New methods for structural biology by NMR spectroscopy
Creators: name: Loh, Choy Theng
Publisher: name: The Australian National University
Year: 2012
Subjects: subject: QP519.9.N83 L64 2012
subject: Nuclear magnetic resonance spectroscopy
subject: Proteins Analysis

description: Selective isotope labelling is a commonly used approach to improve the spectral resolution in NMR spectra. In the present thesis, a new method was developed to suppress the isotope scrambling and isotope dilution of selectively 15N-labelled proteins as a result of metabolic amino acid conversions. Chapter 2 demonstrates how the suppression of pyridoxal-5'-phosphate (PLP) dependent enzyme activity suppresses isotope scrambling. Broadband inactivation of the PLP enzymes present in an E. coli cell extract was achieved by irreversible reduction of the Schiff-base linkages formed between the aldehyde group of PLP and amino groups of the enzyme using sodium borohydride, and used in cell-free protein synthesis resulted in greatly improved selectivity in isotope labelling. Furthermore non-PLP enzymes can be suppressed using known inhibitors. As PLP enzymes readily exchange the alpha-protons of amino acids, the broadband inactivation of PLP enzymes opens the door to the cell-free synthesis of perdeuterated proteins in aqueous solution from perdeuterated amino acids, where all amides are fully protonated while the alpha-positions remain deuterated. This is of particular interest if the protein cannot be reversibly unfolded to exchange its amide protons following in vivo expression in deuterium oxide. The study of protein-protein and protein-ligand complexes is greatly facilitated if long-range structure restraints derived from pseudocontact shifts (PCSs) are available. Here, a new DOTA-amide lanthanide tag with an activated thiol group was evaluated that binds to a single cysteine residue in the target protein via a disulfide bond. Chapter 3 shows that this tag, which binds lanthanides tightly, generates PCSs reliably with minimal tag motions, which was attributed to its rigidity, bulkiness and short linker length to the protein. The present thesis also developed a new approach for site-specific tagging of unnatural amino acids that were incorporated into the target protein using orthogonal aminoacyl-tRNA synthetase/tRNA systems. Specifically, p-azido-L-phenylalanine and p-propargyloxy-L-phenylalanine were site-specifically incorporated into different proteins by cell-free methods (described in Chapter 7) and by in vivo protein expression (Chapters 4-6). The azido and alkyne groups of the unnatural amino acids are capable of undergoing Cu(I)-catalyzed azide-alkyne cycloaddition with reagents that bear the complementary group required for the cycloaddition reaction. The results showed that by using lanthanide tags with the requisite reactive groups, lanthanide-induced PCSs can indeed be observed in all proteins that were successfully reacted with the tags (Chapters 4 and 5). In general, the reaction yields critically depended on the presence of ligands that are thought to form complexes with the Cu(I) ions. Unexpectedly, however, one of the lanthanide tags produced the highest yields in a Cu(II)acetate-catalyzed azide-alkyne cycloaddition in the absence of any added reducing agent or Cu(I)-chelating agent (Chapter 6). This reaction involved the alkyne group of a p-propargyloxy-L-phenylalanyl residue in the protein and the azide group of picolinic acid derivatives. The present data are the first demonstration that PCSs can be generated reliably by introducing unnatural amino acids into the target protein and performing a selective chemical reaction with the unnatural amino acids to attach a lanthanide tag in a site-specific manner.