Transient structural features of Glycogen phosphorylase revealed by millisecond hydrogen deuterium exchange mass spectrometry

Abstract

Allostery is a fundamental mechanism of protein activation/inhibition, intricately linked to the structural dynamics of proteins upon stimulation by changes in their environment. Proteins have evolved to regulate their function by allostery from a variety of inputs, such as pH, metal-binding, ligand-binding, light and even the Earth’s weak magnetic field. However, a complete understanding of the exact mechanisms responsible for long-range intra-molecular signal transmission remains elusive, due to the limited availability of advanced biophysical techniques capable of capturing these signals across temporal and spatial dimensions. Glycogen phosphorylase (GlyP), the first allosteric enzyme described, demonstrates this challenge. The precise mechanisms controlling GlyP activity are unclear, particularly regarding the key regulatory loops of the active site 250′ (amino acid (248-260) and 280s (278-288), which are weakly stable and often lack density in structural models. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an extremely sensitive technique that quantifies backbone amide hydrogen-to-deuterium exchange, which is directly linked to local structural dynamics. Compared to structural elucidation methods, such as X-ray crystallography, HDX-MS offers higher sensitivity in detecting conformational bias, as low as a few percent difference and can probe structural dynamics with minimal sample requirements of pico-nanomole quantities. Protein amide HDX occurs with a vast dynamic range from µs up to at least days. It is this large dynamic range that presents the opportunity for information about the protein dynamics. Hence, HDX can bridge the gap between protein structure and biological function. Conventional HDX-MS sample preparation methods can reproducibly measure labelling times of >10s, leaving weakly stable intrinsically disordered regions (IDR)/proteins (IDP) intractable. Thus, development of reproducible and reliable millisecond HDX instruments is needed, as it can uncover the structural dynamics of IDP/IDR, crucial for protein function, as well as paving the way for potential non-equilibrium measurements of protein structural dynamics. The work in this thesis reports on the development of a novel millisecond HDX instrument and associated methods and softwares, revealing the allosteric mechanism of GlyP transition in greater detail. In Manuscript I, the construction, development and validation of an automated, rapid-mixing quench-flow HDX instrument is demonstrated. The broad applicability of this instrument is also shown by measuring rapid exchange kinetics of small peptides, furthermore, allowing for the analysis of an entire protein. In Manuscript II structural dynamic differences between the active and inactive form of GlyP were analysed. Structural motifs that critically regulate the activity of GlyP are weakly stable and therefore not often revealed by conventional HDX and often absent in X-ray crystallographic studies. In manuscript III, we were able to quantitatively measure the changes in local stability throughout GlyP, including these weakly stable sites. The key regulatory loops were probed by millisecond HDX and their site-localized stability differences quantified, supporting a longstanding hypothesis for the mechanism of allosteric regulation. In Manuscript IV a rapid, non-equilibrium millisecond HDX-MS method was demonstrated and used to precisely locate dynamic structural changes during glycogen phosphorylase's allosteric activation and inhibition. This approach revealed transient changes in localized structure, common to both allosteric activation by AMP and inhibition by caffeine. Altogether, the achievements in this work enhance our understanding of the complex mechanisms underpinning allostery. The mass spectrometry approach developed here allows for precise quantitative measurements and has the potential to be used in the future to observe dynamic changes in protein structure triggered by nearly any environmental stimulus.