Determination of conformational changes in the structure of human calmodulin-1 using electron paramagnetic resonance spectroscopy
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Double electron electron resonance spectroscopy is a versatile and powerful tool for probing paramagnetic systems such metalloproteins and proteins with conjugated spin labels. Chapter 1 introduces EPR theory and the molecular biological concepts that this work is based on. This lays the groundwork with regard to basic knowledge and principles that the following chapters rely on. Chapter 2 collates the methodology used in all aspects of the work conducted in this body of work so that the techniques used may be critically assessed and the work reproduced under the same conditions in future experimentation. Chapter 3 focuses on the in-silico investigation of human calmodulin1 and applies a combinatorial approach utilising bioinformatic and computational techniques towards site selection for site-directed spin labelling and the elucidation of calmodulin1 conformational states through production of simulated models and predictions for expected DEER distance distributions. This ranges from literature reviews, structural region identification and degree of conservation assessment through to AI generated predicted models for secondary and tertiary structures and spin label modelling across different predicted conformational states and predicted distances. Chapter 4 provides a detailed explanation of the experimental work conducted to produce the variants of interest and the subsequent in-vitro process of data acquisition with details on a case-by-case basis. This focuses on the interactions between calmodulin1, Ca²⁺ and the M13 peptide. Chapter 5 takes a look at a comparative labelling technique that takes advantage of a rigid diHis motif for Cu²⁺ spin labelling in combination with MTSL (R1 when peptide-conjugated) for Cu²⁺NTA-R1 measurements that have been previously conducted in chapter 4 and utilises a different technique for distance measurements, RIDME. This chapter also applies other applications of RIDME to different spin systems, this time measuring distances between HisTag-bound Mn²⁺ and R1, taking advantage of another type of spin labelling technique.
Thesis, PhD Doctor of Philosophy
Creative Commons Attribution-ShareAlike 4.0 Internationalhttp://creativecommons.org/licenses/by-sa/4.0/
Embargo Date: 2024-08-04
Embargo Reason: Thesis restricted in accordance with University regulations. Restricted until 4th August 2024
Description of related resourcesDEER and RIDME Datasets for Calmodulin1 Variants (thesis dataset) Fisher, S. R., University of St Andrews, 4 Aug 2024. DOI: https://doi.org/10.17630/90d0e0d0-e972-4a03-996a-3ef9e075db1a
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