Direct Observation of Chain Lengths and Conformations in Oligofluorene Distributions from Controlled Polymerization by Double Electron-Electron Resonance.

Synthetic polymers are mixtures of chains with different lengths, and their chain length and chain conformation are often experimentally characterized by ensemble averages. We demonstrate that double electron-electron resonance (DEER) spectroscopy can reveal the chain length distribution and the chain conformation and flexibility of the individual n-mers in oligo-(9,9-dioctylfluorene) from controlled Suzuki-Miyaura coupling polymerization. The required spin-labeled chain ends were introduced efficiently via a TEMPO-substituted initiator and chain-terminating agent, respectively, with an in situ catalyst system. Individual precise chain length oligomers as reference materials were obtained by a stepwise approach. Chain length distribution, chain conformation, and flexibility can also be accessed within poly(fluorene) nanoparticles.

Light-induced dipolar spectroscopy - A quantitative comparison between LiDEER and LaserIMD.

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.

Laser-Induced Magnetic Dipole Spectroscopy.

Pulse electron paramagnetic resonance measurements of nanometer scale distance distributions have proven highly effective in structural studies. They exploit the magnetic dipole-dipole coupling between spin labels site-specifically attached to macromolecules. The most commonly applied technique is double electron-electron resonance (DEER, also called pulsed electron double resonance (PELDOR)). Here we present the new technique of laser-induced magnetic dipole (LaserIMD) spectroscopy based on optical switching of the dipole-dipole coupling. In a proof of concept experiment on a model peptide, we find, already at a low quantum yield of triplet excitation, the same sensitivity for measuring the distance between a porphyrin and a nitroxide label as in a DEER measurement between two nitroxide labels. On the heme protein cytochrome C, we demonstrate that LaserIMD allows for distance measurements between a heme prosthetic group and a nitroxide label, although the heme triplet state is not directly observable by an electron spin echo.

EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels.

The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.