# New Nitroxide Spin Labels for Distance Measurements in Biological Systems

> **NIH NIH R01** · UNIVERSITY OF NEBRASKA LINCOLN · 2020 · $412,926

## Abstract

Project Summary
Nitroxide spin labels are useful probes for investigation of biomacromolecules such as proteins DNA, and RNA.
In a typical approach, doubly-labeled proteins are prepared by site-directed spin labeling (SDSL) and a set of
distances between spin labels is measured using pulsed electron paramagnetic resonance (EPR) dipolar
spectroscopy (PDS). Double electron electron resonance (DEER) is the most widely used PDS method. The
temperatures at which the DEER measurements currently can be made are limited by the dynamic averaging
effects associated with methyl group rotation in the typical nitroxide spin label, and therefore have to be
performed at about 50 – 70 K, requiring use of liquid helium, or with lower sensitivity at 80 K with liquid
nitrogen. In addition, application of the SDSL methodology to in vivo measurements is hampered by the short
in vivo lifetime of the currently available nitroxide spin labels. The objective of the proposed work is to
synthesize and evaluate new nitroxide spin labels for DEER distance measurements in proteins at
physiological temperature and for in vivo DEER at cryogenic temperature. The proposed project has three
specific aims. Aim 1: synthesize ultra-rigid, small-sized, and polar nitroxide spin labels for distance
measurements at physiological temperatures (D-Labels). The target labels are novel nitroxides devoid of
methyl groups with structure motifs that address the problem of conformational flexibility, molecular size and
hydrophobicity. Aim 2: synthesize nitroxide spin labels for in vivo DEER distance measurements (C-Labels).
The targets labels are biostable gem-diethyl and gem-dicarboxylate nitroxide spin labels. Aim 3: evaluate
effectiveness of the synthesized spin labels for SDSL-DEER distance measurements at physiological
temperatures and for in vivo DEER distance measurements. All labels synthesized in Aims 1 and 2 will be
tested for their electron spin relaxation times (Tm and T1) as a function of temperature up to 310 K and for the
rate of reduction in ascorbate/glutathione solutions. SDSL-DEER distance measurements at physiological
temperature will be calibrated using T4 Lysozyme (T4L) that is doubly spin labelled with D-Labels and
immobilized in carbohydrate matrix. C-Labels will be tested for in vivo DEER distance measurements at
cryogenic temperature, via SDSL of selected proteins in live mitochondria and in E. coli. Collaborating
laboratories will carry out these studies and set the stage for the general adoption of the proposed spin labels
by highlighting their properties and defining the range of their applications. These studies will provide structural
information at physiological temperature and/or in the native cellular environment, which is characterized by
factors and conditions that may have a crucial role in determining the biologically relevant conformation of the
proteins under investigation. The wealth of information enabled by the proposed labels will be close to ideal for
...

## Key facts

- **NIH application ID:** 9922926
- **Project number:** 5R01GM124310-04
- **Recipient organization:** UNIVERSITY OF NEBRASKA LINCOLN
- **Principal Investigator:** ANDRZEJ RAJCA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $412,926
- **Award type:** 5
- **Project period:** 2017-08-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9922926

## Citation

> US National Institutes of Health, RePORTER application 9922926, New Nitroxide Spin Labels for Distance Measurements in Biological Systems (5R01GM124310-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9922926. Licensed CC0.

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