UBC Theses and Dissertations
Characterizing labile protons by NMR spectroscopy Brockerman, Jacob Arthur
Labile hydrogens in proteins, including those on ionizable functional groups, undergo rapid exchange with water and thus are typically difficult to characterize by current techniques in structural biology. Their properties are often inferred from biophysical arguments rather than direct experimental determination. Here I present new methods for studying these hydrogens by NMR spectroscopy using two glycoside hydrolases as model protein systems. Chapter 2 centres on characterizing the hydroxyl protons of serine and threonine residues in proteins by NMR spectroscopy. Using auxtrophic E. coli strains, I produced Bacillus circulans xylanase (BcX) selectively labeled with ¹³C/¹⁵N-serine or ¹³C/¹⁵N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed using long-range heteronuclear scalar correlation experiments. Their dihedral angle-dependent scalar couplings with adjacent sidechain protons were determined via a quantitative ¹³C/1¹⁵N-filtered spin-echo difference experiment. The hydrogen exchange kinetics of these hydroxyls were measured using a ¹³C/¹⁵N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provided insights into the structural and dynamic properties of this model protein. Chapter 3 characterizes a mutant of T4 phage lysozyme (T4L) with an altered catalytic mechanism due to the substitution of residue Thr26 with a histidine (T26H). It has been proposed that T26H-T4L hydrolyzes peptidoglycan via a double displacement mechanism with His26 serving the unusual role of a nucleophile. To gain further insights into this or alternative mechanisms, I used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or ionization states of all the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the proposed nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway where only a minor population of protein is in its catalytically competent ionization state (His26 deprotonated and Glu11 protonated). I also demonstrated that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH. This stands in contrast to a recent neutron crystallographic study of T26H-T4L in which, perplexingly, Arg145 was proposed to have a deprotonated guanidine sidechain.
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