Location of tryptophans in membrane-bound annexins

Abstract

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.

Amino acid sequence analysis of the annexin super‐gene family of proteins

“…These observations show that the a -b loop of repeat 3 is characteristically different from the a-b loops in repeats 1, 2 and 4 and suggests that the loop may perform a specific function in the intact annexin. Further support for this hypothesis comes from recent tryptophan fluorescence studies [38] that indicate the Trp residue at position 22 of AIB3 and AVB3 associates closely with the phospholipid in membranebound annexins. These data suggest that the uniqueness of the repeat 3 a -b loop may be due to a specific phospholipid binding function.…”

Phosphatidylserine Is Not the Cell Surface Receptor for Vesicular Stomatitis Virus

“…The binding of annexin V to PS is both specific and strong, with a K d of 9 to 15 nM on cells (23,35) and 40 pM on phospholipid vesicles (33). Importantly, annexin V can also detect and bind to very low levels of PS (16,17).…”

Membrane‐bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins

“…Given the above model, it is expected that chelation of Ca 2÷ will result in the complete reversal of annexin binding. While this has been proven true whenever annexin binding to liposomes or proteins had been investigated in reconstitution experiments in vitro [6][7][8][9][10][11][12][13][14][15], different results were obtained with certain annexins whenever tissue or cell subfractionation was used to investigate the binding of endogenous annexins to natural membranes. So, fractions ofannexins I, 1I, IV-VII were reported to exist in several cell types in a membrane-bound form, to resist extraction with EGTA, and to require detergents for their solubilization [10,[16][17][18][19][20].…”