Projects

EPR spectroscopy of diamagnetic bio-macromolecules is based on site-directed spin labeling (SDSL). Here, we present a novel labeling strategy for proteins. We developed and synthesized a nitroxide-based spin label that can be ligated to proteins by an inverse-electron-demand Diels-Alder (DAinv) cycloaddition to genetically encoded non-canonical amino acids (ncAA). The nitroxide moiety is shielded by a photoremovable protecting group (PPG) with an attached tetraethylene glycol unit to achieve water solubility. We demonstrate SDSL of two model proteins with the PaNDA (Photoactivatable Nitroxide for DAinv reaction) label. Our strategy features high reaction rates combined with high selectivity, and the possibility to deprotect the nitroxide in E. coli lysate.

Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling (SDSL) is an important tool to obtain long-range distance restraints for protein structural research. We here study a variety of azide- and alkyne-bearing noncanonical amino acids (ncAA) in terms of protein single- and double-incorporation efficiency via nonsense suppression, metabolic stability, yields of nitroxide labeling via copper-catalyzed [3 + 2] azide–alkyne cycloadditions (CuAAC), and spectroscopic properties in continuous-wave and double electron–electron resonance measurements. We identify para-ethynyl-l-phenylalanine and para-propargyloxy-l-phenylalanine as suitable ncAA for CuAAC-based SDSL that will complement current SDSL approaches, particularly in cases in which essential cysteines of a target protein prevent the use of sulfhydryl-reactive spin labels.

In-cell distance determination by electron paramagnetic resonance (EPR) spectroscopy reveals essential structural information about biomacromolecules under native conditions. We demonstrate that the pulsed EPR technique RIDME (relaxation induced dipolar modulation enhancement) can be utilized for such distance determination. The performance of in-cell RIDME has been assessed at Q-band using stiff molecular rulers labeled with Gd(III)-PyMTA and microinjected into Xenopus laevis oocytes. The overtone coefficients are determined to be the same for protonated aqueous solutions and inside cells. As compared to in-cell DEER (double electron–electron resonance, also abbreviated as PELDOR), in-cell RIDME features approximately 5 times larger modulation depth and does not show artificial broadening in the distance distributions due to the effect of pseudosecular terms.

We report site-directed protein spin labelling via Suzuki–Miyaura coupling of a nitroxide boronic acid label with the genetically encoded amino acid 4-iodo-L-phenylalanine. The resulting spin label bears a rigid biphenyl linkage with lower flexibility than spin label R1. It is suitable to obtain defined electron paramagnetic resonance distance distributions and to report protein–membrane interactions and conformational transitions of α-synuclein.

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSL⁻EPR experiments crucially depends on the employed labeling strategy, with key aspects being labeling chemoselectivity and biocompatibility, as well as stability and spectroscopic properties of the resulting label. The use of genetically encoded noncanonical amino acids (ncAA) is an emerging strategy for SDSL that holds great promise for providing excellent chemoselectivity and potential for experiments in complex biological environments such as living cells. We here give a focused overview of recent advancements in this field and discuss their potentials and challenges for advancing SDSL⁻EPR studies.

Nanometric distance measurements with EPR spectroscopy yield crucial information on the structure and interactions of macromolecules in complex systems. The range of suitable spin labels for such measurements was recently expanded with a new class of light-inducible labels: the triplet state of porphyrins. Importantly, accurate distance measurements between a triplet label and a nitroxide have been reported with two distinct light-induced spectroscopy techniques, (light-induced) triplet-nitroxide DEER (LiDEER) and laser-induced magnetic dipole spectroscopy (LaserIMD). In this work, we set out to quantitatively compare the two techniques under equivalent conditions at Q band. Since we find that LiDEER using a rectangular pump pulse does not reach the high modulation depth that can be achieved with LaserIMD, we further explore the possibility of improving the LiDEER experiment with chirp inversion pulses. LiDEER employing a broadband pump pulse results in a drastic improvement of the modulation depth. The relative performance of chirp LiDEER and Laser-IMD in terms of modulation-to-noise ratio is found to depend on the dipolar evolution time: While LaserIMD yields higher modulation-to-noise ratios than LiDEER at short dipolar evolution times (τ=2μs), the high phase memory time of the triplet spins causes the situation to revert at τ=6μs.