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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Cleavage-Resistant Protein Labeling With Hydrophilic Trityl Enables Distance Measurements In-Cell
Sensitive in-cell distance measurements in proteins using pulsed-Electron Spin Resonance (ESR) require reduction-resistant and cleavage-resistant spin-labels. Among the reduction-resistant moieties, the hydrophilic trityl core known as OX063 is promising due to its long phase-memory relaxation time (T_m). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the T_m of OX063 remains sufficiently long at higher temperatures, opening the possibility for measurements at temperatures above 50 K. In this work, we synthesized deuterated OX063 with a maleimide linker (mOX063-d24). We show that the combination of the hydrophilicity of the label and the maleimide linker enables high protein labeling that is cleavage-resistant in-cells. Distance measurements performed at 150 K using this label are more sensitive than the measurements at 80 K. The sensitivity gain is due to the significantly short longitudinal relaxation time (T_1) at higher temperatures, which enables more data collection per unit of time. In addition to in vitro experiments, we perform distance measurements in Xenopus laevis oocytes. Interestingly, the T_m of mOX063-d24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d24 provides highly sensitive distance measurements both in vitro and in-cells.
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- Award ID(s):
- 2006154
- NSF-PAR ID:
- 10230891
- Date Published:
- Journal Name:
- The Journal of Physical Chemistry B
- ISSN:
- 1520-6106
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed in
E. coli , most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.This article was corrected on 19 July 2022. See the end of the full text for details.
Basic Protocol 1 : Lentivirus production and expression line creationSupport Protocol 1 : Six‐well assay for estimation of production cell line yieldSupport Protocol 2 : Universal ELISA for quantifying proteins with fused leucine zippers and His‐tagsBasic Protocol 2 : Cultures for production of Class II MHC proteinsBasic Protocol 3 : Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatographyAlternate Protocol 1 : IMAC purification of His‐tagged Class II MHCSupport Protocol 3 : Protein concentration measurements and adjustmentsSupport Protocol 4 : Polishing purification by anion‐exchange chromatographySupport Protocol 5 : Estimating biotinylation percentage by streptavidin precipitationBasic Protocol 4 : Peptide exchangeBasic Protocol 5 : Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometryAlternate Protocol 2 : Native isoelectric focusing to validate MHC‐II peptide loadingBasic Protocol 6 : MultimerizationBasic Protocol 7 : Staining cells with Class II MHC tetramers
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acs.jpcb.1c02371
