Abstract
Fluorescence correlation spectroscopy (FCS) has proven to be a powerful technique with single-molecule sensitivity. Recently, it has found a complement in the form of fluorescence intensity distribution analysis (FIDA). Here we introduce a fluorescence fluctuation method that combines the features of both techniques. It is based on the global analysis of a set of photon count number histograms, recorded with multiple widths of counting time intervals simultaneously. This fluorescence intensity multiple distributions analysis (FIMDA) distinguishes fluorescent species on the basis of both the specific molecular brightness and the translational diffusion time. The combined information, extracted from a single measurement, increases the readout effectively by one dimension and thus breaks the individual limits of FCS and FIDA. In this paper a theory is introduced that describes the dependence of photon count number distributions on diffusion coefficients. The theory is applied to a series of photon count number histograms corresponding to different widths of counting time intervals. Although the ability of the method to determine specific brightness values, diffusion times, and concentrations from mixtures is demonstrated on simulated data, its experimental utilization is shown by the determination of the binding constant of a protein-ligand interaction exemplifying its broad applicability in the life sciences.
Full Text
The Full Text of this article is available as a PDF (108.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alessandro R., Spoonster J., Wersto R. P., Kohn E. C. Signal transduction as a therapeutic target. Curr Top Microbiol Immunol. 1996;213(Pt 3):167–188. doi: 10.1007/978-3-642-80071-9_11. [DOI] [PubMed] [Google Scholar]
- Baumann G., Maier D., Freuler F., Tschopp C., Baudisch K., Wienands J. In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells. Eur J Immunol. 1994 Aug;24(8):1799–1807. doi: 10.1002/eji.1830240812. [DOI] [PubMed] [Google Scholar]
- Booker G. W., Breeze A. L., Downing A. K., Panayotou G., Gout I., Waterfield M. D., Campbell I. D. Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase. Nature. 1992 Aug 20;358(6388):684–687. doi: 10.1038/358684a0. [DOI] [PubMed] [Google Scholar]
- Chen Y., Müller J. D., So P. T., Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J. 1999 Jul;77(1):553–567. doi: 10.1016/S0006-3495(99)76912-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eggeling C., Fries J. R., Brand L., Günther R., Seidel C. A. Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1556–1561. doi: 10.1073/pnas.95.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eigen M., Rigler R. Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5740–5747. doi: 10.1073/pnas.91.13.5740. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Furet P., Gay B., Caravatti G., García-Echeverría C., Rahuel J., Schoepfer J., Fretz H. Structure-based design and synthesis of high affinity tripeptide ligands of the Grb2-SH2 domain. J Med Chem. 1998 Aug 27;41(18):3442–3449. doi: 10.1021/jm980159a. [DOI] [PubMed] [Google Scholar]
- Gram H., Schmitz R., Zuber J. F., Baumann G. Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display. Eur J Biochem. 1997 Jun 15;246(3):633–637. doi: 10.1111/j.1432-1033.1997.00633.x. [DOI] [PubMed] [Google Scholar]
- Kask P., Palo K., Fay N., Brand L., Mets U., Ullmann D., Jungmann J., Pschorr J., Gall K. Two-dimensional fluorescence intensity distribution analysis: theory and applications. Biophys J. 2000 Apr;78(4):1703–1713. doi: 10.1016/S0006-3495(00)76722-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kask P., Palo K., Ullmann D., Gall K. Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13756–13761. doi: 10.1073/pnas.96.24.13756. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koppel D. E., Axelrod D., Schlessinger J., Elson E. L., Webb W. W. Dynamics of fluorescence marker concentration as a probe of mobility. Biophys J. 1976 Nov;16(11):1315–1329. doi: 10.1016/S0006-3495(76)85776-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levitzki A. Signal-transduction therapy. A novel approach to disease management. Eur J Biochem. 1994 Nov 15;226(1):1–13. doi: 10.1111/j.1432-1033.1994.tb20020.x. [DOI] [PubMed] [Google Scholar]
- Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
- Müller K., Gombert F. O., Manning U., Grossmüller F., Graff P., Zaegel H., Zuber J. F., Freuler F., Tschopp C., Baumann G. Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting. J Biol Chem. 1996 Jul 12;271(28):16500–16505. [PubMed] [Google Scholar]
- Overduin M., Rios C. B., Mayer B. J., Baltimore D., Cowburn D. Three-dimensional solution structure of the src homology 2 domain of c-abl. Cell. 1992 Aug 21;70(4):697–704. doi: 10.1016/0092-8674(92)90437-h. [DOI] [PubMed] [Google Scholar]
- Qian H., Elson E. L. On the analysis of high order moments of fluorescence fluctuations. Biophys J. 1990 Feb;57(2):375–380. doi: 10.1016/S0006-3495(90)82539-X. [DOI] [PMC free article] [PubMed] [Google Scholar]