Rational design of FRET-based sensor proteins

Real-time imaging of molecular events inside living cells is important for understanding the basis of physiological processes and diseases. Genetically encoded sensors that use fluorescence resonance energy transfer (FRET) between two fluorescent proteins are attractive in this respect because they do not require cell-invasive procedures, can be targeted to different locations in the cell and are easily adapted through mutagenesis and directed evolution approaches. Most FRET sensors developed so far show a relatively small difference in emission ratio upon activation, which severely limits their application in high throughput cell-based screening applications. In our work, we try to develop strategies that allow design of FRET-based sensors with intrinsically large ratiometric changes. This rational design approach requires a better understanding and quantitative description of the conformational changes in these fusion proteins. In this chapter, I first discuss some of the key factors and strategies that determine the ratiometric response of FRET sensors, followed by an overview of our recent work in this area. Important concepts that will be discussed are (1) the conformational behavior of flexible peptide linkers to quantitatively describe the dependence of energy transfer on linker length and (2) the control of intramolecular domain interactions using the concept of effective molecular concentration

Rational design of FRET-based sensor proteins

Real-time imaging of molecular events inside living cells is important for understanding the basis of physiological processes and diseases. Genetically encoded sensors that use fluorescence resonance energy transfer (FRET) between two fluorescent proteins are attractive in this respect because they do not require cell-invasive procedures, can be targeted to different locations in the cell and are easily adapted through mutagenesis and directed evolution approaches. Most FRET sensors developed so far show a relatively small difference in emission ratio upon activation, which severely limits their application in high throughput cell-based screening applications. In our work, we try to develop strategies that allow design of FRET-based sensors with intrinsically large ratiometric changes. This rational design approach requires a better understanding and quantitative description of the conformational changes in these fusion proteins. In this chapter, I first discuss some of the key factors and strategies that determine the ratiometric response of FRET sensors, followed by an overview of our recent work in this area. Important concepts that will be discussed are (1) the conformational behavior of flexible peptide linkers to quantitatively describe the dependence of energy transfer on linker length and (2) the control of intramolecular domain interactions using the concept of effective molecular concentration

Frugivoria em morcegos (Mammalia, Chiroptera) no Parque Estadual Intervales, sudeste do Brasil

<abstract language="eng">This study was carried out at the Intervales State Park, an Atlantic Rain Forest area in Southeastern Brazil. Bats were monthly mist netted over a full year, and fecal samples were collected for dietary analysis. The seeds found in each sample were identified in the laboratory under a stereoscopic microscope by comparison with seeds taken from ripe fruits collected in the study area. Three hundred and seventy one bats were collected, of which 316 (85.2%) were frugivorous. The total number of fecal samples with seeds and/or pulp was 121. Sturnira lilium (E. Geoffroy, 1810) was the most abundant species in the study area (n = 157 captures) and Solanaceae fruits accounted for 78.5% of the fecal samples with seeds (n = 56). Artibeus fimbriatus Gray, 1838 (n = 21 samples) fed mostly on Cecropiaceae (38%) and Moraceae fruits (24%), and Artibeus lituratus (Olfers, 1818) (n = 7 samples) on Cecropiaceae (57%) and Moraceae (29%). Carollia perspicillata (Linnaeus, 1758) (n = 16 samples) fed mostly on Piperaceae fruits (56,3%), but Solanaceae (31,3%) and Rosaceae seeds (12,5%) were also found in feces. Overall, seeds found in bat feces belong to eight plant families: Solanaceae (n = 67 samples); Cecropiaceae (n = 14); Piperaceae (n = 14); Moraceae (n = 8); Rosaceae (n = 3); Cucurbitaceae (n = 3); Cluseaceae (n = 1), and Araceae (n = 1). The close association of different bat species with fruits of certain plant families and genus may be related to a possible mechanism of resource partitioning that shapes the structure of the community