Photophysics of the red chromophore of HcRed: evidence for cis-trans isomerization and protonation-state changes.

HcRed is a dimeric intrinsically fluorescent protein with origins in the sea anemone Heteractis crispa. This protein exhibits deep red absorption and emission properties. Using a combination of ensemble and single molecule methods and by varying environmental parameters such as temperature and pH, we found spectroscopic evidence for the presence of two ground state conformers, trans and cis chromophores that are in thermal equilibrium and that follow different excited-state pathways upon exposure to light. The photocycle of HcRed appears to be a combination of both kindling proteins and bright emitting GFP/GFP-like proteins: the trans chromophore undergoes light driven isomerization followed by radiative relaxation with a fluorescence lifetime of 0.5 ns. The cis chromophore exhibits a photocycle similar to bright GFPs and GFP-like proteins such as enhanced GFP, enhanced YFP or DsRed, with radiative relaxation with a fluorescence lifetime of 1.5 ns, singlet-triplet deactivation on a microsecond time scale and solvent controlled protonation/deprotonation in tens of microseconds. Using single molecule spectroscopy, we identify trans and cis conformers at the level of individual moieties and show that it is possible that the two conformers can coexist in a single protein due to the dimeric nature of HcRed.

Thermochromism of a poly(phenylene vinylene): untangling the roles of polymer aggregate and chain conformation.

We report reversible thermochromism of a conjugated polymer, poly{2,5-bis[3-(N,N-diethylamino)-1-oxapropyl]-1,4-phenylenevinylene} (DAO-PPV), in diluted solutions of toluene and 1,2-dichlorobenzene. By means of temperature- and solvent-dependent steady-state spectroscopy, picosecond time-resolved photoluminescence spectroscopy, and dynamic light scattering, we provide new insights into the role of polymer aggregates in defining the thermochromic behavior of PPVs. We find DAO-PPV to exhibit a low temperature state with vibronically structured red visible absorption and emission spectra. Structurally, this low temperature state is a densely packed and disordered polymer aggregate, which contains a fraction of well-ordered, packed polymer chains. These ordered regions serve as low energy trap sites for the more disordered regions in the aggregate, thus regulating the final emission of the aggregate and imposing a vibronically resolved emission spectrum, which is usually associated with emission from one or a few chromophores. The high temperature state of DAO-PPV is a loose aggregate, with structureless absorption and emission spectra in the green visible range. Structurally, the loose aggregate is a well-solvated aggregate retaining the physical dimension of the dense aggregate but for which interchain interactions are diminished with the increase of temperature. As a result, the spectroscopic behavior of the loose aggregate is very similar if not identical to that of the single polymer chain. Increased solubility untangles polymer aggregates into single, dispersed, polymer chains, as we demonstrate here for DAO-PPV in 1,2-dichlorobenzene and at high temperature.

An intrinsically fluorescent recognition ligand scaffold based on chaperonin protein and semiconductor quantum-dot conjugates.

Genetic engineering of a novel protein-nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. The hybrid system is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon Sulfolobus shibatae. This chaperonin is an 18-subunit double ring, which self-assembles in the presence of Mg ions and ATP. Described here is a mutant chaperonin (His-beta-loopless, HBLL) with increased access to the central cavity and His-tags on each subunit extending into the central cavity. This mutant binds water-soluble semiconductor quantum dots, creating a protein-encapsulated fluorescent nanoparticle. The new bioconjugate has high affinity, in the order of strong antibody-antigen interactions, a one-to-one protein-nanoparticle stoichiometry, and high stability. By adding selective binding sites to the solvent-exposed regions of the chaperonin, this protein-nanoparticle bioconjugate becomes a sensor for specific targets.