Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures

Electron paramagnetic resonance spectroscopy (EPR) is mostly used in structural biology in conjunction with pulsed dipolar spectroscopy (PDS) methods to monitor interspin distances in biomacromolecules at cryogenic temperatures both in vitro and in cells. In this context, spectroscopically orthogonal spin labels were shown to increase the information content that can be gained per sample. Here, we exploit the characteristic properties of gadolinium and nitroxide spin labels at physiological temperatures to study side chain dynamics via continuous wave (cw) EPR at X band, surface water dynamics via Overhauser dynamic nuclear polarization at X band and short-range distances via cw EPR at high fields. The presented approaches further increase the accessible information content on biomolecules tagged with orthogonal labels providing insights into molecular interactions and dynamic equilibria that are only revealed under physiological conditions.


Protein preparation
All details related to the solid state peptide synthesis and the spin labeling procedures are published as supplementary material in 1 . The peptide concentration was determined by UV/vis spectroscopy (Eppendorf BioSpectrometer Basic) using a 280 nm extinction coefficient of 8480 M -1 cm -1 for the long Bim (26-27aa) peptides and 5500 M -1 cm -1 for the short Bim peptides (16-17aa). Full length human Bax was expressed in E. coli, purified, and labelled with MTSL at the two natural cysteines C62 and C126 as described in 2,3 . The concentration was determined by UV/vis using an extinction coefficient of 36940 M -1 cm -1 4 .The Bcl-xL protein used in this study lacks the C-terminal helix 9 and was expressed and purified as described previously 1 . The concentration was determined by UV/vis using a theoretical extinction coefficient of 47440 M -1 cm -1 calculated via ExPASy program (https://www.expasy.org/).

CW EPR kinetics measurements
All samples were prepared at a final volume of 20 µL, containing 4 µL of large unilamellar vesicles (LUVs) and a variable protein/peptide volume to reach a final concentration of 20 µM for each reaction partner. The void volume to reach 20 µL was filled using SMART buffer (20 mM, NaCl 150 mM, pH 7.5). Subsequently, the sample was placed in a glass capillary (Blaubrand) and sealed with Critoseal (VWR). CW EPR kinetics were recorded using an Elexsys E580 X-band (9.45GHz) spectrometer (Bruker, Germany) equipped with an ER 4122 SHQ (Bruker, Germany) and a nitrogen flow cryostat. The temperature was stabilized with a temperature controller (Eurotherm) at 37°C. The following measurement parameters were used: mod. amplitude -0.15 mT, mod. frequency -100 kHz, sweep width -10 mT, sweep time -20.5 s, microwave power 9.5 mW.

ODNP
ODNP measurements were performed on the home-made ODNP setup at RUB. The setup consisted of an Elexsys E580 X-band spectrometer equipped with a SHQ cavity (Bruker), a microwave power source Bridge12 (Bridge12), a NMR Avance III HD spectrometer (Bruker) and a home-made NMR coil based on S. Han design. ODNP measurements were performed using an automated home-developed Python package "TopDNP" running from Top-Spin software (Bruker). The code with description of usage is freely available on GitHub: https://github.com/haditim/TopDNP. ODNP data were analyzed using an automated homedeveloped Python package "DNPy", the code with the detailed information about the evaluation procedure is free available on https://github.com/haditim/DNPy.

S4
For ODNP measurements 4µL of corresponding BIM-peptide (Cfinal=15µM (spin)) or its mixture with BclXL (1:1 protein:protein) was transferred into pre-cut 0.6 mm ID x 0.84 mm OD round quartz capillary (VitroCom) and sealed with Critoseal (VWR) from both sides. For competition experiment, BIM-peptide pre-mixed and pre-incubated for 1h at room temperature was mixed with BIM26 or Gd-BIM27 in 1:3 ratio. The same samples in capillaries were put into a bigger 3 mm OD quarz capillary (Quarzglas Heinrich) for the subsequent CW EPR measurements on a Miniscope MS5000 (Magnettech, Freiberg Instruments) using 10 mW microwave power and 0.1 mT modulation amplitude (100 kHz mod. frequency) at room temperature.

GHz CW EPR
All room temperature samples were prepared at a final volume of 6 µL with a variable protein/peptide volume to reach a final concentration of 100 µM for each reaction partner. The void volume to reach 6 µL was filled depending on the sample type with SMART buffer and/or TFE (2,2,2-Trifluorethanol). The low temperature samples (30 K) were prepared by at a higher sample volume of 9 µL due to the added 33% final glycerol-d8 concentration. Accordingly, these samples have a 67 µM final protein/peptide concentration.
The high-field cw EPR experiments were carried out using a home-built 240 GHz EPR spectrometer at the Institute for Terahertz Science and Technology at UCSB described S9 Figure S4. High-field cw EPR spectra at cryogenic temperature. Comparison of the 240 GHz cw EPR spectra acquired at room temperature (from Fig. 5) and at 30 K. The spectra are narrower at low temperature, but the addition of TFE is shown to decrease the linewidth both at room and at 30 K due to the disappearance of the dipolar broadening arising from inter-peptide interactions. S10