DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.

Claire L Motion,Sabine Van Doorslaer,David R Bolton,Hassane El Mkami,Janet E Lovett,Paul A S Cruickshank,Stacey Bell,Graham M Smith,Robert I Hunter,Scott L Cassidy

doi:10.1021/acs.jpclett.6b00456

Abstract

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron–electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

Highlights

Heme proteins contain a metalloporphyrin chelating an iron atom
One very useful method for investigating elements of the structure of proteins, conformational changes, or protein− protein binding is double electron−electron resonance (DEER, known as PELDOR).[3−6] This is a pulsed electron paramagnetic resonance (EPR) spectroscopic method, which measures the dipolar interaction between two paramagnetic centers
The dipolar interaction is distance dependent and DEER can measure distances in the nanometer range.[7−12] Often the paramagnetic centers have been engineered into the protein of interest by using site-directed mutagenesis to give a protein with the desired number of cysteine amino acids

Summary

■ EXPERIMENTAL METHODS

All W-band 94 GHz experiments were conducted using a home-built high-power (1 kW) pulsed spectrometer, with integrated vector modulator phase box. Full experimental details and sample preparation can be found in the Supporting Information. Descriptions of sample preparation, including UV−vis experiments; EPR experiment parameters and pulsed EPR data; DEER fitting parameters; T1 and Tm results; signal-to-noise analysis; and Q-band DEER. Descriptions of sample preparation, including UV−vis experiments; EPR experiment parameters and pulsed EPR data; DEER fitting parameters; T1 and Tm results; signal-to-noise analysis; and Q-band DEER. (PDF)

■ ACKNOWLEDGMENTS

Findings

■ REFERENCES