Fluorescence studies of spectrin and its subunits

Abstract

To better understand the solution structure of spectrin, the environment of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its alpha and beta subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the beta subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated beta chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2-p-toluidinyl napthalene-6-sulfonic acid (TNS). It therefore appears that low ionic strength, alpha-beta association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solution does not involve exposure of significant numbers of hydrophobic sites.

Chaperone Activity and Prodan Binding at the Self-associating Domain of Erythroid Spectrin

“…The ␤-spectrin came out in the flow-through, and the ␣-spectrin was eluted with the same buffer containing 0.5 M NaCl as described earlier (47). The subunits obtained were renatured by dialyzing first against 5 mM Tris-HCl, 0.1 mM EDTA, 4 mM ␤-mercaptoethanol at pH 7.4, followed by dialysis against the same buffer without the ␤-mercaptoethanol (48). Before all of the fluorescence experiments, the protein was dialyzed extensively against the buffer containing 10 mM Tris-HCl, 20 mM NaCl, pH 7.8, to remove DTT.…”

Spectrin Organization and Dynamics: New Insights

“…A plot of the ratio of (I 1 /I 3 ) of 0.2 lM pyrene fluorescence as a function of increasing spectrin concentration is shown in (c). The plots in (b) and (c) are adapted and modified from Haque et al (2000) distributed over the entire molecule and yet are localized in the same position in each domain makes them convenient intrinsic fluorescence reporter groups for monitoring conformational changes in spectrin that contribute to its elastic deformability exhibited in physiological conditions (Subbarao and MacDonald 1994;Kelkar et al 2005). Many of these tryptophans are at or in the vicinity of hydrophobic patches in spectrin, which can bind hydrophobic ligands such as fatty acids and phospholipids and cause quenching of tryptophan fluorescence (Sikorski et al 1987;Kahana et al 1992).…”

“…In addition, there are five tryptophans in the actin binding domain at the amino terminus and two tryptophans at the carboxy terminus in the b subunit of the spectrin dimer. Some of these conserved tryptophans have been shown to promote folding of spectrin domains ) and contribute to their thermodynamic stability (Subbarao and MacDonald 1994;Pantazatos and MacDonald 1997). The fact that tryptophans are (14), and (6) diethylether (3).…”

“…Spectrin is amphiphilic in nature and is characterized by hydrophobic stretches in its polypeptide sequence containing a large number of hydrophobic sites which can bind hydrophobic molecules, fatty acids, detergents, and phospholipids (Isenberg et al 1981;Cohen et al 1986;Streichman et al 1991;Kahana et al 1992;Subbarao and MacDonald 1994;Sikorski 1995, An et al 2004;Chakrabarti 2003, 2004). The hydrophobic binding sites in spectrin are crucial since this region is believed to facilitate the interaction of spectrin with membranes (see later).…”

“…Interestingly, many of these sites contain or are close to tryptophan residues in spectrin (see later). The interaction of spectrin with hydrophobic ligands can therefore be conveniently monitored utilizing fluorescence-based approaches (Isenberg et al 1981;Sikorski et al 1987;Kahana et al 1992;Subbarao and MacDonald 1994;Chakrabarti 2003, 2004). In addition, the binding of unrelated hydrophobic ligands such as hemin, protoporphyrin, DNA-binding drugs and local anesthetics to spectrin has been reported (Beaven and Gratzer 1978;Cassoly 1978;Majee and Chakrabarti 1995;Majee et al 1999;Mondal and Chakrabarti 2002).…”