Optical Sciences

Biomolecules and nanostructures

The Optical Sciences group studies the interaction of light and matter. Our current focus is on detection and sensing/imaging with an emphasis on the development of integrated photonics. We are part of Twente University's Department of Science and Technology and member of the MESA+ institute.


Exciting photophysics in autofluorescent proteins from red corals

  • María García-Parajó - Former member

  • Now in Proceedings of the National Academy of Sciences (PNAS) USA 98, 14392 (2001).

    A major breakthrough in Biotechnology occurred approximately seven years ago when scientists could isolate and clone a green autofluorescence protein (GFP) from the jellyfish Aequorea victoria. Cloning of this protein has revolutionised the field of Molecular and Cell Biology allowing biologists to follow gene expression and a multitude of processes in living cells. In addition to the naturally occurring GFP biochemists created different mutants of the GFP so that nowadays autofluorescent proteins with emission maxima ranging from 450nm to 530nm are readily available. However, despite multiple attempts to extend the palette of colours further into the red, no GFP has been produced with emission maxima longer than 530nm. In October 1999 a group of researchers from the Russian Academy of Sciences succeeded in cloning six fluorescent proteins from tropical corals. One particular mutant known as the DsRed has bright red fluorescence with emission maxima at 583nm, the longest wavelength emission reported so far in a wild type species.

    Recent studies on the DsRed have shown its tremendous advantages: bright red fluorescence and resistance against photobleaching. However, it appeared that the protein aggregates when expressed in cells and there has been indication for some residual green fluorescence which initially has been attributed to incomplete maturation of the protein. We applied single molecule detection to elucidate the nature of the fluorescence emission in the DsRed. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Dual colour excitation (at 488 nm and 568 nm) single molecule microscopy revealed the existence of a considerable number of green species in each tetramer. Even in nominally mature proteins, we found that 86% of the DsRed contained at least one green species with a red-to-green ratio of 1.2 - 1.5. Based on our findings, oligomer suppression would not only be advantageous for protein fusion but it will also increase the fluorescence emission of individual monomers.

    Figure 1. DsRed, a recently cloned red autofluorescent protein from tropical corals (left) has attracted great interest as an expression tracer and fusion partner for multicolor imaging. The colored spots on the right image correspond to the fluorescence emission of individual DsRed proteins immobilized in a water-pore gel. The real-time multi-step behavior of the fluorescence emission (below) reflects its tetrameric nature.
    Figure 2. (a) Fluorescence intensity distribution for 122 DsRed molecules excited with circularly polarized light at 568 nm. To compare the intensity levels between different trajectories, each recorded signal has been normalized to the excitation intensity with background subtraction. The histogram has been constructed after analyzing one by one all the trajectories and building independent distributions for each intensity level. In the case of molecules showing only one or two levels, the assignment to a particular distribution was done by looking at its normalized intensity level after background subtraction, and including it in the best fitting distribution. The peak intensities of the four count distributions are: 966 (w = 338), 633 (w = 267), 357 (w = 220) and 163 (w = 170) for 4th, 3rd, 2nd and 1st levels respectively. The inset in (a) shows the percentage of molecules displaying four, three, two and only one intensity level. (b) P distribution of all four different levels for the same number of molecules as in (a). For comparison, the inset in (b) shows the P distribution for 232 DiI molecules embedded in a thin polymer film. The width of the distribution reflects the random orientation of individual transition dipoles as expected for single molecules embedded in an amorphous matrix. The P distributions for the 1st and the 2nd level display similar width, while for the 3rd and the 4th levels a dip develops around P = 0.
    Figure 3. (a) Distribution of red and green species within the DsRed, as derived from single molecule data. (b) The cartoon illustrates the mechanism of fluorescence in the DsRed. For simplicity only red species are considered. Left side: energy transfer occurs between all chromophores, and emission results with equal probability in time from any of the four chromophores. Right side: a damaged chromophore absorbs efficiently, but partially quenches the fluorescence of the remaining undamaged chromophores. h 1, h 2, and h 3 are the quenching fractions for the nearest, intermediate and most distant pairs respectively.


    The following articles have been published regarding this project:

    Single molecule studies of the red autofluorescent protein DsRed

    (full pdf)
    Garcia-Parajo MF, Koopman M, van Dijk EMHP, Subramaniam V, van Hulst NF
    vol 82 issue 1: p234 part 2 JAN 2002

    The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

    (abstract) (full pdf)
    Garcia-Parajo MF, Koopman M, van Dijk EMHP, Subramaniam V, van Hulst NF
    vol 98 issue 25: p14392-p14397 DEC 4 2001
    Printable version