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Title: The application of scattering techniques at complementary length scales to structural studies of clathrin complexes in vitro
Author: Jones, Joseph
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2016
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Abstract:
Clathrin is a well-studied molecule yet its assembly properties have been characterised only in fairly simple biochemical terms to date. When clathrin is isolated for study in vitro, certain important aspects of its in vivo functionality are conserved: • purified clathrin units can be induced to assemble, reversibly, into cage-like forms that exhibit an array of polygonal binding motifs, similar to those observed in a vesicle's coat complex; • the artificial clathrin cages so produced are found to be heterogeneous, resulting in particles of different size according to the number of units each incorporates. Laboratory methods that investigate mechanisms of cage disassembly have become integral to the supply of knowledge about clathrin's structural biology. Yet there has been little progress towards a quantitative approach for exploring the assembly mechanisms that might explain clathrin cage dispersity; indeed, for some biophysical techniques the latter is an unfortunate property that might even preclude a meaningful analysis. This thesis shows that when purified clathrin is made to assemble in vitro by an overnight dialysis method (x2.8.3), the distribution of coalescent particle sizes that results can be predicted according to the pH and composition of the buffer solution and the final concentration of the clathrin sample: • Within the confines of a certain parameter space, variation in the distribution of particle sizes is found to be consistent with the predictions of a Becker-D�oring model for reversible coalescence: the empirical evidence suggests that a dynamic population of supramolecular cage structures provides a mechanism for energy minimisation towards achieving thermodynamic equilibrium. • Addition to, or substitution for, the basic compounds that constitute the assembly buffer solution exerts detectable effects upon the clathrin particle size distribution; the nature and magnitude of these effects may be expressed in terms of the state function Gibbs energy. To demonstrate these, a methodology was developed to focus on the preparation of samples suitable for analysis by the dynamic light scattering (DLS) technique, thereby aiming to provide an extensible and far richer analysis than was a afforded previously by the somewhat erroneous, certainly tedious, task of manually counting and classifying particles by size to construct a histogram from electron microscopy images. DLS determines the frequency profile of fluctuations in the electromagnetic field issued by a bulk sample to measure the diffusion processes of its particles. To then derive a number distribution relies upon exploiting prior knowledge of the physical system or else assumptions for the same. So, the research presented here has involved both a theoretical and an experimental component: over the course of the project there has been a process of iteration between developing experimental methods at the bench and interpreting the data recovered, until the physical picture and the theoretical model that have emerged appear to be consistent. And the results described in this thesis do indeed encourage further work. A number of very promising themes have not been advanced further here due only to the limitations of time: • 3. By developing a quantitative approach that relates the physical properties of the clathrin particle in solution to thermodynamic measures, the empirical results that have been collected over the course of this project are expressed in terms that translate more readily to the language of simulation and dynamical systems modelling. • 4. Analysis of DLS makes a valuable contribution to the project that aims to resolve accessory proteins bound to clathrin cages using the SANS contrast variation technique, with auxilin as a model. • Perhaps the most important conclusion to be drawn, however, is that: 5. The additional insight made available by analysis of DLS allows greater control to be exercised over the directed assembly of clathrin in vitro, which in turn provides fresh opportunities for experimental design. Chapter 1: The scientific questions addressed by this thesis; key concepts; review of the academic literature; project aims and objectives. Chapter 2: Details of the practical methods and materials used to conduct experiments in the laboratory with clathrin; software resources. Chapter 3: Description of the theoretical framework that has been developed over the course of the project; mathematical methods. Chapter 4: The empirical results for clathrin that have been recovered over the course of the project; statistical methods and models. Chapter 5: How the main findings of Part II have been used to solve a problem for clathrin studies when using a high resolution application (SANS). Chapter 6: Discussion of the main findings with respect to the project aims and objectives; directions for further work and unresolved questions. Figure 1: The narrative of this thesis. Part IV consists of appendices: statistical diagnostics, examples of programming and supplementary images.
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Council ; Wellcome Trust ; Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.723107  DOI: Not available
Keywords: QH426 Genetics
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