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Multidimensional heteronuclear NMR spectroscopy of high molecular weight proteins in solution : Application to a bacterial dimethylarginine dimethylaminohydrolase

Plevin, Michael John; (2003) Multidimensional heteronuclear NMR spectroscopy of high molecular weight proteins in solution : Application to a bacterial dimethylarginine dimethylaminohydrolase. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

In higher mammals, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for regulating the levels of the asymmetric methylarginine family of nitric oxide synthase (NOS) inhibitors. In this thesis a bacterial DDAH from Psendomorias aeruginosa (PaDDAH) is characterised by multidimensional heteronuclear NMR spectroscopy. A model system was developed to mimic heteronuclear NMR investigations of larger proteins. 15N R1, 15N R2 and heteronuclear NOE experiments were conducted on a sample of isotope-labelled perdeuterated ubiquitin in 50 % (v/v) glycerol at five different temperatures (17.5°C < T < 34.3°C). Isotropic rotational correlation times (tc) were estimated at each temperature. Between 17.5°C and 34.3°C a 21.2 ns range of Tc was observed. TROSY-based 3D HNCA and 3D HN(CA)CB spectra were recorded of [2H,13C,15N]-labelled ubiquitin in 50 % (v/v) glycerol at 25°C (tc = 34.6 ns). Sequence specific backbone 1HN, 15N, 13C? and 13C? resonance assignment of ubiquitin was performed under these conditions. 2D [1H, 15N]-TROSY spectra of [15N]-labelled WT PaDDAH showed the protein to be folded with superior resolution and signal-to-noise obtained on perdeuteration. The hydrodynamic properties of WT PaDDAH were analysed by solution NMR spectroscopy. Estimates of the translational diffusion coefficient Dz (7.6x10-7 cm s-1) and Tc (35 ns) indicated the enzyme to be a homodimer in solution. A series of TROSY-based 3D triple resonance spectra were recorded of [2H, 13C, 15N]-labelled WT PaDDAH. A full complement of 1HN, 15N cross peaks and 13C correlations was not observed in these spectra. Therefore, unambiguous resonance assignments were not obtained. The self-association properties of WT PaDDAH were investigated using analytical size exclusion chromatography (SEC). The results suggested WT PaDDAH exists in a dynamic equilibrium between monomer and homodimer species in solution. A dissociation constant Kd for this equilibrium was estimated (50nM < Kd < 250 nM). Residues contributing to the homodimer interface were identified from the previously reported crystal structure of PaDDAH. A series of point mutants at the protomer- interface were produced and each assayed for solubility, enzymatic activity and hydrodynamic properties. Each soluble PaDDAH mutant retained enzymatic activity but exhibited modified solution properties in comparison to the wild-type enzyme. A subset of PaDDAH mutants were further analysed by ID 1H NMR and 2D heteronuclear NMR methods. 2D NH correlation spectra of N36W, R40E and R98H PaDDAH were highly comparable to WT PaDDAH. Estimations of Tc suggested N36W PaDDAH is a strongly self-associated homodimer variant (Tc = 31 ns) and R40E PaDDAH tends towards a monomeric species (Tc = 21.9 ns). The lower Tc for R40E PaDDAH suggests this protein may be more amenable for future attempts at sequence-specific backbone resonance assignment. The investigations and results presented in this thesis are discussed with reference to recent advances in heteronuclear NMR studies of higher molecular weight proteins.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Multidimensional heteronuclear NMR spectroscopy of high molecular weight proteins in solution : Application to a bacterial dimethylarginine dimethylaminohydrolase
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; NMR spectroscopy
URI: https://discovery.ucl.ac.uk/id/eprint/10097621
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