Alkyne-Tagged Raman Probes for Local Environmental Sensing by Hydrogen-Deuterium Exchange.

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 pKas 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.

Bond-selective fluorescence imaging with single-molecule sensitivity.

Bioimaging harnessing optical contrasts and chemical specificity is of vital importance in probing complex biology. Vibrational spectroscopy based on mid-infrared (mid-IR) excitation can reveal rich chemical information about molecular distributions. However, its full potential for bioimaging is hindered by the achievable sensitivity. Here, we report bond selective fluorescence-detected infrared-excited (BonFIRE) spectral microscopy. BonFIRE employs two-photon excitation in the mid-IR and near-IR to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence. The system utilizes tuneable narrowband picosecond pulses to ensure high sensitivity, biocompatibility, and robustness for bond-selective biological interrogations over a wide spectrum of reporter molecules. We demonstrate BonFIRE spectral imaging in both fingerprint and cell-silent spectroscopic windows with single-molecule sensitivity for common fluorescent dyes. We then demonstrate BonFIRE imaging on various intracellular targets in fixed and live cells, neurons, and tissues, with promises for further vibrational multiplexing. For dynamic bioanalysis in living systems, we implement a high-frequency modulation scheme and demonstrate time-lapse BonFIRE microscopy of live HeLa cells. We expect BonFIRE to expand the bioimaging toolbox by providing a new level of bond-specific vibrational information and facilitate functional imaging and sensing for biological investigations.

Receptor compaction and GTPase rearrangement drive SRP-mediated cotranslational protein translocation into the ER.

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.