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 Mn2+-EDTA tags. The distance distribution obtained is in excellent agreement with model calculations based on the known solution NMR structure. Thus, site-specific labeling with Mn2+ tags opens a highly promising approach to nanometer distance measurements in biological macromolecules. SECTION: Biophysical Chemistry Long-range (nm scale) distance measurements betweenspecific 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 nitroxide-labeled 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.6 The distance between the unpaired electrons of the nitroxides is determined by measurement of their dipolar interaction, ωdd, following the relation ω = μ β πℏ θ − = ω θ − g r4 (3 cos 1) (3 cos 1) dd 0