Single-molecule fluorescence pulsed interleaved excitation, a novel tool to study biomolecular interactions.. (SMF-PIE)
During the last few years, single-molecule fluorescence (SMF) methodologies have revolutionized the accessible information on biomolecular complexes, molecular dynamics, and biological mechanisms.
Novel and important structural and dynamic insights can be obtained by studying molecules one by one, particularly into biological processes. In this field, the development of new methodologies and tools, on one hand, and of new improved fluorophores, on the other, are two major lines of research.
In the current proposal, a cutting edge SMF methodology will be implemented: SMF Pulsed Interleaved Excitation (PIE) with two-color coincidence detection, a technique that allows alternating excitation of fluorophores of different colors acting as donor and acceptor in fluorescence resonance energy transfer (FRET) processes.
SMF-PIE has several advantages over the conventional SMF schemes to study biological systems, such as accurate determination of FRET efficiencies and inter-fluorophore distances, effective removal of the spectral, or distinction between molecules that have a low FRET efficiency from those that lack an acceptor tag.
The main goals of this project are, hence, as follows:
* Implementation, calibration, and validation of a state-of-the-art SMF-PIE instrumentation. Subsequently, SMF-PIE will be applied in three multidisciplinary biophysical approaches.
* Testing the feasibility and performance of newly synthesized fluorescein derivatives for SMF studies, especially those derivatives belonging to the Tokyo Green family of dyes.
* Providing new insights into the intercalation mechanism of DNA intercalators at the single molecule level, by means of single-pair FRET between fluorescein derivatives, acting energy donors, and DNA intercalator fluorophores as acceptors.
* Studying at the single molecule level the early pre-fibrillar aggregates from an amyloidogenic protein, the SH3 domain of alpha-spectrin. SMF-PIE will be employed to provide insights into the amyloid fibril formation mechanisms, a major biophysical and biomedical challenge nowadays.
Universidad de Granada
During the last few years, single-molecule fluorescence (SMF) methodologies have revolutionized the accessible information on biomolecular complexes, molecular dynamics, and biological mechanisms.
Novel and important structural and dynamic insights can be obtained by studying molecules one by one, particularly into biological processes. In this field, the development of new methodologies and tools, on one hand, and of new improved fluorophores, on the other, are two major lines of research.
In the current proposal, a cutting edge SMF methodology will be implemented: SMF Pulsed Interleaved Excitation (PIE) with two-color coincidence detection, a technique that allows alternating excitation of fluorophores of different colors acting as donor and acceptor in fluorescence resonance energy transfer (FRET) processes.
SMF-PIE has several advantages over the conventional SMF schemes to study biological systems, such as accurate determination of FRET efficiencies and inter-fluorophore distances, effective removal of the spectral, or distinction between molecules that have a low FRET efficiency from those that lack an acceptor tag.
The main goals of this project are, hence, as follows:
* Implementation, calibration, and validation of a state-of-the-art SMF-PIE instrumentation. Subsequently, SMF-PIE will be applied in three multidisciplinary biophysical approaches.
* Testing the feasibility and performance of newly synthesized fluorescein derivatives for SMF studies, especially those derivatives belonging to the Tokyo Green family of dyes.
* Providing new insights into the intercalation mechanism of DNA intercalators at the single molecule level, by means of single-pair FRET between fluorescein derivatives, acting energy donors, and DNA intercalator fluorophores as acceptors.
* Studying at the single molecule level the early pre-fibrillar aggregates from an amyloidogenic protein, the SH3 domain of alpha-spectrin. SMF-PIE will be employed to provide insights into the amyloid fibril formation mechanisms, a major biophysical and biomedical challenge nowadays.
The Reintegration Grant has been crucial in two ways. For the fellow to obtain a mature academic position at the University, as well as being able to start an independent research career. For the research group and the University, the ERG has been helpful to increase the international visibility of the research group, achieving several publications of impact, as well as leaving the group in an excellent position to fulfill future, more ambitious research projects.
publicación de artículos en revistas científicas de alto índice de impacto, y presentación de resultados en congresos y reuniones científicas internacionales
On one hand, several publications in journals of impact in the field of Physical Chemistry, and Biophysical Chemistry have been achieved, as well as many contributions in international conferences, have supported the achievements of the scientific goals of the project. In the other hand, at the end of the project, the research group FQM-247, and the University of Granada will count with a state-of-the-art single-molecule fluorescence instrumentation with capabilities comparable to world-leading research groups in the field. This should enhance the future prospects of the University of Granada regarding research in single molecule fluorescence and biophysics.
Fotoquímica y Fotobiología
Code PAIDI: FQM-247
Jose Mª Álvarez Pez / Ángel Orte Gutiérrez.
Universidad de Granada
Budget of Andalusian group: € 45,000.00
- Angel Orte Gutierrez;
- Jose Mª Alvarez Pez
Keywords: Single Molecule spectroscopy; DNA intercalators; amyloids
Duration: December, 10th 2008 to December, 9th 2011
Project cost: € 45,000.00
