Fluorescence quenching of spectrin and other red cell membrane cytoskeletal proteins. Relation to hydrophobic binding sites

Abstract

The intrinsic fluorescence of spectrin is strongly quenched by low concentrations of 2-bromostearate. This results from binding at a series of hydrophobic sites. Analysis of dynamic fluorescence quenching by acrylamide, iodide and caesium ions, separately and in conjunction with 2-bromostearate, leads to the conclusion that most of the tryptophan side-chains are exposed to solvent. The sites at which the fatty-acid-quenched tryptophans are located apparently interact with the lipid bilayer in the cell, as judged by quenching by bromostearate dissolved in the lipid phase. A minor proportion of the side-chains in native spectrin give rise to sharp proton magnetic resonance signals, indicative of segmental mobility; these chain elements contain some tryptophan residues, as revealed by weak downfield signals from the heterocyclic ring protons. These signals are not appreciably perturbed by stearic acid or by phosphatidylserine liposomes, suggesting that the hydrophobic binding sites are not in mobile chain elements. By contrast with a series of globular proteins which, with the exception of serum albumins, show little or no quenching by 2-bromostearate, the peripheral red cell membrane skeletal proteins ankyrin (and its spectrin-binding domain), protein 4.1 and (to a lesser extent) actin show evidence of a high affinity for the hydrophobic ligand and may, like spectrin, interact directly with the bilayer in situ.

Fluorescence studies of spectrin and its subunits

“…This ligand has not been studied extensively and it cannot be ruled out that the high accessibility is due to denaturation of the protein. Kahana et al [1992] report a KS-, of 0.8 M-' for Cs+ , whereas we observed virtually no quenching (Table I). It is not clear from their report whether the ionic strength of the spectrin solution was maintained constant when the concentration of the quencher ions was increased.…”

Invariant tryptophan at a shielded site promotesfolding of the conformational unit of spectrin.

“…Sci. USA 91 (1994) acrylamide, iodide, and brominated fatty acid quenching of the steady-state fluorescence of tryptophans in intact spectrin were interpreted to indicate exposure of the tryptophans to the bulk phase (39). The relevance of these findings for the location of the invariant tryptophan is uncertain, however, since an intact spectrin dimer contains 80 tryptophans, only about half of which are in the invariant position.…”

Brain spectrin (fodrin) interacts with phospholipids as revealed by intrinsic fluorescence quenching and monolayer experiments

“…The ability of spectrin to bind hydrophobic ligands such as brominated stearic acid [14,15] fatty acids, or anionic, cationic and zwitterionic detergents [16] lends support to the view that spectrin contains a number of hydrophobic sites. In particular, the βII domain of molecular mass 65 kDa (containing the ankyrin-binding site) [17] was suggested to be rich in such regions [18].…”