Measuring distances in proteins by saturation-recovery EPR

Abstract

We describe a protocol for detecting electron spin-spin interactions between a radical and a metal ion in a protein or protein complex by saturation-recovery electron paramagnetic resonance (EPR). This protocol can be used with a protein containing an endogenous metal center and either an endogenous or synthetic radical species. We suggest a two-step approach whereby dipole-dipole or exchange interactions are first detected by continuous-wave EPR experiments and then quantified by saturation-recovery EPR. The latter measurements make it possible to measure long distances to within a few Å ngstroms. The protocol for making distance measurements by saturation-recovery EPR will take approximately 6 days to complete. INTRODUCTIONThe techniques that first come to mind when thinking about protein structure determination are usually solution-state NMR or X-ray crystallography. However, a complete protein structure is not always available or necessary to answer some of the most interesting biophysical questions. Sometimes, the measurement of one distance (or a few) is sufficient to establish the plausibility of a mechanism or corroborate a proposed structure. If a protein or protein complex contains a metal-binding site and a radical species, the magnetic interaction between the unpaired electrons at these two sites can be detected and quantified by saturation-recovery EPR and the resulting data used to measure distances of B10-40 Å (refs. 1,2). Enzymes with multiple redox-active cofactors such as photosystem II (PSII) and ribonucleotide reductase (RNR) are obvious targets for this methodology 3,4 . Indeed, in these systems, saturation-recovery EPR may also be used to measure exchange couplings between redox partners 4 . However, saturation-recovery EPR can also be used to measure distances between an endogenous metal-binding site and a strategically placed spin label to yield structural information 5 .Long distances in macromolecules have been successfully measured using fluorescence resonance energy transfer [6][7][8] and by pulsed EPR methods such as double quantum coherence, ''2 + 1,'' and DEER sequences that use a pair of spin labels (radicals) [9][10][11] . Distance measurement by saturation recovery has an advantage over these other methods when the protein contains an endogenous paramagnetic metal center in that the protein has a built-in ''label.'' The other label, that is, the radical, may be either endogenous or introduced through spin-labeling methods [12][13][14] . Additionally, in the case where the paramagnetic metal center is itself of interest, the radical can serve as a reporter of its magnetic properties. For example, in our experiments with the B2 subunit of RNR, the stable tyrosine radical was used to determine the exchange coupling within the dinuclear iron center 4 .As pulsed EPR instruments are not yet as common as pulsed (Fourier-transform) NMR instruments, we recommend that the investigator first attempts to detect the presence of electron spinspin interactions using the more common continuous...

Synthesis, Optical Properties and Magnetic Studies of 2,6‐Bis(pyrazolylmethyl)pyridine Functionalized with Two Nitronyl Nitroxide Radicals

“…It should be noted that in the case of both exchange-coupled systems and in the presence of dipolar interactions, the relaxation factor b is usually found to be less than 1, as observed here for B. [24,25] From the microwave saturation results it is assumed that the uncoupled biradical component in B (with |J/a N | ϽϽ 1) should relax similarly to the PzNN monoradical, hence it saturates much faster than the dominant component under a strong exchange limit (|J/a N | ϾϾ 1). The comparison between the relaxation envelopes of B and PzNN at different microwave powers (see the Supporting Information) allows the subtraction from the observed ∆m s www.eurjoc.org= 1 resonance envelope of B a weighted percentage of uncoupled biradical (ca.…”

“…When dipolar and/or exchange interactions are present, the product (T 1 T 2 ) is no longer constant and b is found to be smaller than 1. [24,25] …”

Structure and dynamics of a DNA-based model system for the study of electron spin–spin interactions

“…Even though the microwave power used to record the EPR spectrum causes more spins to transition into the parallel than anti-parallel state, at low observe powers the equilibrium (Boltzmann) distribution of spins in the two states is main- Fig. 3. The interplay between the spin-lattice relaxation rate constant, k 1 , and the microwave observe power in the microwave progressive power saturation experiment.…”

“…[1][2][3] Sample preparation for saturation-recovery EPR may be easier than that for fluorescence resonance energy transfer (FRET), [4] if either or both of the required ''labels", a paramagnetic metal center and a radical, are intrinsic species. If labeling with a radical is required for saturation-recovery EPR, a wide variety of nitroxide spin-labels are available.…”

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

“…This latter method has been used repeatedly with proteins (e.g., [336][337][338][339][340]). It will be fascinating to see what other methods will come up in the near and also more distant future to help to solve the "mysteries" of metal ion binding to nucleic acids.…”