Thermodynamic Selection of Steric Zipper Patterns in the Amyloid Cross-β Spine

At the core of amyloid fibrils is the cross-β spine, a long tape of β-sheets formed by the constituent proteins. Recent high-resolution x-ray studies show that the unit of this filamentous structure is a β-sheet bilayer with side chains within the bilayer forming a tightly interdigitating “steric zipper” interface. However, for a given peptide, different bilayer patterns are possible, and no quantitative explanation exists regarding which pattern is selected or under what condition there can be more than one pattern observed, exhibiting molecular polymorphism. We address the structural selection mechanism by performing molecular dynamics simulations to calculate the free energy of incorporating a peptide monomer into a β-sheet bilayer. We test filaments formed by several types of peptides including GNNQQNY, NNQQ, VEALYL, KLVFFAE and STVIIE, and find that the patterns with the lowest binding free energy correspond to available atomistic structures with high accuracy. Molecular polymorphism, as exhibited by NNQQ, is likely because there are more than one most stable structures whose binding free energies differ by less than the thermal energy. Detailed analysis of individual energy terms reveals that these short peptides are not strained nor do they lose much conformational entropy upon incorporating into a β-sheet bilayer. The selection of a bilayer pattern is determined mainly by the van der Waals and hydrophobic forces as a quantitative measure of shape complementarity among side chains between the β-sheets. The requirement for self-complementary steric zipper formation supports that amyloid fibrils form more easily among similar or same sequences, and it also makes parallel β-sheets generally preferred over anti-parallel ones. But the presence of charged side chains appears to kinetically drive anti-parallel β-sheets to form at early stages of assembly, after which the bilayer formation is likely driven by energetics

The Physical and Chemical Origins of Amyloid at Interfaces

Amyloidosis is of serious concern within the modern world with an ageing global population, there is a significant drive to understand how amyloid forms in order to research and develop new therapeutics to combat this threat. This thesis proposes using a cross-disciplinary collaborative approach to determine multiple aspects within the amyloid-like fibrillation pathway for human insulin (HI) at acidic and neutral pH conditions. Chapter 2 uses Reflection Anisotropy Spectroscopy to obtain fibrillar orientation information with the goal to investigate whether structural morphology can direct fibril growth. Lack of reproducibility at the surfaces and the observation of a ‘blue-haze’ on the silicon wafers led to the method development in Chapter 3. Chapter 4 uses conventional biophysical techniques, fluorescence spectroscopy and electron microscopy to study hydrophobic functionalised mesoporous silica microparticles provides a scaffold for human insulin fibrillation to occur. These results show that the microparticles induced fibril morphology changes and inhibited or enhanced the fibrillation process, with respect to the human insulin control. Chapter 5 explores the mass and structural changes of adsorbed human insulin at a hydrophobic surface, by QCM-D and Raman spectroscopy. The results of which showed variable human insulin adsorption to the two differing hydrophobically functionalised surfaces at pH 2 and pH 7 conditions. Chapter 6 is the result of a collaboration with the Department of Physics and provides the first images obtained from infrared scanning near-field optical microscopy in reflection with a quantum cascade laser source of human insulin adsorbed to gold surfaces to obtain both structural and locational information. These preliminary results provide exciting opportunities for future development of this technique at other surfaces and proteins. Chapter 7 uses fluorescence excitation – emission matrices to uncover the origin of the deep-blue autofluorescence phenomenon, with human insulin, α-synuclein and other peptides