Can label or protein deuteration extend the phase relaxation time of Gd(III) spin labels?
<p>Pulse-dipolar electron paramagnetic resonance (PD-EPR) has emerged as an effective tool in structural biology, enabling distance measurements between spin labels attached to biomolecules. The sensitivity and accessible distance range of these measurements are governed by the phase memory ti...
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| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Copernicus Publications
2025-08-01
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| Series: | Magnetic Resonance |
| Online Access: | https://mr.copernicus.org/articles/6/211/2025/mr-6-211-2025.pdf |
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| Summary: | <p>Pulse-dipolar electron paramagnetic resonance (PD-EPR) has emerged as an effective tool in structural biology, enabling distance measurements between spin labels attached to biomolecules. The sensitivity and accessible distance range of these measurements are governed by the phase memory time (<span class="inline-formula"><i>T</i><sub>m</sub></span>) of the spin labels. Understanding the decoherence mechanisms affecting <span class="inline-formula"><i>T</i><sub>m</sub></span> is crucial for optimizing sample preparation and spin-label design. This study investigates the phase relaxation behavior of two Gd(III) spin-label complexes, Gd-PyMTA and Gd-TPMTA, with various degrees of deuteration. These two complexes have significantly different zero-field-splitting (ZFS) parameters. Hahn echo decay and dynamical decoupling (DD) measurements were performed at W-band (95 GHz) in deuterated solvents (D<span class="inline-formula"><sub>2</sub></span>O<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M4" display="inline" overflow="scroll" dspmath="mathml"><mo>/</mo></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="14pt" class="svg-formula" dspmath="mathimg" md5hash="57ee8123d9c9aefcf23d9c7f6463c158"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-6-211-2025-ie00001.svg" width="8pt" height="14pt" src="mr-6-211-2025-ie00001.png"/></svg:svg></span></span>glycerol-d<span class="inline-formula"><sub>8</sub></span>), both for the free complexes and when conjugated to proteins. The impact of temperature, concentration, and field position within the EPR spectrum on <span class="inline-formula"><i>T</i><sub>m</sub></span> was examined. Results indicate that protons within 5 Å of the Gd(III) ion do not contribute to nuclear spin diffusion (NSD), and protein deuteration offers minimal enhancement in <span class="inline-formula"><i>T</i><sub>m</sub></span>. The dominant phase relaxation mechanisms identified at low concentrations were direct spin-lattice relaxation (<span class="inline-formula"><i>T</i><sub>1</sub></span>) and transient ZFS (tZFS) fluctuations. Dynamical decoupling (DD) measurements, using the Carr–Purcell sequence with <span class="inline-formula">∼</span> 140 refocusing pulses, resolved the presence of two populations: one with a long phase relaxation time, <span class="inline-formula"><i>T</i><sub>m,s</sub></span>, and the other with a short one, <span class="inline-formula"><i>T</i><sub>m,f</sub></span>. The dominating mechanism for the slowly relaxing population is direct-<span class="inline-formula"><i>T</i><sub>1</sub></span>. <span class="inline-formula"><i>T</i><sub>m,s</sub></span> showed no concentration dependence and was longer by a factor of about 2 than <span class="inline-formula"><i>T</i><sub>m</sub></span> for both complexes. We tentatively assign the increase in <span class="inline-formula"><i>T</i><sub>m,s</sub></span> to full suppression of the residual indirect-<span class="inline-formula"><i>T</i><sub>1</sub></span>-induced spectral diffusion and NSD mechanisms. For the fast-relaxing population, <span class="inline-formula"><i>T</i><sub>m,f</sub></span> is shorter for Gd-TPMTA; therefore, we assign it to populations for which the tZFS mechanism dominates. Because of the relatively short <span class="inline-formula"><i>T</i><sub>1</sub></span> and the contribution of the tZFS mechanism, protein deuteration does not significantly affect <span class="inline-formula"><i>T</i><sub>m</sub></span>.</p> |
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| ISSN: | 2699-0016 |