Allosteric modifiers of hemoglobin. 2. Crystallographically determined binding sites and hydrophobic binding/interaction analysis of novel hemoglobin oxygen effectors

1991 ◽  
Vol 34 (2) ◽  
pp. 758-767 ◽  
Author(s):  
Fred C. Wireko ◽  
Glen E. Kellogg ◽  
Donald J. Abraham
Keyword(s):  
Binding Sites ◽  
Interaction Analysis ◽  
Binding Interaction ◽  
Hydrophobic Binding

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

1992 ◽  
Vol 282 (1) ◽  
pp. 75-80 ◽  
Author(s):  
E Kahana ◽  
J C Pinder ◽  
K S Smith ◽  
W B Gratzer
Keyword(s):  
Cell Membrane ◽  
Fluorescence Quenching ◽  
Binding Sites ◽  
Cytoskeletal Proteins ◽  
Lipid Phase ◽  
Side Chains ◽  
Red Cell ◽  
Segmental Mobility ◽  
Red Cell Membrane ◽  
Hydrophobic Binding

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.

Prothrombin Residues 473 to 487 Contribute To Factor Va Binding in the Prothrombinase Complex.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1720-1720
Author(s):  
Subramanian Yegneswaran ◽  
Rolf M. Mesters ◽  
Jose A. Fernandez ◽  
John H. Griffin
Keyword(s):  
Binding Sites ◽  
Synthetic Peptides ◽  
Spectroscopic Data ◽  
Active Sites ◽  
Inhibitory Peptide ◽  
Binding Interaction ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Direct Binding ◽  
Prothrombinase Activity

Abstract To identify sequences in prothrombin (fII) involved in assembly of the prothrombinase complex (fXa:factor Va:fII:phospholipids), synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. The fII peptide comprising residues 473–487 (designated PT473–487 that are homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity with 50% inhibition observed at 12 microM and 10 microM peptide, respectively. Prothrombinase inhibition by PT473–487 was fVa-dependent and sequence-specific since the peptide did not inhibit fII activation in the absence of fVa and a scrambled sequence peptide, PT473–487SCR, was not inhibitory. Peptide PT473–487 also inhibited cleavage at Arg271 in meizo-thrombin, showing that it similarly inhibited fXa cleavage at both Arg320 and Arg271. Peptide PT473–487 did not inhibit the amidolytic activities of fXa or thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine if PT473–487 interacted directly with fVa, fluorescein labeled-fVa (Fl-fVa) was prepared. When PT473–487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC50 at 3 microM peptide) whereas the control peptide PT473–487SCR peptide did not alter the anisotropy, suggesting a direct binding interaction between PT473–487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473–487 provide fVa binding sites and mediate interactions between fVa and fII in the prothrombinase complex that contribute to cleavages at both Arg271 and Arg320 by fXa.

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