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Conference: Bucharest University Faculty of Physics 2017 Meeting

Section: Biophysics; Medical Physics

Title:

Fluorescence lifetime of tryptophan - a tool to assess protein microenvironment. Design and optimization of a time resolved fluorimeter.

Authors:

Bogdan ZORILA (1, 2), Mihaela BACALUM (1), Mihai RADU (1), Aurel I. POPESCU (2)

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Affiliation:

1) Department of Life and Environmental Physics, “Horia Hulubei” National Institute of Physics and Nuclear Engineering, Măgurele, Romania

2) Department of Electricity, Solid Physics and Biophysics, Faculty of Physics, University of Bucharest, Măgurele, Romania

E-mail

bzorila@nipne.ro

Keywords:

Fluorescence lifetime, single and multi-exponential decay, tryptophan, proteins, peptides

Abstract:

From the three fluorescent amino acids, tryptophan (the other two being tyrosine and phenylalanine) is the main fluorophore that is part of some protein structure. Its fluorescence properties have been studied and applied to understand protein structure and dynamics as well as the interaction of macromolecules with different ligands [1, 2, 3]. Tryptophan possesses intrinsic specific fluorescence properties. These properties (e.g., fluorescence anisotropy, emission and excitation spectra, excited state lifetimes) can be measured and characterized [4]. The tryptophan fluorescence properties are sensitive to its microenvironment. For example, when a tryptophan is buried inside the hydrophobic core of a protein, its fluorescence is blue-shifted compared to the fluorescence observed when tryptophan is located at the protein surface. In this work, we describe the steps of development of a simple, custom setup, time resolved fluorimeter, the calibration and verifying steps using fluorescence lifetime standards. The nanosecond lifetime standards used for fluorimeter optimization, calibration, and verifying steps were the standard fluorophores eliciting single-exponential decays [5, 6]: indole, N-acetyl-L-tryptophanamide (NATA), p-terphenyl, and 2.5-diphenyl-oxazole (PPO). Lifetime of free tryptophan in water, at different temperatures and concentrations, was also measured. Some practical applications of this time resolved fluorimeter are also presented. These applications are: fluorescence quenching and water-to-lipid partition free energy measurements for antimicrobial peptide, indolicidin, in relation to neutral synthetic phosphocholine, DOPC, and negatively charged DOPC-DPPG (85:15 mol) large unilamelar vesicles.

References:

[1] Tayeh N., Rungassamy T., Albani J. R. (2009) Fluorescence spectral resolution of tryptophan residues in bovine and human serum albumins. J. Pharm. Biomed. Anal., 50: 107-116

[2] Albani J. R. (2007) Principles and applications of fluorescence spectroscopy. Blackwell, London

[3] Albani J. R. (2009) Fluorescence lifetimes of tryptophan: structural origin and relation with So → 1Lb and So → 1La transition. J. Fluoresc., 19: 1061-1071

[4] Albani J. R. (2014) Origin of Tryptophan Fluorescence Lifetimes Part 1. Fluorescence Lifetimes Origin of Tryptophan Free in Solution J. Fluoresc., 24: 93-104

[5] Lakowicz J. R. (2006) Principles of fluorescence spectroscopy. New York, Springer Science

[6] Valeur B. (2001) Molecular Fluorescence: Principles and Applications. Wiley-VCH Verlag GmbH

Acknowledgement:

Bogdan ZORILA is preparing a PhD at Faculty of Physics, University of Bucharest. This work was supported by the Romanian Ministry of Education and Research through Grants: PN 09370301, PN 16420203, PNII-123/2012, PNII-98/2012 and PN-II-ID-PCCE-2011-2-0027.