Structure restraints from heteronuclear pseudocontact shifts generated by lanthanide tags at two different sites

Pseudocontact shifts (PCS) encode long-range information on 3D structures of protein backbones and side-chains. The level of structural detail that can be obtained increases with the number of different sites tagged with a paramagnetic metal ion to generate PCSs. Here we show that PCSs from two different sites can suffice to determine the structure of polypeptide chains and their location and orientation relative to the magnetic susceptibility tensor χ, provided that PCSs are available for 1H as well as heteronuclear spins. In addition, PCSs from two different sites are shown to provide detailed structural information on the conformation of methyl group-bearing amino-acid side-chains. A previously published ensemble structure of ubiquitin is shown to explain the magnetic susceptibility and alignment tensors slightly better than structures that try to explain the experimental data by a single conformation, illustrating the potential of PCSs as a tool to investigate small conformational changes.Financial support by the Australian Research Council is gratefully acknowledged

New methods for structural biology by NMR spectroscopy

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

Site-specific incorporation of unnatural amino acids into proteins by cell-free protein synthesis

Cell-free protein synthesis (CFPS) offers a fast and inexpensive means to incorporate unnatural amino acids (UAAs) site specifically into proteins. This enables engineering of proteins and allows production of protein-based probes for analysis of their interactions with other molecules. Using dialysis Escherichia coli CFPS system in combination with aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii high expression yield of proteins with site specifically incorporated UAAs can be achieved. Typically the target protein can be prepared at concentrations of about 1 mg/mL, which is generally sufficient for subsequent applications

High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNACUA opt and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyanophenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract

Using paramagnetism to slow down nuclear relaxation in protein NMR

Paramagnetic metal ions accelerate nuclear spin relaxation; this effect is widely used for distance measurement and called paramagnetic relaxation enhancement (PRE). Theoretical predictions established that, under special circumstances, it is also possible to achieve a reduction in nuclear relaxation rates (negative PRE). This situation would occur if the mechanism of nuclear relaxation in the diamagnetic state is counterbalanced by a paramagnetic relaxation mechanism caused by the metal ion. Here we report the first experimental evidence for such a cross-correlation effect. Using a uniformly 15N-labeled mutant of calbindin D9k loaded with either Tm3+ or Tb3+, reduced R1 and R2 relaxation rates of backbone 15N spins were observed compared with the diamagnetic reference (the same protein loaded with Y3+). The effect arises from the compensation of the chemical shift anisotropy tensor by the anisotropic dipolar shielding generated by the unpaired electron spin

Generation of pseudocontact shifts in proteins with lanthanides using small “clickable” nitrilotriacetic acid and iminodiacetic acid tags

Pseudocontact shifts (PCS) induced by paramagnetic lanthanide ions provide unique long-range structural information in nuclear magnetic resonance (NMR) spectra, but the site-specific attachment of lanthanide tags to proteins remains a challenge. Here we incorporated p-azido-phenylalanine (AzF) site-specifically into the proteins ubiquitin and GB1, and ligated the AzF residue with alkyne derivatives of small nitrilotriacetic acid and iminodiacetic acid tags using the Cu(I) -catalysed "click" reaction. These tags form lanthanide complexes with no or only a small net charge and produced sizeable PCSs with paramagnetic lanthanide ions in all mutants tested. The PCSs were readily fitted by single magnetic susceptibility anisotropy tensors. Protein precipitation during the click reaction was greatly alleviated by the presence of 150 mM NaCl.Financial support by the Australian Research Council is gratefully acknowledged

Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide-alkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies

DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra

Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin