Location of tryptophans in membrane-bound annexins.
The annexins are a novel class of calcium-dependent membrane binding proteins with highly homologous sequences and similar binding characteristics. In order to better define structural parameters of the membrane-bound form, the localization of tryptophan residues in several of these proteins was studied by use of quenchers of their intrinsic fluorescence. Lipocortin I contains a single tryptophan located near its N-terminus, while the single tryptophan in lipocortin V is located in a repeated consensus sequence. Calcium-dependent binding to vesicles composed of 50% egg phosphatidylcholine and 50% bovine brain phosphatidylserine was accompanied by an increase in emission intensity resulting from a relief of internal quenching. The tryptophan fluorescence of bound lipocortin I was nearly unaffected by substituting the quencher 1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphocholine (5-PC) for egg phosphatidylcholine, while that of the lipocortin V tryptophan was quenched significantly. With the quenching doxyl spin-label located deeper in the bilayer at the 12- and 16-positions of the acyl chain, the quenching was progressively weaker, suggesting an interfacial location for the tryptophan of lipocortin V. The same experiments with lipocortin I show almost no quenching in any case, suggesting that this tryptophan near the amino terminus is protected or oriented away from the membrane surface. Data on the bovine liver calelectrins are also presented showing that endonexin also has a tryptophan residue that interacts strongly with phospholipids.
Meers
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Department of Pathology, William B. Castle Hematology Laboratory, Boston University School of Medicine, Massachusetts 02118.
D000818:Animals; D017304:Annexin A5; D017302:Annexins; D002135:Calcium-Binding Proteins / Q000032:analysis; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000032:analysis; D014364:Tryptophan / Q000032:analysis*
D017304:Annexin A5; D017302:Annexins; D002135:Calcium-Binding Proteins; D014364:Tryptophan