High resolution electron microscopy of biological systems

This thesis demonstrates the relevance of advanced transmission electron microscopy (TEM) techniques such as aberration corrected scanning transmission electron microscopy (STEM) to the study of biological samples. By developing the application of these techniques to biologically relevant systems, this study shows how advanced EM can be an effective tool by providing insight into the structure of biological systems at the highest (i.e. atomic) resolutions. High angle annular dark field (HAADF) STEM has been used to gain insight into the core structure and iron loading mechanisms of the iron storage protein, ferritin. The iron content of ferritin was quantified using size-selected gold clusters as a mass balance, the first application of this technique to a biological sample. Preliminary structural studies of a novel colorectal cancer therapy have been undertaken, where polymeric alginate molecules chelate chemotoxic luminal iron in the colon. In particular the nano-structures built when alginates bind iron under physiological conditions have been established and investigated for the first time, using HAADF-STEM. Finally using TEM, it has been revealed for the first time the structures formed, and morphology changes induced, when proteins are encapsulated by membrane mimicking nano-discs

Morphology of the ferritin iron core by aberration corrected scanning transmission electron microscopy

As the major iron storage protein, ferritin stores and releases iron for maintaining the balance of iron in fauna, flora, and bacteria. We present an investigation of the morphology and iron loading of ferritin (from equine spleen) using aberration-corrected high angle annular dark field scanning transmission electron microscopy. Atom counting method, with size selected Au clusters as mass standards, was employed to determine the number of iron atoms in the nanoparticle core of each ferritin protein. Quantitative analysis shows that the nuclearity of iron atoms in the mineral core varies from a few hundred iron atoms to around 5000 atoms. Moreover, a relationship between the iron loading and iron core morphology is established, in which mineral core nucleates from a single nanoparticle, then grows along the protein shell before finally forming either a solid or hollow core structure

Alginate-iron speciation and its effect on in vitro cellular iron metabolism

Alginates are a class of biopolymers with known iron binding properties which are routinely used in the fabrication of iron-oxide nanoparticles. In addition, alginates have been implicated in influencing human iron absorption. However, the synthesis of iron oxide nanoparticles employs non-physiological pH conditions and whether nanoparticle formation in vivo is responsible for influencing cellular iron metabolism is unclear. Thus the aims of this study were to determine how alginate and iron interact at gastric-comparable pH conditions and how this influences iron metabolism. Employing a range of spectroscopic techniques under physiological conditions alginate-iron complexation was confirmed and, in conjunction with aberration corrected scanning transmission electron microscopy, nanoparticles were observed. The results infer a nucleation-type model of iron binding whereby alginate is templating the condensation of iron-hydroxide complexes to form iron oxide centred nanoparticles. The interaction of alginate and iron at a cellular level was found to decrease cellular iron acquisition by 37% (p < 0.05) and in combination with confocal microscopy the alginate inhibits cellular iron transport through extracellular iron chelation with the resulting complexes not internalised. These results infer alginate as being useful in the chelation of excess iron, especially in the context of inflammatory bowel disease and colorectal cancer where excess unabsorbed luminal iron is thought to be a driver of disease

Cellular localisation of alginate with confocal microscopy.

<p>Cells were treated with iron alone (control) or iron and FITC alginate with or without cell-membrane permeabilisation. (A) Cells treated with iron alone as expected showed no FITC signal. (B) Cells treated with iron and FITC alginate showed negligible punctate FITC staining on the cell periphery (C) Cells permeabilised with Saponin and then cultured with iron and FITC alginate showed an abundance of intracellular FITC signal which was mostly cytoplasmic in localisation.</p

Effects of alginate on cellular iron transport.

<p>(A) Intracellular iron concentration decreases when RKO cells were incubated with iron-59 and alginate (0.3% w/v) compared to iron only control (B) Treatment of RKO cells with iron increases ferritin expression whilst co-incubation with alginate (0.3% w/v) significantly suppressed the iron mediated ferritin induction. All experiments were performed in triplicate with error bars representing +/- SEM and * denotes statistical significance at p < 0.05.</p

Synthesis of FITC alginate.

<p>(Ai) Reaction coupling scheme of FITC onto alginate under peptide coupling conditions. (Aii) Image of fluorescent alginate in normal light (left) and exposed to λ = 365 nm UV light (right). (B) Absorption and emission (red and blue lines respectively) spectra of the fluorescent alginate (FlAlg) product. The native alginate reactant has no absorption or emission profile, however, upon conjugation with FITC a highly absorption and emission peaks are observed.</p

Physical characterisation of alginate iron composites.

<p>(Ai) Low magnification STEM images of alginate-iron composites revealed the alginate network ‘decorated’ in iron (denoted by arrows) with a single highly dense iron nucleation site (denoted with an asterisk). (Aii) A higher magnification image of the nucleation centre revealed nanoparticles of approximately 2–5 nm in diameter. (B) Fast Fourier transform analysis of HAADF-STEM images of two individual nanoparticles. (C) EDX mapping of iron-alginate composites with oxygen, iron and sodium localisation shown in the sample area. The copper from the copper TEM grid functions as a control.</p

Chemical analysis of iron alginate binding.

<p>(Ai) Isothermal titration microcalorimetry thermogram of 8 μl injectants of 5 mM Fe(III) into 0.04 mM alginate at 37°C. (Aii) Corresponding isotherm. (Bi) UV-Visible difference spectra of iron (III) titrated into alginate with a clear absorbance change at ca. 280 nm (Bii) absorbance change at 274nm vs final Fe concentration (M) with binding curve) (C) CD spectra of alginate-iron composites isolated via equilibrium dialysis. An induced CD signal is evident at ca. 280 nm. This correlates to the iron-hydroxide species bonded to the alginate as indicated from the UV-Visible spectra.</p

Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins

In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer (poly(styrene-co-maleic acid), SMA) to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.[Figure not available: see fulltext.] © 2015, Tsinghua University Press and Springer-Verlag Berlin Heidelberg.SCOPUS: ar.jinfo:eu-repo/semantics/publishe