An investigation of the nanostructural features of avian eggshell

The avian eggshell is a highly ordered bioceramic with both inorganic and organic constituents. The eggshell takes approximately 18 hours to form in the shell gland region of the hen's oviduct, a process which is repeated every 24 hours in modern hybrid laying hens, which are capable of laying in excess of 300 eggs per annum. Although the formation of the eggshell is rapid, it still results in a structure that is highly organised and which displays unique functional properties that depend on the interplay between its mineral and protein phases. The interactions between the inorganic and organic components during the formation of the eggshell however are poorly understood but it is likely that they occur at the nanometer level. Thus, it is hypothesised that structural variation at the nanostructural level will impinge on the overall structural integrity and mechanical performance of the eggshell. In this thesis. X-ray diffraction (XRD), small angle X-ray scattering (SAXS) and microfocus small angle X-ray scattering (?SAXS) were applied to investigate the nanostructure of the eggshell. A description of these different techniques is given in Chapter 2 along with theoretical considerations as to how the resulting data presented in subsequent chapters was analysed. In Chapter 3, thin sections of normal and abnormal eggshells, where the layers were structurally intact, were analysed using ?SAXS. The results of this experiment suggest that there are nanostructural features within the different layers of normal eggshells, especially in the mammillary layer. The size dimensions of these nanopores/nanovoids were subsequently estimated to be between 3 - 5 nm in both visually normal and abnormal eggshells. However, despite being of similar size, the distribution of these nanopores or voids was found to be disrupted in the abnormal eggshell samples. In Chapter 4, XRD and SAXS were used to analyse powdered eggshell samples which represented different stages of gestation. In this case, the diffraction and scattering data produced were used to calculate an average size measurement of the structural features within bulk eggshell samples. The X-ray diffraction data indicated that the crystallite sizes were large, between 54-232 nm. However, size dimensions of approximately 5.7 - 7.2 nm were observed from the analysis of the SAXS data confirming the hypothesis that SAXS was measuring nanovoids within the crystallites arising from the presence of embedded protein. A further study using SAXS to compare the nanostructural features of eggshells of varying mechanical strengths is presented in Chapter 5. Here, powdered eggshells from young and aged hens were compared and found to contain an average nanovoid size value of approximately 5.9 nm and 5.8 nm, respectively. These size dimensions were confirmed by a parallel study in which mercury intrusion porosimetry was used to investigate pore size distribution. It was concluded that the average size of nanovoids is comparable in eggs of different mechanical strength. Chapter 6 describes the results of an investigation in which SAXS was used to monitor the nucleation events which take place when calcium carbonate is grown in vitro with and without the presence of eggshell proteins. The results of this study although preliminary suggest that the initial nucleation event is extremely rapid and provides a unique insight into the size of the calcium carbonate crystals which initially form. The main conclusions from this thesis are summarised in Chapter 7, in relation to the effect that changes in eggshell nanostructure could have on the overall structure and function of this complex biomineralised structure. Potential further studies are also discussed

The interaction of unfolding α-lactalbumin and malate dehydrogenase with the molecular chaperone αB-crystallin: a light and X-ray scattering investigation

Purpose: The molecular chaperone αB-crystallin is found in high concentrations in the lens and is present in all major body tissues. Its structure and the mechanism by which it protects its target protein from aggregating and precipitating are not known. Methods: Dynamic light scattering and X-ray solution scattering techniques were used to investigate structural features of the αB-crystallin oligomer when complexed with target proteins under mild stress conditions, i.e., reduction of α- lactalbumin at 37 °C and malate dehydrogenase when heated at 42 °C. In this investigation, the size, shape and particle distribution of the complexes were determined in real-time following the induction of stress. Results: Overall, it is observed that the mass distribution, hydrodynamic radius, and spherical shape of the αB-crystallin oligomer do not alter significantly when it complexes with its target protein. Conclusions: The data are consistent with the target protein being located in the outer protein shell of the αB-crystallin oligomer where it is readily accessible for possible refolding via the action of other molecular chaperones. © 2010 Molecular Vision

The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil

Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection

Rapid shape determination of tissue transglutaminase using high-throughput computing

Small-angle X-ray scattering can be used to determine the molecular shape of macromolecules in solution which are otherwise refractory to conventional high-resolution studies. DAMMIN and GASBOR are applications that utilize ab initio methods to build models of proteins using simulated annealing; both DAMMIN and GASBOR have to be run numerous times on the same input data to generate the most likely protein shape. Condor is a specialized workload-management system for PC computation-intensive tasks. Using Condor, DAMMIN and GASBOR can be run a number of times simultaneously on the same input data, allowing the shape of proteins to be determined in a fraction of the time it would have taken to have run DAMMIN and GASBOR sequentially. The main advantage of this approach is that it allows quicker data processing; therefore, results are obtained promptly and without undue delay. Tissue transglutaminase is a multidomain enzyme that catalyses the formation of isopeptide bonds between polypeptide chains. This reaction requires the enzyme to undergo a series of conformational changes that are not well understood in order to allow the sequential interaction with the two substrate proteins and their subsequent release when cross-linked. Condor was applied to determine the solution shape of tissue transglutaminase in a rapid fashion. Eventually, the next step will be to move towards online analysis at synchrotron sources by developing a graphical user interface that will enable remote access, allowing users to submit jobs to Condor whilst at synchrotrons

The interaction of unfolding α-lactalbumin and malate dehydrogenase with the molecular chaperone αB-crystallin: a light and X-ray scattering investigation

Purpose: The molecular chaperone αB-crystallin is found in high concentrations in the lens and is present in all major body tissues. Its structure and the mechanism by which it protects its target protein from aggregating and precipitating are not known

The interaction of unfolding alpha-lactalbumin and malate dehydrogenase with the molecular chaperone alpha B-crystallin: a light and X-ray scattering investigation

Purpose: The molecular chaperone αB-crystallin is found in high concentrations in the lens and is present in all major body tissues. Its structure and the mechanism by which it protects its target protein from aggregating and precipitating are not known. Methods: Dynamic light scattering and X-ray solution scattering techniques were used to investigate structural features of the αB-crystallin oligomer when complexed with target proteins under mild stress conditions, i.e., reduction of α- lactalbumin at 37 °C and malate dehydrogenase when heated at 42 °C. In this investigation, the size, shape and particle distribution of the complexes were determined in real-time following the induction of stress. Results: Overall, it is observed that the mass distribution, hydrodynamic radius, and spherical shape of the αB-crystallin oligomer do not alter significantly when it complexes with its target protein. Conclusions: The data are consistent with the target protein being located in the outer protein shell of the αB-crystallin oligomer where it is readily accessible for possible refolding via the action of other molecular chaperones

Microfocus X-ray Diffraction of Historical Parchment Reveals Variations in Structural Features through Parchment Cross Sections

We propose a new method of investigating variation of preservation within a parchment sample, which allows a more detailed analysis of alteration of the material structure. X-ray diffraction analysis of parchment typically involves the sample aligned with the plane of the parchment perpendicular to the direction of the X-ray beam, with a beam size of approximately 200 um and an image consisting of the composite diffraction features from the entire thickness of the sample. Here we describe the use of microfocus X-ray beams, with a beam size of 1.5 um vertically x 15 um horizontally, to carry out surface-to-surface scans of thin sections of parchment. Up to 200 images can be taken in a single cross-sectional scan of a 300 ím thick parchment section. This allows for X-ray diffraction analysis of features present only in specific areas of the parchment, such as at the surface. The orientation of collagen fibrils in the plane of the parchment, the effects of laser cleaning (including possible laser induced damage), mineral phases and crystalline lipids present in samples, and parchment structure under an inked region are investigated. It is shown that the long collagen fibril axis lies parallel to the parchment surface throughout the sections. Laser cleaning appears not to damage the collagen in parchment, while laser-damaged samples display gelatinization of the collagen at the surface. Polymorphs of calcium carbonate were detected in several samples but in most cases were not confined to the surfaces, as would be expected if the chalk finishing process was the main source of mineral phases in parchment. Crystalline lipid is found in most samples and appears to exhibit a preferential alignment with the plane of the phospholipid bilayer arranged parallel to the long fibril axis of collagen. The d spacing of the lipid is variable throughout a parchment section, indicating fluctuations in the hydration state, phase, or biochemical composition of the lipid. Ink affects the parchment to a depth of approximately 90 um, as measured by principal components analysis, disrupting the structure of the collagen to this depth. These features demonstrate the ability of this technique to examine diagenesis of individual components of parchment on a scale not previously studied