# NMR Methods to decipher the structural and dynamics aspects of TCR mechanobiology

> **NIH NIH P01** · DANA-FARBER CANCER INST · 2022 · $399,976

## Abstract

ABSTRACT
 To protect us from myriad diseases, adaptive immunity requires T cell recognition of protein-derived peptides
of foreign, mutant, or otherwise anomalous origin expressed on the surface of aberrant cells. Central to T cell
function is the cell surface  T cell receptor (TCR), which recognizes these various peptides bound to major
histocompatibility molecules (pMHC). While a great deal is known about the static conformations of TCR
molecules and their pMHC ligands as well as the resultant ligation complexes, it is still unknown precisely what
happens within the TCR-pMHC complex to generate the diverse signaling outcomes which drive T cell
responses. Recent experiments have highlighted the dynamic nature of TCR-pMHC ligation and signaling, with
a critical input of force necessary to generate T cell responses. This implies a dynamic system, poised to signal
with the input of piconewton (pN) amounts of force to generate signaling-ready TCR proteins. We propose to
develop NMR methods for studies of large extracellular domains involved in TCR mechanobiology, including the
TCR and its developmental precursor, the preTCR, as well as the pMHC ligands that are at the limit of NMR
observation. This includes protein domains that cannot be expressed in bacterial systems and thus cannot
readily be perdeuterated, a current standard labeling strategy for addressing high molecular weight protein
systems. Thus, in Aim 1 we employ direct 15N-detection methods, which do not require protein perdeuteration
for backbone resonance assignment. Further, we will develop new 13C-detected experiments with TROSY
enhancement that yield highly resolved spectra of aromatic side chains. To augment the state of the art NMR
technology above, in Aim 2 we will establish new labeling schemes to aid in deciphering the structure and
dynamics of preTCR, TCR and pMHC. To tackle the resonance assignment of these large proteins we will pursue
the use of “mixed pyruvate” as a carbon source to label proteins to obtain residue specific patterns. In tandem
with Aim 1 we will produce isolated 13C and 13C-19F labeled aromatic amino acids through chemoenzymatic
synthesis. Since some protein components of the TCR systems we intend to study cannot be recombinantly
expressed in E.Coli, we will pursue expression in eukaryotic systems, where complete deuteration is a challenge.
The labeling technology developed here will be transferred to Core B. In Aim 3, we will use the NMR technologies
and labeling methods, described above, to obtain information about structure and dynamics of TCR-pMHC
complexes. In particular relaxation dispersion and CEST, we will leverage 19F nuclei as probe to access dynamics
in the low microsecond time scale. The extracted dynamics information will be utilized with the MD Core C to
observe dynamics in silico and link the dynamics and conformational states measured in NMR to those that are
observed under force. Functional impact of the atomistic findings will be assess...

## Key facts

- **NIH application ID:** 10438680
- **Project number:** 5P01AI143565-03
- **Recipient organization:** DANA-FARBER CANCER INST
- **Principal Investigator:** GERHARD WAGNER
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $399,976
- **Award type:** 5
- **Project period:** 2020-07-29 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10438680

## Citation

> US National Institutes of Health, RePORTER application 10438680, NMR Methods to decipher the structural and dynamics aspects of TCR mechanobiology (5P01AI143565-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10438680. Licensed CC0.

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