title: Nuclear magnetic resonance spectroscopy of the thioredoxin-like protein ERp18 : solution structure and dynamics
Creators: name: Louise Rowe, Michelle
Publisher: name: University of Kent
Year: 2023

description: A great many proteins essential for life contain one or more intra-molecular disulphide bonds. These disulphide bonds help stabilise the protein and are important for the functionality of some proteins. During formation of these disulphide bonds it is important for the correct cysteine residues to be paired together in order to achieve the correct three- dimensional (3D) structure. Within the endoplasmic reticulum (ER) there are a number of proteins that are thought to help in the formation (oxidation), breakage (reduction) and isomerisation of disulphide bonds so that the correct fold can be achieved. These reactions are all catalysed by members of the thioredoxin superfamily, and each have a thioredoxin- like 3D fold and a thioredoxin-like active site motif of the form Cys-Xxx-Xxx-Cys. This study focuses on the ER resident protein ERpl8 and attempts to provide insight about its function by the elucidation of the solution structure and analysis of backbone dynamics using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N and ¹³C/¹⁵N isotopically labelled ERpl8 was recombinantly expressed with an N-terminal His-tag using E. coliBL21 (DE3) pLysS cells with yields of up to 60 mg/1. Purification using metal affinity chromatography followed by anion exchange chromatography produced ERpl8 with a purity level greater than 95 %. Resonance assignment has been completes for the oxidised form of ERpl8 as well as extensive backbone resonance assignment of the reduced form of ERpl8 using a range of heteronuclear, multidimensional NMR experiments. These assignment lists have led to the calculation of the solution structure of oxidised ERpl 8, and allowed comparative NMR dynamics and amide hydrogen exchange rates for oxidised versus reduced ERpl8 to be collected. Through-space NMR experiments were used to obtain structural information of oxidised ERpl 8 and solution structure ensembles were calculated using the crystallography and NMR system (CNS). The solution structure of oxidised ERpl8 agrees well with a recently deposited crystal structure of ERpl 8 and has allowed identification of a long loop located in the vicinity of the active site. Investigation into the dynamic behaviour of the backbone of both oxidised and reduced ERpl 8 has revealed that only oxidised ERpl8 displays motion in the milli- to microsecond timescale. This suggests that conformational exchange is limited to the oxidised form and was found to be predominantly located in the helix immediately following the active site. Increased flexibility, indicated by motion in the pico- to nanosecond timescale, is observed in both oxidation states of ERpl8 and is restricted mainly to residues in loops located close to the active site in the 3D structure. Along with the dynamics, hydrogen exchange rates of backbone amide hydrogens illustrated that reduced ERpl 8 forms a more compact, stable structure than that of oxidised ERpl 8. This increased stability of the reduced form of ERpl 8 suggests that its function is to oxidise disulphide bonds in other proteins and is consistent with similar findings using guanadinium chloride denaturation studies of both oxidised and reduced ERpl 8