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Content archived on 2024-06-18

Dissecting the fluorescence properties of the Enhanced Cyan Fluorescent Protein by computational analysis of its structure and dynamics

Objective

Due to its intrinsic visible fluorescence, the Green Fluorescent Protein (GFP) is used to monitor cellular and biomolecular functions. Its chromophore is formed by cyclization of three aminoacid residues: Ser65, Tyr66 and Gly67. The substitution of Tyr66 b y Trp and few additional mutations lead to the Enhanced Cyan Fluorescent Protein (ECFP), characterized by cyan emission.

One unexplained feature is that ECFP has double humped rather than conventional single excitation and emission peaks. NMR and crystallo-graphic studies suggested that these two spectral peaks of ECFP originate from two conformational states of the protein. However, a preliminary time-resolved fluorescence study indicates that the photophysics of ECFP is more complicated. At least four lifetimes, which depend differently on pH and temperature are required to describe the fluorescence decay and suggest more than two fluorescent species.

Molecular calculations and simulations are particularly valuable for relating properties of biomolecules t o their structure. Our project will be the first study which will combine a variety of theoretical techniques and relate the results to spectral data, in order to understand the intriguing photophysics of ECFP. From electrostatic calculations we will obtain the population probabilities of the different states as a function of pH.

The analysis of molecular dynamics and of quantum mechanical calculations will provide information on the behaviour of each state and the temperature dependence of this behaviour. T he combination of these two approaches, together with the results of the excited state dynamics analysis, will lead to a quantitative mechanistic model of the dependence of the fluorescence signal on pH and temperature.

Understanding the structural basis o f the photophysical process will lead to the design of ECFP mutants with simplified or modified photophysical properties improving the fluorescence tools for cellular biology and medical diagnostics.

Fields of science (EuroSciVoc)

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Keywords

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Topic(s)

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Call for proposal

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FP6-2004-MOBILITY-5
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Funding Scheme

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EIF - Marie Curie actions-Intra-European Fellowships

Coordinator

UNIVERSITAET BAYREUTH
EU contribution
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Total cost

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