Elucidating protein stabilization with fluorescence techniques

Student thesis: Doctoral Thesis

Abstract

Insulin is an important protein that controls blood glucose levels, but its function becomes ineffective during protein aggregation. Insulin readily undergoes protein aggregation and it contributes to hyperglycaemia. In long-term hyperglycaemia, glucose can alter the structure and stability of insulin via glycation.In this thesis, fluorescence techniques have been used to monitor protein dynamics that are crucial to our understanding of protein aggregation and glycation. A synthetic protein that readily forms fibrous structures with homogenous growth kinetics is synthesized to simplify the interpretation of fluorescence kinetics. The conformational changes that both insulin and the protein model undergo throughout 700 hours have been monitored with a range of fluorescence techniques.For the fibrillogenesis model, time-resolved emission spectra (TRES) measurements revealed two fluorescent species occurring around 33,000 and 31,500 cm-1 at 0.33 hours after preparation. After this time, the second component decreases to 30,500 cm-1and continues to emit at this range throughout 700 hours. This suggests that fibril formation occurs within a few hours (confirmed by structured illumination microscopy (SIM)) and remains stable. Long term stability of the model provides a measurement-supported basis for its use as a reference material in fluorescence studies of protein aggregation.For free hexameric insulin, TRES measurements show drastic conformational changes. Two fluorescent components are shown at 33,000 cm-1 and 32,000 cm-1.The second component decreases to 31,000 cm-1 after 196 hours. This shows that the protein aggregates increase in size during this time. For free monomeric insulin, there is a lack of fluorescence intensity or TRES spectral changes, which show a lack of conformational change throughout 700 hours. In ThT studies and dSTORM images, there was no evidence to show amyloid fibrils were formed in both hexameric and monomeric insulin.Spectral similarities are shown in TRES measurements between free hexameric insulin and insulin with glucose until 196 hours after preparation. At least two fluorescent species were observed around 33,000 and 31,500 cm-1. The second component decreases to 30,500 cm-1 at 196 hours and does not experience spectral shifts in 10 ns, which shows that glycation begin to cross link insulin aggregates, making them more rigid.In dSTORMimages show there are an abundance of aggregates compared to free hexameric insulin which confirms cross linkage observed in the TRES measurements. For monomeric insulin with glucose, no significant changes were shown in the spectroscopy measurements and dSTORM images show smaller aggregates compared to free monomeric insulin. This demonstrates that glycation inhibits monomeric insulin aggregation.The findings in this thesis hold promise for monitoring the storage of insulin and its application in the control of diabetes and may support the development of more effective therapeutics against amyloidosis.
Date of Award31 Mar 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorDavid Birch (Supervisor) & Olaf Rolinski (Supervisor)

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