Subset of Fluorophores Is Responsible for Radiation Brightening in Viromimetic Particles

Abstract

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 virussupported 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.

Ultrafast Collective Excited State Dynamics of a Virus-supported Fluorophore Antenna

“…However, from previous work it was clear that the nature of the template is important for the radiation brightening effect. 11,12 Below we provide further evidence for it from a study of relaxation dynamics.…”

“…Figure 1A shows a molecular model of BMV, with the dye-accessible external and internal lysines colored in red and green respectively. 12 Here "internal" means located between the lumenal or outer capsid surfaces. The minimum nearest neighbour distance between lysines was estimated to be 1.9 nm.…”

“…For quantitative time-domain analysis and comparison, TA spectra were integrated from 520 to 530 nm and fitted with an exponential model from 400 fs to 2.6 ns (Figure SI8- 12). For analysis, the wavelength and time-delay range were chosen past the ground state bleach minimum to avoid artifacts from pump laser scattering as well to remove coherent artifacts which arise around the zero delay time where the pump and probe beams are temporally overlapping.…”

“…local viscosity and temperature). [24][25][26] From previous work 11,12 we know that the radiation brightening from multi-fluorophore VLPs is very sensitive to the local environment, unlike ASE. Therefore, the significant shortening of excited state lifetime observed in TA and streak camera experiments, the delayed intense pulse in fluorescence emission, the broad fluorescence spectrum, the sensitivity to the fluorophore local environment, all point to a superradiance-like mechanism responsible for radiation brightening.…”

Viruses: A Physical Chemistry Perspective

“…Finally, Anil Sushma et al remind us that viruses are not only streamlined selfish genes apt of wreaking havoc in organismal processes but also molecular architects of unparalleled skill . The latter, less familiar and explored characteristic can be harnessed to create hybrid nanoparticles, which blend biological and abiotic properties for enhanced function.…”