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Improved "optical highlighter" probes derived from discosoma red fluorescent protein.

ABSTRACT: The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda < 760 nm) femtosecond irradiation--a phenomenon that underpins the application of DsRed as an "optical highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.

SUBMITTER: Robinson LC

PROVIDER: S-EPMC1305146 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Two structurally related fluorescent imaging probes allow optical imaging of bacterial leg infection models in living athymic and immunocompetent mice. Structurally, the probes are comprised of a deep-red fluorescent squaraine rotaxane scaffold with two appended bis(zinc(II)-dicolylamine) (bis(Zn-DPA)) targeting ligands. The bis(Zn-DPA) ligands have high affinity for the anionic phospholipids and related biomolecules that reside within the bacterial envelope, and they are known to selectively target bacterial cells over the nearly uncharged membrane surfaces of healthy mammalian cells. Planar, whole-animal optical imaging studies showed that intravenous dosing of either probe (10 nmol) allowed imaging of localized infections of Gram-positive Staphylococcus aureus and Gram-negative Salmonella enterica serovar typhimurium. High selectivity for the infected target leg (T) over the contralateral nontarget leg (NT) was reflected by T/NT ratios up to six. The infection imaging signal was independent of mouse humoral immune status, and there was essentially no targeting at a site of sterile inflammation induced by injection of lambda-carrageenan. Furthermore, the fluorescent probe imaging signal colocalized with the bioluminescence signal from a genetically engineered strain of S. enterica serovar typhimurium. Although not highly sensitive (the localized infection must contain at least approximately 10(6) colony forming units for fluorescence visualization), the probes are remarkably selective for bacterial cells considering their low molecular weight (<1.5 kDa) and simple structural design. The more hydrophilic of the two probes produced a higher T/NT ratio in the early stages of the imaging experiment and washed out more rapidly from the blood clearance organs (liver, kidney). Therefore, it is best suited for longitudinal studies that require repeated dosing and imaging of the same animal. The results indicate that fluorescent probes based on squaraine rotaxanes should be broadly useful for in vivo animal imaging studies, and they further validate the ability of imaging probes with bis(Zn-DPA) ligands to selectively target bacterial infections in living animals.

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Improved "optical highlighter" probes derived from discosoma red fluorescent protein.

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