Site-Specific Tagging of Proteins with Paramagnetic Ions for Determination of Protein Structures in Solution and in Cells

Abstract

High-resolution NMR spectroscopy is sensitive to local structural variations and subtle dynamics of biomolecules and is an important technique for studying the structures, dynamics, and interactions of these molecules. Smallmolecule probes, including paramagnetic tags, have been developed for this purpose. Paramagnetic effects manifested in magnetic resonance spectra have long been recognized as valuable tools for chemical analysis of small molecules, and these effects were later applied in the fields of chemical biology and structural biology. However, such applications require the installation of a paramagnetic center in the biomolecules of interest. Paramagnetic metal ions and stable free radicals are the most widely used paramagnetic probes for biological magnetic resonance spectroscopy, and therefore mild, high-yielding approaches for chemically attaching paramagnetic tags to biomolecules are in high demand.In this Account, we begin by discussing paramagnetic species, especially transition metal ions and lanthanide ions, that are suitable for NMR and EPR studies, particularly for in-cell applications. Thereafter, we describe approaches for site-specific tagging of proteins with paramagnetic ions and discuss considerations involved in designing high-quality paramagnetic tags, including the strength of the binding between the metal-chelating moiety and the paramagnetic ion, the chemical stability, and the flexibility of the tether between the paramagnetic tag and the target protein. The flexibility of a tag correlates strongly with the averaging of paramagnetic effects observed in NMR spectra, and we describe methods for increasing tag rigidity and applications of such tags in biological systems. We also describe specific applications of established site-specific tagging approaches and newly developed paramagnetic tags for the elucidation of protein structures and dynamics at atomic resolution both in solution and in cells. First, we describe the determination of the 3D structure of a short-lived, low-abundance enzyme intermediate complex in real time by using pseudocontact shifts as structural restraints. Second, we demonstrate the utility of stable paramagnetic tags for determining 3D structures of proteins in live cells, and pseudocontact shifts are shown to be valuable structural restraints for in-cell protein analysis. Third, we show that a NMR optimized paramagnetic tag allows one to determine distance restraints on proteins by double electron−electron resonance (DEER) measurements with high spatial resolution both in vitro and in cells. Finally, we summarize recent advances in site-specific tagging of proteins to achieve atomic-resolution information about structural changes of proteins, and the advantages and challenges of magnetic resonance spectroscopy in biological systems.

Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones

“…Recently, the need to prepare homogeneous antibody-drug conjugates has inspired a wave of interest in exploring cysteine-specific bioconjugation reagents with improved reactivity, specificity, stability, and biocompatibility 6,7 . To this end, maleimide derivatives [8][9][10] , dehydroalanine 11,12 , perfluoroaromatic reagents [13][14][15][16] , organometallic palladium reagents 17,18 , and many others [19][20][21][22][23] have been developed, and each of these reagents shows unique merits. However, only a few of them permit multifunctionalization of a single Cys residue 22,24,25 , which would be advantageous in certain situations.…”

Design and applications of lanthanide chelating tags for pseudocontact shift NMR spectroscopy with biomacromolecules

“…Comprehensive reviews about lanthanide chelating tags and their applications are available in the literature [1,7,[85][86][87][88][89][90][91]. A very recent article by Su et al documents his contributions to the field [92]. Furthermore, reviews were published that give an insight into strategies for measurements of PCSs in proteins [93], prospects of lanthanides in biomolecular NMR including LCTs [94], analysis of protein-ligand [20] as well as protein-protein complexes [95,96] using PCSs and RDCs, the combination of paramagnetic NMR long-range restraints and crystallography [97,98], sparse-labelling in combination with long-range restraints obtained by paramagnetic NMR [99] and LCTs used for the conformational analysis and interactions of oligosaccharides [100].…”

Improving Metal Adsorption on Triethylenetetramine (TETA) Functionalized SBA‐15 Mesoporous Silica Using Potentiometry, EPR and ssNMR

“…EPR known also as electron spin resonance is a potent tool to probe species with unpaired electrons such as the organic free radicals [15,39] and the paramagnetic transition metals. [40][41][42] EPR is an analog of NMR as it provides chemical and structural information by probing the electron spin rather than the nuclear spin. The existence of unpaired electrons is common as electrons usually paired forming diamagnetic molecules, making EPR superior for selective sites studies.…”

“…For example one can study the specific binding site of macromolecules such as the heme group in hemoglobin and myoglobin offering a powerful tool to monitor only the active sites in the macromolecules. [42,43] Moreover, EPR can be used to screen reactions that involve paramagnetic reactive intermediates, elucidate the structure of paramagnetic inorganic molecules, and to determine the oxidation state of transition metal ions. [41,44] For example, copper can have different oxidation states such as Cu + and Cu 2+ where Cu + is d 10 system with no unpaired elector hence no EPR signal can be observed while Cu +2 is d 9 with one unpaired electron that can be detected by EPR spectroscopy.…”