Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding. 1994

B J Thevenin, and N Periasamy, and S B Shohet, and A S Verkman
Department of Laboratory Medicine, University of California, San Francisco 94143-0134.

Interactions between the erythrocyte membrane and its skeleton are mediated primarily by binding of cytoskeletal components to a conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To examine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3 about which pH-dependent conformational changes occur. Time-resolved anisotropy of labeled cdb3 in isolated form and in stripped erythrocyte membranes was measured by parallel-acquisition frequency-domain microfluorimetry. Samples had a single-component fluorescein lifetime of approximately 4 ns. Multifrequency phase and modulation data (5-200 MHz) fitted well to a segmental motion model containing two correlation times (tau 1c and tau 2c) and two limiting anisotropies (r1infinity and r2infinity). Measurements in protease-cleaved and denatured samples indicated that tau 1c (100-150 ps) corresponded to rapid rotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmental motion of cdb3. Both motions were hindered as quantified by nonzero r1infinity and r2infinity. The strong pH dependence of segmental motion correlated with that of cdb3 conformation measured by intrinsic tryptophan fluorescence. Significant changes in cdb3 segmental motion occurred upon interactions with the small ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymes known to bind to the N terminus of band 3. These time-resolved fluorescence measurements of the nanosecond segmental dynamics of a labeled membrane protein provide evidence for the sensitivity of cdb3 conformation to ligand binding and suggest long-range structural communication through cdb3.

UI MeSH Term Description Entries
D008024 Ligands A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed) Ligand
D008956 Models, Chemical Theoretical representations that simulate the behavior or activity of chemical processes or phenomena; includes the use of mathematical equations, computers, and other electronic equipment. Chemical Models,Chemical Model,Model, Chemical
D010732 Phosphofructokinase-1 An allosteric enzyme that regulates glycolysis by catalyzing the transfer of a phosphate group from ATP to fructose-6-phosphate to yield fructose-1,6-bisphosphate. D-tagatose- 6-phosphate and sedoheptulose-7-phosphate also are acceptors. UTP, CTP, and ITP also are donors. In human phosphofructokinase-1, three types of subunits have been identified. They are PHOSPHOFRUCTOKINASE-1, MUSCLE TYPE; PHOSPHOFRUCTOKINASE-1, LIVER TYPE; and PHOSPHOFRUCTOKINASE-1, TYPE C; found in platelets, brain, and other tissues. 6-Phosphofructokinase,6-Phosphofructo-1-kinase,Fructose-6-P 1-Kinase,Fructose-6-phosphate 1-Phosphotransferase,6 Phosphofructokinase,Phosphofructokinase 1
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D002118 Calcium A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes. Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D003545 Cysteine A thiol-containing non-essential amino acid that is oxidized to form CYSTINE. Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D003593 Cytoplasm The part of a cell that contains the CYTOSOL and small structures excluding the CELL NUCLEUS; MITOCHONDRIA; and large VACUOLES. (Glick, Glossary of Biochemistry and Molecular Biology, 1990) Protoplasm,Cytoplasms,Protoplasms
D004163 Diphosphoglyceric Acids Glyceric acids where two of the hydroxyl groups have been replaced by phosphates. Bisphosphoglycerates,Acids, Diphosphoglyceric
D005454 Fluorescence Polarization Measurement of the polarization of fluorescent light from solutions or microscopic specimens. It is used to provide information concerning molecular size, shape, and conformation, molecular anisotropy, electronic energy transfer, molecular interaction, including dye and coenzyme binding, and the antigen-antibody reaction. Anisotropy, Fluorescence,Fluorescence Anisotropy,Polarization, Fluorescence,Anisotropies, Fluorescence,Fluorescence Anisotropies,Fluorescence Polarizations,Polarizations, Fluorescence
D005634 Fructose-Bisphosphate Aldolase An enzyme of the lyase class that catalyzes the cleavage of fructose 1,6-biphosphate to form dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The enzyme also acts on (3S,4R)-ketose 1-phosphates. The yeast and bacterial enzymes are zinc proteins. (Enzyme Nomenclature, 1992) E.C. 4.1.2.13. Aldolase,Fructosediphosphate Aldolase,Aldolase A,Aldolase B,Aldolase C,Fructose 1,6-Bisphosphate Aldolase,Fructose 1,6-Bisphosphate Aldolase, Class II,Fructose 1-Phosphate Aldolase,Fructose Biphosphate Aldolase,Fructosemonophosphate Aldolase,1,6-Bisphosphate Aldolase, Fructose,Aldolase, Fructose 1,6-Bisphosphate,Aldolase, Fructose 1-Phosphate,Aldolase, Fructose Biphosphate,Aldolase, Fructose-Bisphosphate,Aldolase, Fructosediphosphate,Aldolase, Fructosemonophosphate,Fructose 1 Phosphate Aldolase,Fructose 1,6 Bisphosphate Aldolase,Fructose Bisphosphate Aldolase

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