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I. Mechanism and Optimization of CALI in vitro

I. Mechanism and Optimization of CALI in vitro. D.Humphrey 1 , Z. Rajfur 1 , J. Wilson 1 , W. Smith 1 , L. Cai 1 , M. McLean 2 , S. Sligar 2 , and K. Jacobson 1 1 Cell and Developmental Biology Department, University of North Carolina, Chapel Hill, NC 27599

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I. Mechanism and Optimization of CALI in vitro

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  1. I. Mechanism and Optimization of CALI in vitro D.Humphrey1, Z. Rajfur1, J. Wilson1, W. Smith1, L. Cai1, M. McLean2, S. Sligar2, and K. Jacobson1 1Cell and Developmental Biology Department, University of North Carolina, Chapel Hill, NC 27599 2Department of Biochemistry, University of Illinois, Urbana, IL 61801

  2. Cell Laser Chr protein chromophore • Chromophores • Malachite green antibody • FP fusion protein • FlAsH/ReAsH

  3. -galactosidase activity was measured using a high sensitivity spectrophotometric assay whereby beta-gal converts the yellow-orange galactoside analog, CPRG, into galactose and chlorophenol red, yielding a dark red solution. • -gal activity after irradiation • Before irradiation Relative Activity = X 100% Beam size ~ 3 mm Mirror Argon Ion Laser Sample Teflon chamber METHOD Irradiation of Sample -galactosidase assay

  4. RESULTS

  5. Lysate CALI/photobleaching as a function of irradiation dose

  6. Purified EGFP-b-Gal CALI/photobleaching

  7. CALI decreases in the absence of oxygen

  8. CALI effect is decreased when samples are irradiated in the presence of singlet oxygen quenchers, and free radical traps

  9. C terminus 55 Å N terminus 35 Å Active Site Structure of a single -gal monomer indicating the relative distance from the N and C terminus to the active site using Swiss Deep View (1BGL.pdb from RCSB Data Bank).

  10. EGFP-b-Gal b-Gal- EGFP CALI of EGFP--gal is more effective than -gal-EGFP using both purified proteins and lysates

  11. The order of CALI effectiveness is FlAsH>EBFP>ECFP>EGFP>EYFP normalized to equal number of photons absorbed.

  12. Future Work Complete and publish CALI work on Ena/VASP proteins in collaboration with the Bear lab at UNC-CH. In collaboration with the Sligar lab, screen for variants of EGFP that bleach faster than native EGFP and test those mutants for CALI efficiency by fusing to -galactosidase. Begin design and testing dual FP constructs that are spectrally distinct using one for localization by fluorescence and the more bleachable variant for CALI.

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