RESUMEN
In certain conditions, dye-conjugated icosahedral virus shells exhibit suppression of concentration quenching. The recently observed radiation brightening at high fluorophore densities has been attributed to coherent emission, i.e., to a cooperative process occurring within a subset of the virus-supported fluorophores. Until now, the distribution of fluorophores among potential conjugation sites and the nature of the active subset remained unknown. With the help of mass spectrometry and molecular dynamics simulations, we found which conjugation sites in the brome mosaic virus capsid are accessible to fluorophores. Reactive external surface lysines but also those at the lumenal interface where the coat protein N-termini are located showed virtually unrestricted access to dyes. The third type of labeled lysines was situated at the intercapsomeric interfaces. Through limited proteolysis of flexible N-termini, it was determined that dyes bound to them are unlikely to be involved in the radiation brightening effect. At the same time, specific labeling of genetically inserted cysteines on the exterior capsid surface alone did not lead to radiation brightening. The results suggest that lysines situated within the more rigid structural part of the coat protein provide the chemical environments conducive to radiation brightening, and we discuss some of the characteristics of these environments.
Asunto(s)
Bromovirus , Virus , Cápside , Proteínas de la Cápside , Colorantes FluorescentesRESUMEN
Concentration quenching is a well-known challenge in many fluorescence imaging applications. Here, we show that the optical emission from hundreds of chromophores confined onto the surface of a 28 nm diameter virus particle can be recovered under pulsed irradiation. We have found that as one increases the number of chromophores tightly bound to the virus surface, fluorescence quenching ensues at first, but when the number of chromophores per particle is nearing the maximum number of surface sites allowable, a sudden brightening of the emitted light and a shortening of the excited-state lifetime are observed. This radiation brightening occurs only under short pulse excitation; steady-state excitation is characterized by conventional concentration quenching for any number of chromophores per particle. The observed suppression of fluorescence quenching is consistent with efficient, collective relaxation at room temperature. Interestingly, radiation brightening disappears when the emitters' spatial and/or dynamic heterogeneity is increased, suggesting that the template structural properties may play a role that could be instrumental in developing virus-enabled imaging vectors that have optical properties qualitatively different than those of state-of-the-art biophotonic agents.