Melittin Index FRET

Quenching of Fluorescence to Measure Accesibility of Tryptophan Residues

Fluorescence quenching is a process which decreases the intensity of the fluorescence emission. The accessibility of groups on a protein molecule can be measured by use of quenchers to perturb fluorophores. Quenching by small molecules either in the solvent or bound to the protein in close proximity to the fluorophore can greatly decrease the quantum yield of a protein. Quenching may occur by several mechanism:

• collisional or dynamic quenching
• static quenching
• quencing by energy transfer
• charge transfer reactions

This page discusses collisional quenching only. You will find information on other forms of quenching in References.

When quenching occurs by a collisional mechanism, the quencing is an additional process that deactivates the excited state besides radiative emission. Because dynamic quenching depopulates the excited state without allowing fluorescence emission, the decrease in fluorescence intensity equates to the decrease in fluorescence lifetime. The dependence of the emission intensity, F on quencher concentration [Q] is given by the Stern-volmer equation:

where

• T and T_o is the lifetime in the presence and absence of quencher, respectively
• k_q is the bimolecular rate constant for the dynamic reaction of the quencher with the fluorophore

The product of k_qT_o is referred to as the Stern-Volmer constant or K_SV

Some commonly used quenchers, that are small, aqueous soluble and do not denature proteins by themselves, are:

• Iodide. Because of its charged nature it efficiently quenches surface residues.
• Oxygen. Small and can penetrate the protein to some degree.
• Acrylamide, nitroxides. Neutral.

The accessible fluorophores experience a decrease in fluorescence upon collision with collisional quenchers. Charge versus steric effect is differentiated by quenchers with different size and charges. Accessibility depends on exposure as well as lifetime. Accessdibility of residues are reflected in the Stern-Volmer constant. For single tryptophan containing proteins, low values of k_q indicate residues of low exposure, ie those buried within the protein structure. These residues also have blue shifted emissions indicating a relatively nonpolar environment. The limited quenching of buried residues is mainly due to penetration of the quencher into the protein structure, with the quenching being determined by the opening of pores large enough to accomodate a quencher molecule.

With charged quenchers, electrostatic effects become important, which can be identified by dependence of quenching on ionic strength. Quenching depends strongly on peptide charge. Neighbouring Lys, Arg, His, Asp or Glu residues may greatly affect quenching. If the fluorophores and quenchers are of like charge, then quenching will result in a decrease in k_q.

Accessibility of Trp Residues in Lysozyme

Hen egg white lysozyme, shown below has been a subject of many fluorescence quenching studies. This protein contains six tryptophan residues. The Stern-Volmer equation has been used to determine the portion of these residues that are exposed to the aqueous solvent.

Lyzozyme contains tryptophan residues which are buried in the tertiary structure, b and others that are exposed on the surface, a. Then, the fluorescence intensity can be written as:

In the presence of quencher, the intensity of the accessible fluorophores decreases according to the Stern-Volmer equation:

where f_a is the fraction of accessible fluorophores.

From this, f_a and K_SV can be determined from a plot of F_o/dF versus 1/[Q]. The ordinate intercept provides the fraction of accessible fluorophores at infinite quencher concentration.

Download the coordinate file 1hel (hen egg white lysozyme) from the PDBsum database using the appropriate PDB link.

Examine the structure of lyzozyme in Rasmol. Tryptophan residues are found at positions 28, 62, 63, 108, 111 and 123. At the Ramol command line, type:

select trp

color yellow

spacefill

Determine the tryptophan residues that are exposed on the surface of the molecule. In your opinion, does the number of exposed tryptophan residues correspond to that could be determined by the plot derived from the Stern-Volmer equation?

Orientation of Proteins in Lipid Bilayers

Fluorescence quenching can be used to determine the transmembrane orientation of proteins. Here is an example - alamethicin:

Alamethicin is a 21 residue small protein (or peptide) that forms voltage dependent pores through lipid bilayers. As shown in the figure, it has an all alpha-helical structure. The wild-type protein contains no tryptophan. Five analogues of alamethicin have been synthesized with tryptophan residues at position 5, 7,12, and 16 along the polypeptide chain. Quenching by n-doxyl stearates has confirmed the relative position of the tryptophan residues in the bilayer and the transmembrane orientation of the protein. In addition, the motion of tryptophan residues has been found to be greater at the ends of the protein rather than at the center, in spite of the fact that there is a microviscosity gradient in the lipid bilayer in the opposite direction.

Melittin Index FRET

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