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1. Supramolecular Approach to Electron Paramagnetic Resonance Distance Measurement of Spin-Labeled Proteins

   https://doi.org/10.1021/acs.jpcb.0c00743  

   Yang, Zhimin; Stein, Richard A.; Ngendahimana, Thacien; Pink, Maren; Rajca, Suchada; Jeschke, Gunnar; Eaton, Sandra S; Eaton, Gareth R.; Mchaourab, Hassane S; Rajca, Andrzej (March 2020, The Journal of Physical Chemistry B)

   We demonstrate a host-guest molecular recognition approach to advance double electron-electron resonance (DEER) distance measurements of spin-labeled proteins. We synthesized an iodoacetamide (IA) derivative of 2,6-diazaadamantane nitroxide (DZD) spin label that could be doubly incorporated into T4 Lysozyme (T4L) by site-directed spin labeling (SDSL) with efficiency up to 50% per cysteine. The rigidity of the fused ring structure and absence of mobile methyl groups increase the spin echo dephasing time (Tm) at temperatures above 80 K. This enables DEER measurements of distances >4 nm in DZD labeled-T4L in glycerol/water at temperatures up to 150 K, with increased sensitivity compared to common spin label such as MTSL. Addition of β-cyclodextrin (β-CD) reduces the rotational correlation time of the label, slightly increases Tm, and most importantly, narrows (and slightly lengthens) the inter-spin distance distributions. The distance distributions are in good agreement with simulated distance distributions obtained by rotamer libraries. These results provide a foundation for developing supramolecular recognition to facilitate long-distance DEER measurements at near physiological temperatures. 

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2. Double Quantum Coherence ESR at Q-Band Enhances the Sensitivity of Distance Measurements at Submicromolar Concentrations

   https://doi.org/10.1021/acs.jpclett.3c02372  

   Mandato, Alysia; Hasanbasri, Zikri; Saxena, Sunil (September 2023, The Journal of Physical Chemistry Letters)

   Recently, there have been remarkable improvements in pulsed ESR sensitivity, paving the way for broader applicability of ESR in the measurement of biological distance constraints, for instance, at physiological concentrations and in more complex systems. Nevertheless, submicromolar distance measurements with the commonly used nitroxide spin label take multiple days. Therefore, there remains a need for rapid and reliable methods of measuring distances between spins at nanomolar concentrations. In this work, we demonstrate the power of double quantum coherence (DQC) experiments at Q-band frequencies. With the help of short and intense pulses, we showcase DQC signals on nitroxide-labeled proteins with modulation depths close to 100%. We show that the deep dipolar modulations aid in the resolution of bimodal distance distributions. Finally, we establish that distance measurements with protein concentrations as low as 25 nM are feasible. This limit is approximately 4-fold lower than previously possible. We anticipate that nanomolar concentration measurements will lead to further advancements in the use of ESR, especially in cellular contexts. 

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3. Endogenous Cu(II) Labeling for Distance Measurements on Proteins by EPR

   https://doi.org/10.1002/chem.202403160  

   Hunter, Hannah R; Kankati, Shashank; Hasanbasri, Zikri; Saxena, Sunil Kumar (October 2024, Chemistry – A European Journal)

   In‐cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in‐cell. However, the need to spin label proteins ex‐situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in‐cell spin labeling. We quantify the amount of Cu(II)‐NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat‐shock and hypotonic swelling to deliver spin label, showing that hypotonic swelling is a facile and reproducible method to efficiently deliver Cu(II)‐NTA into E. coli. Notably, over six repeats we accomplish a bulk average of 57 μM spin labeled sites, surpassing existing endogenous labeling methods. The results of this work open the door for endogenous spin labeling that is easily accessible to the broader biophysical community. 

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4. Peptide nanosponges designed for rapid uptake by leukocytes and neural stem cells

   https://doi.org/10.1039/C8RA00717A  

   Yapa, Asanka S.; Wang, Hongwang; Wendel, Sebastian O.; Shrestha, Tej. B.; Kariyawasam, Nilusha L.; Kalubowilage, Madumali; Perera, Ayomi S.; Pyle, Marla; Basel, Matthew T.; Malalasekera, Aruni P.; et al (January 2018, RSC Advances)

   The structure of novel binary nanosponges consisting of (cholesterol-(K/D) n DEVDGC) 3 -trimaleimide units possessing a trigonal maleimide linker, to which either lysine (K) 20 or aspartic acid (D) 20 are tethered, has been elucidated by means of TEM. A high degree of agreement between these findings and structure predictions through explicit solvent and then coarse-grained molecular dynamics (MD) simulations has been found. Based on the nanosponges' structure and dynamics, caspase-6 mediated release of the model drug 5(6)-carboxyfluorescein has been demonstrated. Furthermore, the binary (DK20) nanosponges have been found to be virtually non-toxic in cultures of neural progenitor cells. It is of a special importance for the future development of cell-based therapies that DK20 nanosponges were taken up efficiently by leucocytes (WBC) in peripheral blood within 3 h of exposure. The percentage of live cells among the WBC was not significantly decreased by the DK20 nanosponges. In contrast to stem cell or leucocyte cell cultures, which have to be matched to the patient, autologous cells are optimal for cell-mediated therapy. Therefore, the nanosponges hold great promise for effective cell-based tumor targeting. 

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5. Gd ³⁺ –Gd ³⁺ distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance

   https://doi.org/10.1039/c6cp07119h  

   Clayton, Jessica A.; Qi, Mian; Godt, Adelheid; Goldfarb, Daniella; Han, Songi; Sherwin, Mark S. (January 2017, Physical Chemistry Chemical Physics)

   Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling is a very powerful tool for elucidating the structure and organization of biomolecules. Gd 3+ complexes have recently emerged as a new class of spin labels for distance determination by pulsed EPR spectroscopy at Q- and W-band. We present CW EPR measurements at 240 GHz (8.6 Tesla) on a series of Gd-rulers of the type Gd-PyMTA–spacer–Gd-PyMTA, with Gd–Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these Gd-rulers show that significant dipolar broadening of the central |−1/2〉 → |1/2〉 transition occurs at 30 K for Gd–Gd distances up to ∼3.4 nm with Gd-PyMTA as the spin label. This represents a significant extension for distances accessible by CW EPR, as nitroxide-based spin labels at X-band frequencies can typically only access distances up to ∼2 nm. We show that this broadening persists at biologically relevant temperatures above 200 K, and that this method is further extendable up to room temperature by immobilizing the sample in glassy trehalose. We show that the peak-to-peak broadening of the central transition follows the expected 1/ r 3 dependence for the electron–electron dipolar interaction, from cryogenic temperatures up to room temperature. A simple procedure for simulating the dependence of the lineshape on interspin distance is presented, in which the broadening of the central transition is modeled as an S = 1/2 spin whose CW EPR lineshape is broadened through electron–electron dipolar interactions with a neighboring S = 7/2 spin. 

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