Sequential Two-Photon Delayed Fluorescence Anisotropy for Macromolecular Size Determination.

Time-resolved fluorescence anisotropy (FA) uses the fluorophore depolarization rate to report on rotational diffusion, conformation changes, and intermolecular interactions in solution. Although FA is a rapid, sensitive, and nondestructive tool for biomolecular interaction studies, the short (∼ns) fluorescence lifetime of typical dyes largely prevents the application of FA on larger macromolecular species and complexes. By using triplet shelving and recovery of optical excitation, we introduce optically activated delayed fluorescence anisotropy (OADFA) measurements using sequential two-photon excitation, effectively stretching fluorescence anisotropy measurement times from the nanosecond scale to hundreds of microseconds. We demonstrate this scheme for measuring slow depolarization processes of large macromolecular complexes, derive a quantitative rate model, and perform Monte Carlo simulations to describe the depolarization process of OADFA at the molecular level. This setup has great potential to enable future biomacromolecular and colloidal studies.

Unprecedented Intramolecular Association-Induced Fluorescence in Tryptophan-Conjugated Peptidomimetics.

We report herewith tryptophan (Trp)-conjugated peptidomimetics that show intramolecular through-space association between the Trp units. Our investigation revealed that the proximal placement of Trp can lead to the emergence of a new and unanticipated fluorescent entity constituting a Trp-Trp dimer. Proton-induced modulation of fluorescence is a consequence of this work. Investigations with control compounds unequivocally revealed that the fluorescence property is not originated from the localized excited state but from the unprecedented Trp-Trp intramolecular dimer in the ground state itself. The present findings will initiate the biophysical scientists to have a relook at the fluorescence properties of Trp-containing proteins.