Understanding the mechanism of protein aggregation in thermal and refolding studies

Biopharmaceutical production of protein for therapeutic use is an expanding practice for treatment of numerous diseases in medicine. However the full benefits of this technique have not yet been fully realised due to a number of barriers. The largest of these barriers is that of protein aggregation, where mis-folded protein monomers self associate to form non functional macromolecules; aggregates. Further understanding of protein aggregation may lead to an improvement in the effectiveness and availability of these therapeutic treatments. Here is presented work which utilises novel or under-used techniques to elucidate information on the structure of protein aggregates, their formation mechanisms and the kinetics and thermodynamics of their growth. Results presented in the chapter on aggregate nucleation indicate that the nucleation stage of aggregation in bovine serum albumin has a temperature dependant mechanism, which in the middle of the temperature range follow mechanisms for stable nuclei population postulated in the literature. However at the extremes of this range, it appears that the nucleation mechanisms deviate from this and that there may be clustering of highly reactive nuclei at high temperatures, and continual formation of aggregate nuclei at low temperatures. Possible explanations for this behaviour are discussed. Analysis presented on the growth of particulate aggregates show that the model of monomer addition to the aggregate nuclei appears to be a fitting description of the growth process, which is generic across proteins. Furthermore the detailed analysis from an ultra violet light scattering spectroscopy technique provides a numerical method for examining the efficiency of aggregate preventing additives, and also illustrates the mechanism by which the additives prevent aggregation through stabilising the native state. Finally; results presented in the chapter on aggregation during refolding indicate the use of fluorescence anisotropy to monitor the molten globule state during refolding of proteins. Most strikingly, it is shown there is an obvious relationship between the mobility period of the protein and its propensity to aggregate. It is also shown that the presence of salt and urea can be utilised to moderate the presence of the molten globule state, and therefore the resultant aggregation

Small Angle Neutron Scattering Studies of Protein Aggregation

Understanding protein aggregation is important for many areas, including the improvement of processing and formulation of biopharmaceuticals, elucidating the mechanism of neurodegenerative diseases and manufacturing novel biomaterials. Of particular interest is the nucleation stage of aggregation, which is not well understood. The initial nucleation of protein aggregates has proved elusive due to the small size scales at which they form, and the rapid time period that this occurs in. Here we present the small angle neutron scattering data collected at D22 beamline at Institut Laue-Langevin, Grenoble. This provided time resolved information on the nanoscale on bovine serum albumin (BSA) which was used as a model system for studying aggregation processes. Using a dual population scattering model to characterise the presence of both monomer and aggregate populations, we observed two distinct aggregation stages; a nucleation phase followed by growth phase. During the nucleation phase, a stable population of aggregates was established. This was followed by a growth phase, characterised by aggregates increasing in size. This behaviour is examined across a temperature range, where an increase in temperature increases both the rate and the size of the aggregate nuclei. Work contained here presents a novel approach to examining the process of protein aggregation on the nanoscale

Elucidating the mechanism and thermodynamics of protein aggregation in order to create novel biomaterials

Protein aggregation occurs under certain conditions that cause individual protein sub-units to adhere to one another. Such conditions include extreme pH, high salinity and elevated temperatures. Understanding protein aggregation is important for many areas, including the improvement of processing and formulation of biopharmaceuticals, elucidating the mechanism of neurodegenerative diseases and manufacturing novel biomaterials. The morphology of protein aggregates can be controlled by tuning the environmental conditions that cause the proteins to adhere to one another, leading to the formation of fibrils, spherulites or particulates, which in turn can aggregate to form higher order structures. The ability to control aggregate morphology allows the potential to create novel architectures for new functional materials, such as complex tissue scaffolds, drug delivery systems, nano meshes and more. However, the mechanism and thermodynamics for protein aggregation is not fully understood. Here, we present results from light scattering and microscopy for several model protein systems which have been subjected to conditions that induce the formation of particulate aggregates. This has allowed the formation and structure of these aggregates to be examined in detail, which in turn has led to a better understanding of the mechanism and thermodynamics for their formation. Overall, the work gives us important insights into protein aggregation, which will eventually allow us to control and adapt this process in order to derive a range of benefits

Investigating the mechanisms responsible for and the factors affecting protein aggregation

Protein aggregation plays a huge role in developing new biomaterials and understanding the mechanisms responsible for a number of neurodegenerative diseases, including Alzheimer’s disease. The rate at which proteins aggregate and the way in which they aggregate is dependent on a variety of factors. Variations in conditions such as temperature, pH, protein concentration and the addition of metal ions such as zinc can affect whether the protein will form fibrils or particulates and how fast the aggregates will form. By understanding the mechanisms driving protein aggregation we can begin to investigate new techniques and drug systems for dealing with neurodegenerative disease as well as creating functional biomaterials using proteins. Here we present results gathered using light scattering spectroscopy and infrared analysis for several proteins where the conditions have been altered to either promote the formation of fibrils or particulate aggregates. This research will eventually give us important information about how to control protein aggregation and modify the mechanisms to produce an array of beneficial products including drugs and biological materials

The application of simultaneous electric and magnetic fields to aid protein crystallisation

Electric fields have a beneficial effect on protein crystallisation. This is because of amino acids contributing to an overall charge for the protein once they have bonded together via a peptide link. Taking this into consideration crystals are able to form when an electrical field is applied because of the potential gradient the current created which causes the migration of the protein through its media resulting in the build up of crystals. The downside to using only an electric field however is that different proteins have different resistances to a change in voltage, and that the alignment would only be along one field. Magnetic fields, when applied to proteins induce diamagnetic behaviour. The peptide bond between each individual amino acid plays an important role in this because it has a carbon-oxygen double bond. A double bond has been shown to exhibit a diamagnetic anisotropy and so in turn so would the peptide bond. This diamagnetic behaviour is exhibited in secondary structure proteins. With the application of simultaneous fields the idea is that one field provides alignment on one plane and the second field on another. From that the protein molecules would be aligned and suspended in the solution at equally spaced intervals in a uniform manor. To further promote this a high protein concentration was used to condense the protein molecules together. The purpose of this research is ultimately to provide insights into how best to solve the structures of different proteins, which would provide us with advances from the application of this multi-field technique