Marianna M. Long

Circular dichroism and absorption of the polytetrapeptide of elastin: A polymer model for the β-turn

Summary Circular dichroism and absorption data are reported for the polytetrapeptide of elastin, HCO-(Val-Pro-Gly-Gly) 40 -Val-OMe. The circular dichroism pattern, with a negative band at 224 nm, a positive band at 205 nm and a second negative band below 190 nm, is distinct from the circular dichroism patterns exhibited by the α-helix, β-pleated sheet and random coil conformations. As previous nuclear magnetic resonance studies have demonstrated that the polytetrapeptide is comprised of repeating β-turns, the absorption and circular dichroism data is presented as a model system for the β-turn conformational feature which is commonly found in globular proteins. The absorption and circular dichroism data are resolved into component Gaussian curves.

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

Abstract 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 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) C O and the Val(4) N H (a β-turn), between the Gly(3) N H and the Gly(5) C O (an 11-atom, hydrogen-bonded ring), and a more limited interaction between the Gly(3) C O and the Gly(5) N H (a γ-turn). Arguments are presented that relate the conformational features proposed above to the coacervate, which is a filamentous state.

On the conformation, coacervation and function of polymeric models of elastin.

In a recent review (1) entitled “Conformations of the Repeat Peptides of Elastin in Solution: An Application of Proton and Carbon-13 Magnetic Resonance to the Determination of Polypeptide Secondary Structure”, the secondary structures of the monomers and polymers of the repeat peptides of tropoelastin were described. The repeat peptides, as reported by Foster et al. (2) and Gray et al. (3) but with the permutation given in terms of the conformational unit, are Val1-Pro2-Gly3-Gly4, Val1-Pro2-Gly3-Val4-Gly5, and Ala1-Pro2-Gly3-Val4-Gly5-Val6. Derivatives of these monomers, their permutations, oligomers and high polymers were studied in four different solvents dimethylsulfoxide, methanol, trifluoroethanol and water. More recently each of the three monomers have been studied in chloroform and their possible conformations examined by means of extensive conformational energy calculations (4–6). In each solvent and in the conformational energy calculations, all of the repeat peptides were found to contain the β-turn with Pro2 and Gly3 at the corners as a dominant conformational feature (see Figure 1). This β-turn utilizes a hydrogen bond between the C-O of residue 1 and the N-H of residue 4.

Circular dichroism of biological membranes: Purple membrane of Halobacterium halobium

Abstract Circular dichroism spectra of purple membrane suspensions exhibit distortions which are characteristic of particulate α-helical systems with ellipticities at 224 nm of the order of 10000. We show here that the pseudo reference state approach to the correction of CD data, when applied to the purple membrane system, results in a CD spectrum which, in its entirety, is typical of a protein with about 75% α-helix, e.g. the corrected ellipticity at 224 nm is 24000 and at 190 nm is 45000. This result is consistent with electron microscopy and diffraction data on the purple membrane which indicates 70–80% α-helix. Application to the purple membrane system validates the pseudo reference state approach. We also note that 80% trifluoroethanol solubilizes the membrane but results in a similar average secondary structure of the protein.

Prolyl hydroxylation of the polypentapeptide model of elastin impairs fiber formation

Abstract With elastogenesis described as a process dominated by intermolecular hydrophobic interactions and the polypentapeptide, (Val-Pro-Gly)Val-Gly)n, presented as a model for the dominant dynamic elements of the elastic fiber, it is demonstrated that hydroxylation of proline residues of the polypentapeptide unfavorably affects the inverse temperature transition leading to fiber formation such that even with only 10% of the proline residues hydroxylated very little fiber formation occurs at 37°C. Significantly higher temperatures are required. As prolyl hydroxylase, elaborated during a fibrogenic response, hydroxylates elastin, this result raises the question as to whether hydroxylation of the precursor protein of the elastic fiber may similarly impair in vivo fiber formation.

Spectroscopic and electron micrographic studies on the repeat tetrapeptide of tropoelastin: (Val-Pro-Gly-Gly)n.

Abstract The polytetrapeptide repeat of tropoelastin, Val 1 -Pro 2 -Gly 3 -Gly 4 , coacervates from aqueous solution as temperature increases. The coacervation is a cooperative process which is concentration dependent, moving the temperature profile of coacervation curves to lower temperature with high concentrations. Electron micrographs of uranyl acetate or uranyl acetate-oxalic acid negatively stained coacervated samples are characterized by filamentous structures whose center-to-center diameter is approximately 50 A as measured with optical diffraction. Coacervation thus results in increased intermolecular order. Circular dichroism spectra in methanol and trifluoroethanol solutions are significantly different from the spectrum in water. Once coacervated, though, the polytetrapeptide gave a CD spectrum very similar to that seen in these organic solvents. The data support the point of view that coacervation is the result of hydrophobic association attending an inverse temperature transition.

Synthetic peptide K+ carrier with Ca2+ inhibition.

Abstract Based on a proposed solution conformation of the Ca 2+ ion complex of the repeat hexapeptide of elastin, l -Val- l -Ala- l -Pro-Gly- l -Val-Gly, it is possible to modify the molecule making it more lipophilic for lipid bilayer permeation while retaining its complexation features. Therefore the two peptides, For-MeVal-Ala-Pro-Sar-Pro-Sar-OMe and For-MeVal-Ala-Pro-Sar-Pro-Sar-OH, were synthesized and evaluated for lipid bilayer activity and cation binding (For, N -formyl; Me, N -methyl; Sar, N -methyl glycine). Both peptides bound Ca 2+ preferentially but did not exhibit the properties of a Ca 2+ carrier. They were however active as K + carriers although K + ion titration curves showed a much lower affinity for K + than for Ca 2+ . The addition of Ca 2+ or Mg 2+ to the bilayer system inhibited the peptide K + carrier activity. Three possible explanations of this interesting Ca 2+ inhibition of carrier activity are irreversible complexation of Ca 2+ , mixed ligand complex formation involving Ca 2+ , lipid and peptide, and impermeability of the lipid layer when peptide is complexed with a divalent cation.

Ultraviolet Absorption, Circular Dichroism, and Optical Rotatory Dispersion in Biomembrane Studies

The fundamental concerns in applying optical methods to the study of biomembranes arise from complications introduced by their particulate nature; the basic objective is not solely to describe the actual absorbance by separating it from the effects of light scattering in the heterogeneous system. Moreover, the objective is to correct the spectrum to the absorbance, or difference absorbance, values that would occur if the molecules were uniformly distributed while retaining their membrane conformational state. This, of course, is because the reference state, for which there is much information, is one in which the polypeptide or protein species of interest is molecularly dispersed in solution. In order to proceed in a more sure-footed manner, a brief review of the fundamentals of absorption and the factors that alter absorption intensity and spectral position is warranted. Achieving a satisfactory perspective of the fundamentals allows for a more confident approach to the unique problems of membrane systems.