Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields

Abstract

We present time‐resolved Gd−Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter‐residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd‐sTPATCN spin labels, whose favorable qualities include a spin‐7/2 EPR‐active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C‐terminus and N‐terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light‐activated long‐range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

Order-of-magnitude SNR improvement for high-field EPR spectrometers via 3D printed quasi-optical sample holders

“…The QSH was tested with three different samples and EPR modes (see Table 1 ): a solution of 100 μM gadolinium chloride in D 2 O by cwEPR as a proxy for Gd spin labels used for distance measurements in spin-labeled proteins ( 58 ), a diamond crystal with nitrogen substitutional defects (P1 centers) by pulsed EPR as a proxy for solid-state spin qubit candidates, and a single crystal of lithium phthalocyanine (LiPc) by rapid-scan EPR as a narrow-line radical that is sensitive the local oxygen concentration ( 59 ). The measurements were compared with those done with the SSH, in both the SSH-M and SSH-T positions.…”

“…Furthermore, the QSH may provide an avenue to greatly reduce dead time of pulsed EPR using high-power microwave sources such as ITST’s free electron laser ( 35 ). Last, rapid-scan EPR of proteins doubly labeled with Gd spin tags offers the possibility of “filming” proteins in action by measuring dipolar-broadened EPR spectra as a function of time ( 58 ). Although experiments performed here were done without a cavity, the QSH-RM sample holder should be compatible with a Fabry-Pérot type cavity, which would further enhance SNR and time resolution.…”

Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures

“…The specific dynamics of spin-labeled side chains in biomolecules are encoded in their EPR spectral shape, and any change in the rotational correlation time of the spin label will produce a different averaging of the anisotropic hyperfine interactions, leading to distinct patterns of narrowing or broadening of the EPR spectral manifold . A few studies exist in which gadolinium labels were also employed in the solution state, , most recently to “film”-triggered conformational changes of an optogenetic protein …”

“…Overhauser dynamic nuclear polarization (ODNP) is an established method based on the transfer of electron spin polarization from spin label to nearby water 1 H nuclei via the Overhauser effect 22 to enhance the 1 H NMR signal, which has been applied to study water dynamics and changes in water accessibility in the proximity of nitroxide probes (Figure 1C). 23−25 Finally, cw EPR at high field (240 GHz) at room temperature enables the study of static or time-resolved 21 residual dipolar interactions between a pair of gadolinium (Gd) labels for distances up to about 4 nm (Figure 1D). 26 The biomolecular system investigated in this study is a minimal interactome consisting of three proteins from the Bcl-2 family, which are involved in the mitochondrial pathway of apoptosis in human cells.…”

Equipping 1,4,7-Triazacyclononane with Substituents via Solid-Phase Synthesis

“…The azacycle 1,4,7-triazacyclononane (tacn, NO­(H,H,H)) is a recurring structural motif in ligands because its macrocyclic structure promotes the stability of the metal ion complexes and its three N atoms allow an adjustment of the ligand on demand because they provide comparatively easy access to a manifold of variations through the attachment of substituents. A huge number of tacn-containing ligands and thereof derived complexes with metal cations (M n + ) were designed and prepared for a variety of applications, e.g., as catalysts, thereby often mimicking the active site of a metalloprotein (M n + = e.g., Cu 1+/2+ , Zn 2+ , Ni 2+ , Mn 2+ , and Fe 3+ ), , as paramagnetic labels for the structural elucidation of biomolecules with electron paramagnetic resonance (EPR) spectroscopic techniques (M n + = Gd 3+ ), − as polarizing agents for the structural analysis of biomolecules with dynamic nuclear polarization (DNP) (M n + = Gd 3+ ), , as contrast agents for magnetic resonance imaging (MRI) (M n + = Gd 3+ , Fe 2+ , and Mn 2+ ), − as radiopharmaceuticals for positron emission tomography (PET) (M n + = 64 Cu 2+ , 68 Ga 3+ , and 111 In 3+ ), − as luminescent dyes (M n + = Eu 3+ , Tb 3+ , and Yb 3+ ), ,− and as sensors (M n+ = Eu 3+ , Yb 3+ ). − The structure of the ligand is decisive for the function of the metal ion complex because it largely defines the number, type, and geometrical arrangement of donor atoms. Typically, in addition to the three nitrogen atoms of tacn, the substituents contribute donor atoms for complex formation.…”