Efficient localization of a native metal ion within a protein by Cu-based EPR distance measurements.

Electron paramagnetic resonance (EPR) based distance measurements have been exploited to measure protein-protein docking, protein-DNA interactions, substrate binding and metal coordination sites. Here, we use EPR to locate a native paramagnetic metal binding site in a protein with less than 2 Å resolution. We employ a rigid Cu2+ binding motif, the double histidine (dHis) motif, in conjunction with double electron electron resonance (DEER) spectroscopy.

Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy.

Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage.

Rotameric preferences of a protein spin label at edge-strand β-sheet sites.

Protein spin labeling to yield the nitroxide-based R1 side chain is a powerful method to measure protein dynamics and structure by electron spin resonance. However, R1 measurements are complicated by the flexibility of the side chain. While analysis approaches for solvent-exposed α-helical environment have been developed to partially account for flexibility, similar work in β-sheets is lacking.

Cu²⁺ as an ESR Probe of Protein Structure and Function.

Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling has been widely used to determine the structure and dynamics of proteins and other biomolecules. The most popular spin label is a nitroxide-based radical which can be attached to a protein via a site-specific reaction with either native cysteines or cysteines engineered into the system via site-directed mutagenesis. Paramagnetic transition metals, including Cu(2+), often serve as cofactors of metalloproteins, and have already been realized as ESR probes to report structural information in these proteins.

The double-histidine Cu²⁺-binding motif: a highly rigid, site-specific spin probe for electron spin resonance distance measurements.

The development of ESR methods that measure long-range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein-backbone structure. Herein we present the double-histidine (dHis) Cu(2+)-binding motif as a rigid spin probe for double electron-electron resonance (DEER) distance measurements.

Cysteine-specific Cu2+ chelating tags used as paramagnetic probes in double electron electron resonance.

Double electron electron resonance (DEER) is an attractive technique that is utilized for gaining insight into protein structure and dynamics via nanometer-scale distance measurements. The most commonly used paramagnetic tag in these measurements is a nitroxide spin label, R1. Here, we present the application of two types of high-affinity Cu(2+) chelating tags, based on the EDTA and cyclen metal-binding motifs as alternative X-band DEER probes, using the B1 immunoglobulin-binding domain of protein G (GB1) as a model system.

Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag.

Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu(2+) binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC-Cu(2+) K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ~10 Å of the Cu(2+) center experience considerable transverse PREs leading to severely attenuated resonances in 2D (15)N-(13)C correlation spectra.

Tunable, mixed-resolution modeling using library-based Monte Carlo and graphics processing units.

Building on our recently introduced library-based Monte Carlo (LBMC) approach, we describe a flexible protocol for mixed coarse-grained (CG)/all-atom (AA) simulation of proteins and ligands. In the present implementation of LBMC, protein side chain configurations are pre-calculated and stored in libraries, while bonded interactions along the backbone are treated explicitly. Because the AA side chain coordinates are maintained at minimal run-time cost, arbitrary sites and interaction terms can be turned on to create mixed-resolution models.

High-resolution structure of a protein spin-label in a solvent-exposed β-sheet and comparison with DEER spectroscopy.

X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of α-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions.

Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor.

Herein, we identify the coordination environment of Cu²(+) in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²(+) and Cu²(+) significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo.