Techniques for labelling biological macromolecules for spectroscopic studies




Wu, Zuyan

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The ability to express and purify soluble protein in significant amounts is a prerequisite for the structure analysis of biological macromolecules by spectroscopic techniques. Nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies are powerful biophysical techniques which are widely used in structural biology. This thesis focuses on the use of different fusion constructs and tagging strategies to produce samples for subsequent NMR and EPR measurements. Following the general introduction of Chapter 1, Chapter 2 explores N-terminal fusions based on the nucleotide sequence of the T7 gene 10 which translates into the hexapeptide MASMTG. A systematic comparison of the expression levels with and without MASMTG tag was conducted for five different proteins (E. coli aspartate/glutamate-binding protein (GBP), green fluorescent protein (GFP), MutT Homolog 1 (MTH1), dengue virus type 2 NS2B-NS3 protease (DENp) and Methanosarcina barkeri pyrrolysyl-tRNA synthetase (nbCRS)) in both a cell-free protein synthesis setup and in vivo in E. coli. The expression yields of DENp, GFP and nbCRS were greatly enhanced by the MASMTG tag, barely changed for GBP and decreased for MTH1. This result shows that the N-terminal fusion with a tag from a protein known to express in very high yields can indeed enhance the expression yields for some proteins even if they are already codonoptimized for expression in E. coli. Chapter 3 describes the development of an efficient and inexpensive strategy for site-specific paramagnetic tagging of oligonucleotides, which allowed measurements of pseudocontact shifts (PCS) in the DNA using lanthanide ion tags. The strategy relies on commercially available oligonucleotides synthesized with a phosphorothioate group. HPLC conditions were developed to separate the two phosphorothioate diastereomers and their configurations determined by an enzymatic assay with snake venom phosphodiesterase. The new lanthanide-binding tag C10 was attached by alkylating the phosphorothioate group. PCS measurements were carried out following hybridization with the complementary DNA strand to form DNA duplexes. Although the PCSs were relatively small, they confirmed the site-specific attachment of the tag. Larger PCSs were observed for the SP than the RP diastereomer and good correlations were observed between backcalculated and experimental PCSs, in particular for the SP-phosphorothioate oligonucleotide, indicating that this tagging approach delivers reliable long-range structural information. Chapter 4 describes the preparation of a homeodomain-DNA complex with three different types of spin labels for double electron–electron resonance (DEER) measurements. A Gd3+ tag was introduced into the homeodomain by copper-catalyzed click reaction with a genetically encoded unnatural amino acid (p-azidophenylalanine) and an EDTA-Mn2+ tag was introduced by reaction with Cys39. With a nitroxide tag attached to a phosphorothioate group in the DNA, DEER measurements determined the distances between the three labels in the triple-tagged homeodomain-DNA complex. The experimentally determined Mn2+−nitroxide and Gd3+−Mn2+ distance distributions agreed well with the distances predicted from the NMR structure of the complex, whereas the calculated Gd3+−nitroxide distance was ∼0.5 nm longer than the experimental one. This study demonstrated the potential of three different spin labels to obtain three independent distance restraints in a single sample. Chapter 5 describes experiments for the site-specific incorporation of the unnatural amino acids Boc-lysine and TMS-lysine into proteins using the Methanosarcina mazei pyrrolysine-tRNA synthetase (PylRS) mutants Y384F/Y306A and Y384F/Y306G/I405R in E. coli BL21 (DE3) and E. coli B95-DA. While Boc-lysine could readily be incorporated, the experiments to incorporate TMS-lysine were unsuccessful. Chapter 6 describes strategies explored to produce uniformly 15N-labelled MARCKS peptide.In vivo expression of this peptide had proven notoriously difficult but was successfully achieved by fusion with a trigger-factor–ubiquitin (TF-Ub) construct, which can be cleaved with a ubiquitinase to release the free peptide. 15N-HSQC spectra were recorded of the peptide in complex with the N60D mutant of calmodulin (CaM) loaded with calcium, and chemical shift changes and paramagnetic relaxation enhancements (PRE) detected in the presence of paramagnetic lanthanide ions confirmed specific binding to CaM and interactions with the N-terminal domain of CaM. This establishes the basis for future structural analysis of the binding mode of the MARCKS peptide to CaM. An intein strategy was unsuccessful for the expression of the MARCKS peptide, but the system successfully produced tag-free PylRS and allowed its purification in soluble form. The purified PylRS was inactive in the in-house cell-free protein synthesis (CFPS) system, indicating that the well-known problems with the activity of this enzyme are not associated with the presence of commonly used purification tags.



MSMTG, paramagnetic lanthanide binding tags, DNA oligonucleotides, pseudocontact shift, Site-directed spin labelling, Antp homeodomain, DEER, unnatural amino acid




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