in general to loss of elasticity on aging and more specifically to the development of pulmonary emphysema. Since random chain networks are not the products of inverse temperature transitions and the temperature at which an inverse temperature transition occurs depends on the hydrophobicity of the polypeptide chain, it now becomes possible to consider chemical processes for turning elastomeric force on and off by reversibly changing the hydrophobicity of the polypeptide chain. This is herein called mechanochemical coupling of the first kind; this is the chemical modulation of the temperature for the transition from a less-ordered less elastic state to a more-ordered more elastic state. In the usual considerations to date, development of elastomeric force is the result of a standard transition from a more-ordered less elastic state to a less-ordered more elastic state. When this is chemically modulated, it is herein called mechanochemical coupling of the second kind. For elastin and the polypentapeptide of elastin, since entropic elastomeric force results on formation of a regular nonrandom structure and thermal randomization of chains results in loss of elastic modulus to levels of limited use in protein mechanisms, consideration of regular spiral-like structures rather than ramdom chain networks or random coils are proposed for mechanochemical coupling of the second kind. Chemical processes to effect mechanochemical coupling in biological systems are most obviously phosphorylation-dephosphorylation and changes in calcium ion activity but also changes in pH. These issues are considered in the events attending parturition in muscle contraction and in cell motility.     

Studies on the conformations and interactions of elastin. Proton magnetic resonance of the repeating tetramer

Proton magnetic resonance studies at 220 MHz were carried out on synthetic polymers of the repeating tetrapeptide of elastin. Temperature dependence and solvent-mixture dependence of peptide proton chemical shifts were determined for both linear polymers, N-formyl-(Val-Pro-Gly₁-Gly₂)_n-Val OMe where n ? 8 and 40, and for cyclic polymers (Val-Pro-Gly₁-Gly₂)_n, where n = 3 and 4. The Gly₂ NH was found to be solvent shielded. In addition, by studying the polymers Boc-Val-Pro-Gly₁-Gly₂-OH, H-Pro-Gly₁-Gly₂-Val OMe, H(Pro-Gly₁-Gly₂-Val)₃OH, and others, it was demonstrated that the Val C–O immediately preceding the Gly₂ NH in the sequence was required for solvent shielding. Also the Gly₂ NH resonance is found at higher field than the Gly₁ NH resonance. This provides the basis for proposing a β turn in which the Pro and Gly₁ residues form the corners, i.e., residues i + 1 and i + 2, and in which the Gly₂ NH, residue i + 3 hydrogen bonds to the carbonyl of residue i, the Val residue. Studies on methanol–water solvent systems indicated retention of the β turn as a significant conformational feature. This suggests that the β turn occurs in the elastic fiber, which contains about 60% water but which utilizes association of hydrophobic groups as a primary force in fibrogenesis.     

Elastin coacervate as a matrix for calcification

A calcifying system has been developed which, for the first time, demonstrates the capacity of α-elastin, as the coacervate, to initiate calcification in an $\text{in}\text{vitro}$ system. This is achieved with blocking the amino and carboxyl charged groups by N-formylation and O-methylation. The calcification system is of particular interest as it demonstrates the initiation of calcification after blockage of the charged amino and carboxyl groups. This leaves either neutral sites or the charged pyridinium moieties as the sites of initiation.     

Effects of metal cations on coacervation of α-elastin

The quasi-elastic light scattering studies were carried out to investigate effects of metal cations such as Ca²⁺ and Na⁺ on the early stage of Coacervation process of α-elastin, a chemical fragmentation product originated from the biological elastomeric protein elastin, in aqueous solutions. In particular, our attention was focused on changes of two types of dynamical behaviors found in the earlier work, which are a remarkable increase and a monotonous decrease in the hydrodynamic radius R of molecules with temperature for critical and off-critical concentrations of α-elastin, respectively. For the critical α-elastin concentration, an addition of Ca²⁺ was found to exert little effects on the steep temperature profile of R observed in the absence of Ca²⁺. On the other hand, an addition of a slight amount of Na⁺ resulted in a monotonous decrease in R , but its further addition restored a remarkable increase in R similar to the critical behaviors in the salt-free system. In the case of off-critical sample, the addition of either Ca²⁺ or Na⁺ above a certain concentration induced a change in R from a monotonous decrease to a remarkable increase. For both critical and off-critical concentrations of α-elastin, Ca²⁺ and Na⁺ brought about an elevation and a lowering of the temperature at which the sample started to be turbid, respectively. © 1995 John Wiley & Sons, Inc.     

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate

A method for the determination of elastase activity is described which uses soluble α-[³H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10^?4mN-α-tosyl-l-lysyl chloromethane and 10^?4m tosyl-l-phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.     

Carbon-13 NMR relaxation studies demonstrate an inverse temperature transition in the elastin polypentapeptide

Carbon-13 NMR longitudinal relaxation time and line-width studies are reported on the coacervate concentration (about 60% water by weight) of singly carbonyl carbon enriched polypentapeptides of elastin: specifically, (L-Val1-L-[1-13C]Pro2-Gly3-L-Val4-Gly5)n and (L-Val1-L-Pro2-Gly3-L-Val4-[1-13C]Gly5)n. On raising the temperature from 10 to 25 degrees C and from 40 to 70 degrees C, carbonyl mobility increases, but over the temperature interval from 25 to 40 degrees C, the mobility decreases. The results characterize an inverse temperature transition in the most fundamental sense of temperature being a measure of molecular motion. This transition in the state of the polypentapeptide indicates an increase in order of polypeptide on raising the temperature from 25 degrees C to physiological temperature. This fundamental NMR characterization corresponds with the results of numerous other physical methods, e.g., circular dichroism, dielectric relaxation, and electron microscopy, that correspondingly indicate an increase in order of the polypentapeptide both intramolecularly and intermolecularly for the same temperature increase from 25 to 40 degrees C. Significantly with respect to elastomeric function, thermoelasticity studies on gamma-irradiation cross-linked polypentapeptide coacervate show a dramatic increase in elastomeric force over the same interval that is here characterized by NMR as an inverse temperature transition. The temperature dependence of mobility above 40 degrees C indicates an activation energy of the order of 1.2 kcal/mol, which is the magnitude of barrier expected for elasticity.     

Secondary structure of peptides: 15. 13C n.m.r. CP/MAS study of solid elastin and proline-containing copolyesters

In order to study the chemical shifts and the cis—trans isomerism of prolyl units neighbouring glycine or other amino acids, 75.4 MH_z¹³C nuclear magnetic resonance (n.m.r.) cross-polarization/magic angel spinning (CP/MAS) spectra of the following solid oligopeptides and sequence polypeptides were measured: Z-Gly-Pro-OH,Z-Gly-Pro-Gly-Gly-OEt,Z-Gly-Pro-Ala-Ala-OMe,(Gly-Pro-Gly)_n,(Gly-Pro-Ala)_n,(β-Ala-Pro)_n and $\text{(δ-Ava-Pro)}{}_{\mathrm{<mtext>n</mtext>}}\text{(δ-Ava=δ-}\text{aminovaleric acid}$). Whereas all these oligo- and polypeptided contain exclusively trans X-Pro bonds, both cis and trans peptide bonds were found in a polypeptide prepared by copolymerization of glycine- and $\text{proline-}\text{N}\text{-carboxyanhydrides}$ in pyridine. On the basis of these model compounds, the ¹³C n.m.r. CP/MAS spectra of solid elastin allows the following conclusions. Almost all X-Pro bonds assume the trans conformation, most alanine and leucine units form α-helical chain segments, whereas only a small fraction of β-sheet structure is present. A 30.3 MHz ¹⁵N n.m.r. CP/MAS spectrum of solid elastin confirms that ～25% of all amino acids assume the α-helical structure. A model of elastin is discussed consisting of an amorphous phase, α-helical chain segments and helical segments of still unknown pitch.     

Studies on the interaction of cholesterol with soluble and insoluble elastins

The exchange of ³H-cholesterol in an aqueous solution of taurodeoxycholate with insoluble elastin and kappa-elastin peptides has been quantitatively studied. At higher cholesterol concentrations, and in similar experimental conditions, 0.3 μg and 0.2 μg of cholesterol could be adsorbed, respectively, on to 1 mg insoluble elastin and 1 mg kappa-elastin coacervate. Therefore, the above amounts of cholesterol fixed per mg elastin represent minimal estimates. The replacement of Na⁺ ions by Ca²⁺ ions increased the cholesterol binding both of fibrous and of soluble elastins. The binding curves tend towards a limiting value in the presence of Na⁺, but no saturation effect was observed in the presence of Ca²⁺. The replacement of sodium ions by calcium ions in the taurodeoxycholate solutions also lowered the coacervation temperature of the kappa-elastin peptides. Fibrous elastin retained more cholesterol at 65°C than at 37°C, suggesting the importance of hydrophobic interactions in cholesterol elastin association. These results suggest that conformational changes could be induced in both soluble and insoluble forms of elastin by calcium ions increasing their affinity for cholesterol. This enhancement of cholesterol fixation by elastin in the presence of Ca²⁺ ions may well have a physiopathological importance. So does also the fact that elastin-peptides exhibit a higher affinity in the presence of Ca²⁺ for cholesterol than insoluble elastin, suggesting the possibility of increased cholsterol (and calcium) fixation as elastin is degraded in situ by elastases.