Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572127
Title: Mechanisms of elasticity in elastic proteins
Author: Green, Ellen Marie
Awarding Body: University of Exeter
Current Institution: University of Exeter
Date of Award: 2012
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Abstract:
This thesis investigates the mechanical properties of the elastic proteins isolated by cyanogen bromide digestion from lamprey cartilages and compares them with the mammalian protein, elastin. Thermomechanical testing and measurements of the effects of hydrophobic solvents on mechanics are used to determine the energetic and entropic contributions to the mechanical properties and the role of solvent interactions. Raman microspectrometry is shown to be a valuable tool in determining the secondary structure of the proteins, their interactions with water and molecular-level effects of mechanical strain. The supramolecular structure of the proteins matrices are investigated using nonlinear microscopy and X-ray diffraction. The mechanical properties of fibrous elastin agreed with those previously reported with elastic moduli in the region of 0.2-0.4 MPa. Elastic moduli decrease by approximately 25% with increased temperature, which was accompanied by a small decrease in hysteresis loss. In agreement with earlier findings, an entropic mechanism of elasticity became dominant only at high temperatures with a major contribution from interactions with solvent water. The lamprey proteins can be divided into two broad groups, the 'soft' branchial and pericardial cartilages resembling elastin, with linear stress-strain behaviour over a range of strains, elastic moduli in the range 0.13 MPa to 0.35 MPa, breaking strains of up to 50% and low hysteresis. Annular and piston proteins showed a very different response having much higher elastic moduli (0.27 MPa to 0.75 MPa), higher breaking strains and large hysteresis. Similarities between elastin and the lamprey matrix proteins extended to their thermomechanical behaviour with a decrease in elastic moduli and a drive towards entropic elasticity at high temperatures, although the annulus and piston were less thermally stable. Raman spectroscopy was able to detect differences between the various proteins and between elastin fibres and fragmentation products. Although no vibrational modes associated with cross-linking of the fibres could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from \alpha -elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% \beta -structures, 46% unordered and 18% \alpha -helix. \alpha -Elastin contained 29% \beta -structures, 53% unordered and 18% \alpha -helix. Strains of up to 60% in ligament fibre bundles resulted in no significant shifts in peak positions or in secondary structure. Polarization measurements revealed that the peptide bonds and several of the bulky side-chains re-orientated closer to the fibre axis with strain. Heating nuchal elastin fibres to 60^{\circ} C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of \beta -structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water. The Raman spectra of the lamprey matrix proteins are similar both to each other and when compared to fibrous elastin. Only small differences could be detected in side-chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were also very similar to that of elastin, with a preponderance of unordered structures which probably confer the high degree of conformational flexibility necessary for entropy elasticity. Piston and annular proteins, like elastin, showed a strong interaction with water, suggesting a greater role of hydrophobic interactions in their mechanics compared to the branchial and pericardial proteins. Elastin is well known to exhibit autofluorescence. However, only the branchial protein has been reported to autofluoresce. This study shows that all four lamprey matrix proteins investigated exhibit strong autofluorescence which was subsequently exploited to image these tissues using multiphoton microscopy. Microscopic investigations revealed that the architecture of lamprey proteins differ from that of elastin. Nuchal elastin forms bundles of fibres running predominantly parallel to the direction of applied force. The arrangement in lamprey cartilage is very different forming honeycomb structures, which in the case of annular and piston cartilages, is surrounded by a dense sheath of matrix material. Dye injections revealed that the branchial and pericardial form open systems whereas in piston and annular cartilages a closed system exists. These variations in architecture are reflected in their different mechanical properties and in vivo functions.
Supervisor: Winlove, Charles Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.572127  DOI: Not available
Keywords: elastin ; lamprin ; elasticity ; Raman ; mechanical testing ; lamprey ; elastic proteins
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