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Photoconvertible fluorophores can enable the visualization and tracking of a specific biomolecules, complexes, and cellular compartments with precise spatiotemporal control. The field of photoconvertible probes is dominated by fluorescent protein variants, which can introduce perturbations to the target biomolecules due to their large size. Here, we present a photoconvertible small molecule, termed CPX, that can be conjugated to any target through azide-alkyne cycloaddition ("click" reaction). To demonstrate its utility, we have applied CPX to study (1) trafficking of biologically relevant synthetic vesicles and (2) intracellular processes involved in transmission of α-synuclein (αS) pathology. Our results demonstrate that CPX can serve as a minimally perturbing probe for tracking the dynamics of biomolecules.

Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Förster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 Å range. Furthermore, Mcm/Acd labeling can be combined with tryptophan fluorescence in three color FRET to monitor multiple interactions in one experiment.

Peptide cyclization via chemoselective reactions between side chains has proven a useful strategy to control folded structure. We report here a method for the synthesis of side-chain to side-chain cyclic peptides based on the intermolecular reaction between a linear peptide functionalized with two aminooxy or hydrazide side chains and an organic dialdehyde linker. A family of oxime-based and hydrazone-based cyclic products is prepared in a modular and convergent fashion by combination of unprotected linear peptide precursors and various small molecule linkers in neutral aqueous buffer. The side-chain to side-chain linkages that result can alter peptide folding behavior. The dynamic covalent nature of the Schiff bases in the cyclic products can be utilized to create mixtures where product composition changes in response to experimental conditions. Thus, a linear peptide precursor can select one organic linker from a mixture, and a cyclic product can dynamically exchange the small molecule component of the macrocycle.

The design, synthesis and preliminary activity of small molecular weight modulators of the heat shock protein 70 (Hsp70) are described. The compounds provide a starting point for the synthesis of novel tools to decipher Hsp70 biology.

Fluorescence spectroscopy is a powerful method for monitoring protein folding in real-time with high resolution and sensitivity, but requires the site-specific introduction of labels into the protein. The ability to genetically incorporate unnatural amino acids allows for the efficient synthesis of fluorescently-labeled proteins with minimally perturbing fluorophores. Here, we describe recent uses of labeled proteins in dynamic structure determination experiments and advances in unnatural amino acid incorporation for dual site-specific fluorescent labeling. The advent of increasingly sophisticated bioorthogonal chemistry reactions and the diversity of unnatural amino acids available for incorporation will greatly enable protein folding and stability studies.

We report here a chemoselective peptide “stapling” method that can be performed on ligand–receptor complexes in situ. An appropriately structured macrocyclic bis-oxime linkage is shown to improve the affinity of a peptide ligand for its native protein receptor. The presence of the receptor as a template to preorganize the ligand into its bioactive conformation is found to bias reaction outcomes, suggesting the potential application of the method for receptor-assisted selection of stapled peptides.

Covalent side-chain cross-linking has been shown to be a viable strategy to control peptide folding. We report here that an oxime side-chain linkage can elicit α-helical folds from peptides in aqueous solution. The bio-orthogonal bridge is formed rapidly under neutral buffered conditions, and the resulting cyclic oximes are capable of dynamic covalent exchange.