Academic literature on the topic 'Paramagnetic lanthanide binding tags'

¹⁹F is an efficient reporter in quantifying the individual isomers and assessing the dynamic exchange between the isomers for the lanthanide complexes. ¹⁹F-NMR is a valuable tool in the design of suitable paramagnetic tags for protein NMR analysis.

Abstract. The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Vita.
Includes bibliographical references.
To determine the function of proteins of interest, chemical biologists employ their full panoply of techniques, including X-ray crystallography and NMR spectroscopy for structural information, and luminescence spectroscopy to determine cellular localization and binding interactions. These techniques generally require a spectroscopic handle, and trivalent lanthanide ions (Ln3+) are protean in this regard: an ordered Ln3+ can have many uses. Paramagnetic lanthanide ions can be exploited to align biomolecules in a magnetic field, and the anomalous signal of any lanthanide ion may be used to obtain phase information from X-ray diffraction data. Most lanthanide ions are luminescent upon sensitization by an organic fluorophore; for example, Tb3+ may be sensitized by the side chain of the amino acid tryptophan. Ln3+ emission profiles are distinct and long lived, and therefore ideal for imaging and resonance energy transfer experiments. Lanthanide-binding tags (LBTs) are short peptide sequences developed to tightly and selectively chelate lanthanide ions. LBTs contain an appropriately placed tryptophan residue for sensitizing Tb3+ luminescence, and are composed entirely of encoded amino acids; incorporation at the genetic level into any protein of interest is thus facilitated. Subsequent expression of the tagged protein may be done using standard biochemical techniques, and the resultant protein contains a site for introducing an ordered lanthanide ion. Within this thesis is discussed the further optimization of LBTs for lanthanide affinity and structural stability. A combination of combinatorial peptide libraries and computational studies has resulted in the discovery of peptides that bind Tb3+ with dissociation constants of better than 20 nM.
(cont.) Furthermore, the concatenation of two LBT motifs has enabled the generation of so-called "double lanthanide-binding tags" (dLBTs). These slightly larger tags have additional advantages including the ability to bind two lanthanide ions, reduced mobility with respect to the tagged protein, and comparable or improved affinity for Ln3+ ions. Furthermore, since the lanthanide Gd3+ is a common handle for magnetic resonance imaging, progress has commenced to expand the utility of LBTs to include this type of experiment. Finally, LBT technology has been used to study the protein Calcineurin by uniquely modifying one calcium-binding loop to selectively bind and sensitize Tb3+.
by Langdon James Martin.
Ph.D.

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.

Pulse electron paramagnetic resonance (EPR) distance measurements using double electron-electron resonance (DEER) experiments have been established as a powerful tool in structural biology. DEER experiments have the ability to measure the distance between two paramagnetic centres in biological macromolecules in the range of about 2 to 8 nm. The paramagnetic centres are usually introduced into proteins by site-directed spin labelling (SDSL) of cysteine residues. This thesis is based on the use of new lanthanide binding tags (LBTs) for paramagnetic nuclear magnetic resonance (NMR) spectroscopy (reported in papers 2 and 5), DEER distance measurements (reported in papers 1 and 3) and time-resolved luminescence resonance energy transfer (LRET) experiments (reported in paper 4). In particular, use of two complementary techniques, DEER experiments and paramagnetic NMR spectroscopy, was investigated for the study of conformational changes of proteins as a result of protein-ligand interactions. Two proteins were studied, the E. coli aspartate/glutamate binding protein (DEBP) and human calmodulin (CaM). Both proteins have different ligand binding characteristics: DEBP binds to small organic molecules, while CaM binds to specific peptide sequences. DEBP is a periplasmic binding protein responsible for the transport of aspartic acid and glutamic acid across the cell membrane and widely used in the design of biosensors of glutamate. The protein is composed of two domains, which bind one amino acid molecule at the domain interface. As DEBP contains a disulfide bond, an alternative cysteine-independent approach for site-specific protein tagging was used, which involved the use of genetically encoded unnatural amino acids that were site-specifically incorporated into proteins using orthogonal amber-suppressor tRNA/aminoacyl-tRNA synthetase systems. p-azido-L-phenylalanine (AzF) residues were incorporated into DEBP at different positions and paramagnetic lanthanide tags were attached to AzF via Cu(I)-catalyzed click chemistry (papers 1 and 2). Multiple Gd3+-Gd3+ distances measured by DEER experiments were used to define the metal positions, subsequently allowing deltachi-tensor determinations from sparse sets of pseudocontact shifts (PCSs). Both the DEER data and PCSs were in agreement with the closed conformation observed in the crystal structure of the homologue from S. flexneri. On the other hand, the PCSs indicated that the transition to the substrate-free protein involves a movement of the two domains as rigid entities relative to each other. CaM is a two-domain protein that acts as an intermediate messenger protein and intracellular calcium sensor, which responds to changes in Ca2+ concentrations by large conformational changes that enable binding to a range of different proteins involved in signalling pathways. The conformational changes of CaM upon binding of the myristoylated alanine-rich C-kinase substrate (MARCKS) peptide were studied using DEER experiments and paramagnetic NMR. MARCKS was chosen due to its unique binding mode compared to other CaM-target peptide complexes. The DEER results indicated that the binding of MARCKS peptide to CaM does not lock CaM in a single conformation. Deviations between the crystal and solution structure of the complex were also evident in the measured PCS data, highlighting the conformational flexibility of CaM that allows CaM to bind to diverse target proteins.