Origin of Tryptophan Fluorescence Lifetimes in Solution and in Proteins. Jihad René Albani Université de Lille 1. Laboratoire Biophysique Moléculaire

Transcription

1 Origin of Tryptophan Fluorescence Lifetimes in Solution and in Proteins Jihad René Albani Université de Lille 1. Laboratoire Biophysique Moléculaire

2 Fluorescence instruments Fluorescence lifetimes were measured with a Horiba Jobin Yvon Fluoromax 4P Fluorescence spectra were obtained with a Perkin-Elmer LS5B Absorption data were obtained with a Shimadzu MPS-2000 double-beam spectrophotometer

3 Fluorescence intensity decay of L- Trp in ph 2, 10 mm phosphate buffer observed at 330 nm. λex=296 nm

4 Fluorescence intensity decay parameters Intensity decay can be analyzed with one or more fluorescence lifetimes. Each lifetime contributes with a certain percentage in the intensity decay. This percentage is called the lifetime preexponential or lifetime amplitude and characterizes the population of the emitting species with a specific lifetimes.

5 Tryptophan fluorescence lifetimes in different environments.

6 Values of c 2 of L-Trp decay in 10 mm phosphate buffer at ph 2, 7 and 12 for two and three fluorescence lifetimes.

7 Lifetimes and pre-exponential values of L-Trp with emission wavelength recorded at ph2 (squares), 7 (triangles) and 12 (circles). λex=296 nm

8 Values of c 2 of L-Trp decay in ethanol

9 Lifetimes and pre-exponentials variation of L-Trp in ethanol with emission wavelengths. λex=296 nm.

10 Conclusions on the origin of L-Trp fluorescence lifetimes in solution (1) In ethanol, The two shorter lifetimes are very close to those found for L-Trp in water or buffer. Thus, origin of these two lifetimes is the same whether tryptophan is dissolved in pure polar solvent or in a mixture of hydrophobic/hydrophilic environment such as ethanol. Therefore, the and ns lifetimes originate from two substructures existing in the excited state. These substructures possess different electronic distributions. The third lifetime observed for L-Tryptophan in ethanol is the result of the hydrophobic/hydrophilic functions (CH3 and OH) interactions with the fluorophore. These interactions induce a third substructure with a specific electronic cloud generating an emission with a lifetime equal to about 4.7 ns.

11 Fluorescence lifetimes pre-exponential values of L-Trp in water (squares) and in CHCl3 (circles). λex =296 nm Conclusions on the origin of L-Trp fluorescence lifetimes in solution (2)

12 Conclusions on the origin of L-Trp fluorescence lifetimes in solution (3) In pure water (hydrophilic medium) and in chloroform (hydrophobic medium), longest and smallest populations are the same. Thus, in both solvents, L-Trp emits from two identical substructures. In ethanol, presence of hydrophobic and hydrophilic chemical properties modifies the solvent interaction with L-tryptophan inducing a third emitting population.

13 Populations emitting originates from the excited state. Fluorescence excitation spectra of L-Trp in water (λ max=280 nm) (a) and in CHCl3 (λmax=295 nm) (b). λ em=360 nm

14 Emitting populations are pre-existing, predefined and not arbitrary ones. Fluorescence lifetimes of L-Trp in pure water as a function of emission wavelength, recorded at three excitation wavelength, 266 (squares), 281 (triangles) and 296 nm (circles). Lifetimes values are independent of excitation wavelength

15 Conclusions on the origin of L-Trp fluorescence lifetimes in solution (4) L-Trp emits with two lifetimes when it is dissolved in pure hydrophobic or hydrophilic solvent. The two lifetimes are generated in the excited state and are inherent to the L-Trp itself. It is always the same L-Trp populations that are emitting and not random ones. Internal specific configurations of L-Trp do exist in the excited state and these configuurations are the only ones which can be excited and able to emit. Description of molecules with one structure is oversimplified. Interaction of L-Trp in solution with both hydrophobic and hydrophilic chemical structures such as it is the case in ethanol, generates a third population with a specific lifetime.

16 Origin of tryptophan fluorescence lifetimes in proteins

17 Emitting populations of tryptophan differ from a native protein to another Pre-exponential values of tryptophan residues fluorescence lifetimes of β-lactoglobulin (filled symbols) and of human serum albumin (open symbols), dissolved in 10 mm ph 7 Tris buffer and excited at 296 nm. Squares: α1; Circles: α2; Triangles: α3

18 Emitting populations of tryptophan are homogenized in denatured proteins Pre-exponential values of tryptophan residues fluorescence lifetimes of β-lactoglobulin (filled symbols) and of human serum albumin (open symbols), dissolved 6 M guanidine ph 7.8 buffer and excited at 296 nm. Squares: α1; Circles: α2; Triangles: α3

19 Fluorescence lifetimes and pre-exponentielles values of L-Trp dissolved in 6 M guanidine, ph 7.8 L-Tryptophan fluorescence lifetimes in 6 M guanidine

20 Does the Peptide Bond Induce the Third Fluorescence Lifetime? Values of fluorescence lifetimes measured at 350 nm for different peptides in pure distilled water and in ethanol

21 Fluorescence lifetimes and pre-exponentials of Trp residue in the peptides Leu-Trp-Leu (closed symbols) Ala-Trp-Ala (open symbols) measured in ethanol at 20 C. λex=296 nm. Lifetimes: Squares: τ1. Circles: τ2. Triangles: τ0 Fluorescence lifetimes of tryptophan in peptides

22 Conclusions obtained fom studies with peptides The peptide bond does not induce the third fluorescence lifetime of L-Trp observed in ethanol. As it is the case for L-Trp in water and in ethanol, α2 is much higher than α1. However, values of preexponentials in peptides differ from those observed for free tryptophan in water or ethanol. Thus, tryptophan environment affects the emitting populations.

23 Tryptophan fluorescence lifetimes in proteins (1) In most of the studied proteins, tryptophan residue(s) emit with three lifetimes independently of the number of tryptophan residues and of their positions in the protein. These fluorescence lifetimes are identical or equal to those found for L-Trp in water or ethanol. Thus, presence of these two lifetimes is inherent to the tryptophan structure and is independent of the structure surrounding the fluorophore. Values of preexponentials vary from a protein to another and thus tryptophan residue environment affects emitting populations.

24 Tryptophan fluorescence lifetimes in proteins (2) The only possible explanation of the presence of the third lifetime in proteins is the permanent contact existing between Trp residue and neighboring amino acids. This contact is the result of the protein folding within the proteins (secondary and tertiary structures in the case of a native protein and random folding in the case of a denatured protein). This contact is similar to that of free tryptophan in ethanol where the fluorophore interacts with both chemical hydrophobic and hydrophilic function of ethanol. In proteins, chemical functions of the amino acids radicals neighboring tryptophan residues replace the two chemical functions of ethanol.

25 Tryptophan fluorescence lifetimes in proteins (3) If the third fluorescence lifetime is generated by the contacts existing between tryptophan residue and neighboring amino acids, thus for a same protein this lifetime would be independent of the nature of the solvent where the protein is dissolved or if the protein is lyophilized. This is true only when the protein is not denatured such as it is the case in 6M guanidine.

26 Variation of fluorescence lifetimes and pre-exponentials values of AP UbC (E2) dissolved in PBS buffer, ph 7 (closed symbols), and of lyophilized protein (open symbols) with emission wavelength. λex=296 nm. Squares: α1. Circles: α2. Triangles: α3 Tryptophan fluorescence lifetimes in proteins (4)

27 Fluorescence lifetimes and pre-exponentials values of α1-acid glycoprotein in 100 mm PBS buffer, ph 7, are measured with three excitation wavelengths, 266 nm (black), 281 nm (red) and 296 nm(blue). α1 are represented by the squares, α2 by the circles and α3 by the triangles. Emission of tryptophan in proteins occurs from pre-existing population (1)

28 Emission of tryptophan in proteins occurs from pre-existing population (2) Tryptophan absorbs with two transitions So 1La and So 1Lb. Fluorescence lifetimes and pre-exponentials are identical in presence of the two transitions or in presence of So 1Lb transition alone. Therefore, fluorescence decay parameters are independent of excitation energy. J.R. Albani (2009) Journal of Fluorescence. 19: Fluorescence lifetimes of tryptophan: structural origin and relation with S o 1 L b and S o 1 L a transitions, J. R. Albani (2011) Journal of Fluorescence. 21: Relation between proteins tertiary structure, tryptophan fluorescence lifetimes and tryptophan S o 1 L b and S o 1 L a transitions. Studies on a 1 -acid glycoprotein and b-lactoglobulin.

29 Can we compare fluorescence of tryptophan to that of indole? Fluorescence lifetimes and pre-exponentials values of indole in water (squares) and ethanol (circles) as a function of emission wavelength. λex=296 nm

30 Global conclusion on this study (1) Two identical emission lifetimes are observed for tryptophan in water, ethanol or in proteins. These two lifetimes are inherent to the tryptophan structure and result from two substructures formed in the excited state. Each sub-structure is formed by tryptophan backbone along with a specific electronic distribution.

31 Global conclusion on this study (2) In ethanol, the third lifetime observed for L-Tryptophan is the result of the hydrophobic/hydrophilic functions (CH3 and OH) interactions with the fluorophore. In proteins, the third lifetime is generated by interactions existing between Trp residue with neighboring amino acids as the result of protein folding. The three sub-structures are pre-existing in the excited state and are put into evidence upon excitation. It is always the same tryptophan populations that are emitting and not random ones.

32 Global conclusion on this study (3) In proteins as well as when tryptophan is free in a solvent, internal specific configurations do exist in the excited state and these configurations are the only ones which can be excited and/or able to emit. From a protein to another, in the native state, surrounding tryptophan residue environment affects organization of emitting populations.

33 References J. R. Albani (2014) Journal of Fluorescence. 24: Origin of tryptophan fluorescence lifetimes. Part 1. Fluorescence lifetimes origin of tryptophan free in solution. J. R. Albani (2014) Journal of Fluorescence. 24: Origin of tryptophan fluorescence lifetimes. Part 1. Fluorescence lifetimes origin of tryptophan in proteins. 33

34 References J. R. Albani, J. Vogelaer, L. Bretesche, D. Kmiecik (2014) Journal of Pharmaceutical and Biomedical Analysis. 91: Tryptophan 19 residue is the origin of bovine b-lactoglobulin fluorescence. J. R. Albani (2007) Journal of Fluorescence 17: , New insights in the interpretation of tryptophan fluorescence. Origin of the fluorescence lifetime and characterization of a new fluorescence parameter in proteins: the 34 emission to excitation ratio.

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