Alkyne-tagged Raman probes have shown
high promise for noninvasive
and sensitive visualization of small biomolecules to understand their
functional roles in live cells. However, the potential for alkynes
to sense cellular environments that goes beyond imaging remains to
be further explored. Here, we report a general strategy for Raman
imaging-based local environment sensing by hydrogen–deuterium
exchange (HDX) of terminal alkynes (termed alkyne-HDX). We first demonstrate,
in multiple Raman probes, that deuterations of the alkynyl hydrogens
lead to remarkable shifts of alkyne Raman peaks for about 130 cm–1, providing resolvable signals suited for imaging-based
analysis with high specificity. Both our analytical derivation and
experimental characterizations subsequently establish that HDX kinetics
are linearly proportional to both alkyne pK
as and environmental pDs. After validating the quantitative nature
of this strategy, we apply alkyne-HDX to sensing local chemical and
cellular environments. We establish that alkyne-HDX exhibits high
sensitivity to various DNA structures and demonstrates the capacity
to detect DNA structural changes in situ from UV-induced
damage. We further show that this strategy is also applicable to resolve
subtle pD variations in live cells. Altogether, our work lays the
foundation for utilizing alkyne-HDX strategy to quantitatively sense
the local environments for a broad spectrum of applications in complex
biological systems.
The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)–driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases.
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