Can label or protein deuteration extend the phase relaxation time of Gd(III) spin labels?

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 ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> </mml:math> ) of the spin labels. Understanding the decoherence mechanisms affecting <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> </mml:math> 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 (D₂O <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mo>/</mml:mo></mml:math> glycerol-d₈), both for the free complexes and when conjugated to proteins. The impact of temperature, concentration, and field position within the EPR spectrum on <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> </mml:math> 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 <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> </mml:math> . The dominant phase relaxation mechanisms identified at low concentrations were direct spin-lattice relaxation ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mn>1</mml:mn></mml:msub> </mml:mrow> </mml:math> ) and transient ZFS (tZFS) fluctuations. Dynamical decoupling (DD) measurements, using the Carr-Purcell sequence with <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mo>∼</mml:mo></mml:math> 140 refocusing pulses, resolved the presence of two populations: one with a long phase relaxation time, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mrow><mml:mi>m</mml:mi> <mml:mo>,</mml:mo> <mml:mi>s</mml:mi></mml:mrow> </mml:msub> </mml:mrow> </mml:math> , and the other with a short one, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mrow><mml:mi>m</mml:mi> <mml:mo>,</mml:mo> <mml:mi>f</mml:mi></mml:mrow> </mml:msub> </mml:mrow> </mml:math> . The dominating mechanism for the slowly relaxing population is direct- <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mn>1</mml:mn></mml:msub> </mml:mrow> </mml:math> . <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mrow><mml:mi>m</mml:mi> <mml:mo>,</mml:mo> <mml:mi>s</mml:mi></mml:mrow> </mml:msub> </mml:mrow> </mml:math> showed no concentration dependence and was longer by a factor of about 2 than <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> </mml:math> for both complexes. We tentatively assign the increase in <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mrow><mml:mi>m</mml:mi> <mml:mo>,</mml:mo> <mml:mi>s</mml:mi></mml:mrow> </mml:msub> </mml:mrow> </mml:math> to full suppression of the residual indirect- <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mn>1</mml:mn></mml:msub> </mml:mrow> </mml:math> -induced spectral diffusion and NSD mechanisms. For the fast-relaxing population, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mrow><mml:mi>m</mml:mi> <mml:mo>,</mml:mo> <mml:mi>f</mml:mi></mml:mrow> </mml:msub> </mml:mrow> </mml:math> is shorter for Gd-TPMTA; therefore, we assign it to populations for which the tZFS mechanism dominates. Because of the relatively short <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mn>1</mml:mn></mml:msub> </mml:mrow> </mml:math> and the contribution of the tZFS mechanism, protein deuteration does not significantly affect <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>T</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> </mml:math> .