Nanometer-Range Distance Measurement in a Protein Using Mn²⁺ Tags

Abstract

Pulse electron paramagnetic resonance measurements of long-range (nm scale) distances between spin labels site-specifically attached to biomacromolecules have proven highly effective in structural studies. The most commonly used spin labels are stable nitroxide radicals, and measurements are usually carried out at X-band frequencies (∼9.5 GHz, 0.35 T). Higher magnetic fields open new possibilities for distance measurements with increased sensitivity using alternative spin labels containing half-integer high-spin metal ions. Here we demonstrate W-band (95 GHz) pulse double electron−electron resonance (DEER) distance measurements in a protein labeled with two Mn 2+ -EDTA tags. The distance distribution obtained is in excellent agreement with model calculations based on the known solution NMR structure. Thus, sitespecific labeling with Mn 2+ tags opens a highly promising approach to nanometer distance measurements in biological macromolecules. SECTION: Biophysical Chemistry L ong-range (nm scale) distance measurements between specific sites in biological macromolecules offer important insight into their structure and interactions. In the past decade, such distance measurements by pulse electron paramagnetic resonance (EPR) techniques, often referred to as pulse dipolar spectroscopy (PDS), have proven highly efficient for nitroxidelabeled proteins and nucleic acids.1−3 Distances in the range of 2−5 nm can be routinely accessed, and distances up to 8 nm can be determined under favorable conditions. 4,5 In proteins, site-directed spin labeling is usually achieved by covalent attachment of nitroxide compounds to cysteine residues that are either native or, more commonly, have been introduced at strategically chosen positions by site-directed mutagenesis. where r is the electron−electron distance and θ is the angle between the interelectron vector r and the external magnetic field. The measurements are carried out at low temperatures on frozen solutions. The most popular PDS experiment is the four-pulse double electron−electron resonance (DEER) sequence, 7 often also referred to as PELDOR (pulse electron double resonance). DEER carried out at standard X-band frequencies (∼9.5 GHz, 0.35 T) has become well-established. An order of magnitude increase in sensitivity can be gained by high-frequency/highfield DEER, provided that the microwave (mw) power is sufficient to generate short enough microwave pulses, as recently demonstrated at the W-band (95 GHz, ∼3.5 T). 8,9 In addition, high-field measurements open new opportunities for the use of metal ions as spin labels. The EPR spectra of highspin transition-metal ions with half-filled valence orbitals, such as Gd 3+ (S = 7/2) and Mn 2+ (S = 5/2), become much simpler at high fields, displaying an intense and relatively narrow central |−1/2⟩ → |1/2⟩ transition. 10,11 The width of this transition is proportional to D 2 /ν 0 , where D is the zero-field splitting (ZFS) parameter and ν 0 is the spectrometer frequency, leading to increased sensitivity with increased freque...

Triarylmethyl Labels: Toward Improving the Accuracy of EPR Nanoscale Distance Measurements in DNAs

“…Recently, Cu 2+ , Gd 3+ and Mn 2+ based labels have appeared as alternatives to traditional nitroxides, proven to have advantageous properties in certain cases. 16–24 Even more recently, triarylmethyl (trityl, TAM) radicals began to be used as spin labels for distance measurements in model systems and biomolecules. 25–28 Although synthetic strategies for spin-labeling using TAMs are by far not enough elaborated compared to nitroxides up to date, the advantages of TAMs are already evident.…”

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

“…Thus, the long-distance information encoded in these PRE data is a crucial source of restraints on the global protein fold, and can be used to complement the shorter-distance restraints extracted from conventional, nuclear dipolar coupling based solid-state NMR experiments. Beyond the PRE measurements demonstrated here, applications of TETAC or analogous cyclen-type tags to the solid-state NMR measurements of electron-nucleus distance dependent pseudocontact shifts in Co 2+ proteins (Bertini et al, 2010; Li et al, 2013; Luchinat et al, 2012) and measurements of nanometer range distances involving Cu 2+ and Mn 2+ centers by electron paramagnetic resonance (Narr et al, 2002; van Amsterdam et al, 2003; Banerjee et al, 2012; Yang et al, 2012; van Wonderen et al, 2013; Ji et al, 2014) can be readily envisioned.…”

A simple double quantum coherence ESR sequence that minimizes nuclear modulations in Cu2+-ion based distance measurements

“…However, DEER and DQC experiments on metal ions are challenging given the lower sensitivity. Nevertheless, our group [26,27,29,30] and others [17,23,[31][32][33][34][35][36][37] have made tangible progress. DEER has significant advantages for Cu 2+ based experiments given that nuclear interactions are easier to reduce and due to the ease of implementation on commercial instruments.…”