Abstract

We studied the dynamic behavior of red shifted mutants of the fluorescence of green fluorescent protein (GFP) like S65T and EGFP by FCS and discovered a fast blinking effect dependent on the pH of the used buffer. This indicates that the protein spends a considerable amount of time in a dark state, with transitions back and forth to the bright state on the 100 ?s time scale. The findings can be related to the overall fluorescence that decreases at low pH. Structural knowledge about this protein suggests that the dark state is actually a protonated form of the chromophore that shifts the absorption to smaller values where no excitation by the FCS laser can occur. FCS thus allows to derive precise kinetic and thermodynamic information of this reversible protonation process.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR004224-12
Application #
6206122
Study Section
Project Start
1999-09-01
Project End
2000-08-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
12
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Cornell University
Department
Type
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850

  Publications

Migone, Fernando F; Cowan, Robert G; Williams, Rebecca M et al. (2016) In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation. Proc Natl Acad Sci U S A 113:2294-9
O'Dell, Ryan S; Cameron, David A; Zipfel, Warren R et al. (2015) Reelin Prevents Apical Neurite Retraction during Terminal Translocation and Dendrite Initiation. J Neurosci 35:10659-74
Byrnes, Laura J; Singh, Avtar; Szeto, Kylan et al. (2013) Structural basis for conformational switching and GTP loading of the large G protein atlastin. EMBO J 32:369-84
Jain, Manu; Robinson, Brian D; Scherr, Douglas S et al. (2012) Multiphoton microscopy in the evaluation of human bladder biopsies. Arch Pathol Lab Med 136:517-26
Degala, Satish; Williams, Rebecca; Zipfel, Warren et al. (2012) Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture. J Orthop Res 30:793-9
O'Dell, Ryan S; Ustine, Candida J M; Cameron, David A et al. (2012) Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone. Neural Dev 7:25
McMullen, J D; Kwan, A C; Williams, R M et al. (2011) Enhancing collection efficiency in large field of view multiphoton microscopy. J Microsc 241:119-24
Kim, Sally A; Sanabria, Hugo; Digman, Michelle A et al. (2010) Quantifying translational mobility in neurons: comparison between current optical techniques. J Neurosci 30:16409-16
Bowles, Robby D; Williams, Rebecca M; Zipfel, Warren R et al. (2010) Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disc composite via collagen gel contraction. Tissue Eng Part A 16:1339-48
McMullen, Jesse D; Zipfel, Warren R (2010) A multiphoton objective design with incorporated beam splitter for enhanced fluorescence collection. Opt Express 18:5390-8

Showing the most recent 10 out of 68 publications