Evidence for a phosphorylation‐induced conformational change in phospholamban cytoplasmic domain by CD analysis

Terzi, Evelyne; Poteur, Livia; Trifilieff, Élisabeth

doi:10.1016/0014-5793(92)80819-3

Cited by 31 publications

(44 citation statements)

References 18 publications

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“…The magnitude of the stimulation by 1D11 in the Ca 2ϩ ATPase and 45 Ca uptake assay at each free calcium concentration ranged from about 10-fold at low free calcium to 0.2-fold at 1 M free calcium. At very low free calcium concentrations (Ͻ31 nM) where large stimulation has been reported (7), calcium uptake in antibody-free cardiac SR was not measurable, but activity was discernible in the presence of 1D11.…”

Section: Characterization Of Mab 1d11 and Preparation Of The 1d11mentioning

confidence: 99%

“…32 P]ATP and 45 CaCl 2 were obtained from DuPont NEN. The catalytic subunit of cAMP-dependent protein kinase, phosphoenol pyruvate, NADH, and C 12 E 8 were purchased from Sigma.…”

Section: Materials-[␥-mentioning

confidence: 99%

“…Cardiac SR (100 g/ml) was incubated with TBS (20 mM Tris/HCl pH 7.4, 150 mM NaCl) or 25 g/ml mAb 1D11 in assay buffer (219.4 mM KCl, 5.86 mM MgCl 2 , 10 mM potassium oxalate, 10 mM NaN 3 , 1 mM EGTA, and 40 mM imidazole, pH 7.1) containing varying amounts of 45 CaCl 2 . Free Ca 2ϩ concentrations were calculated using the Fabiato program (31).…”

Section: Camentioning

confidence: 99%

“…Reconstitution of PLB and Skeletal Muscle Ca 2ϩ ATPase-Skeletal muscle Ca 2ϩ ATPase was purified and reconstituted alone or with either native or synthetic PLB into proteoliposomes, and 45 …”

Section: Camentioning

confidence: 99%

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Biochemical and Biophysical Comparison of Native and Chemically Synthesized Phospholamban and a Monomeric Phospholamban Analog

Mayer

McKenna

Garsky

et al. 1996

Journal of Biological Chemistry

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Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (ϳ6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca 2؉ATPase, which is reversed by anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36, 41, and 46) were substituted by serines, is monomeric on SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is recognized by polyclonal antisera. PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-toSer PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% ␣-helical structure, whereas Cys-toSer PLB manifests only about 30%. Because the physiochemical properties of native and synthetic PLB appear identical, the more readily available synthetic protein should be suitable for more extensive structural studies. Phospholamban (PLB)1 is an oligomeric membrane protein present in stoichiometric amounts associated with the Ca 2ϩ ATPase of cardiac sarcoplasmic reticulum (SR). PLB in its unphosphorylated state attenuates the catalytic activity of the Ca 2ϩ ATPase by reducing its apparent calcium sensitivity. Phosphorylation of PLB (1-3), treatment with antibodies directed against PLB (4 -7), or mild trypsin proteolysis of PLB (8) ATPase activity. Data supporting reversible and direct regulation of the calcium pump by PLB are abundant, yet the detailed mechanistic and structural requirements for PLB inhibition remain to be described.The primary structure of PLB has been deduced from its cDNA (15). It is highly conserved among species, has an acetylated amino terminus, and consists of 52 amino acids with a predicted molecular mass of 6123 Da. Several secondary structure models (an example is shown in Fig. 1) have been proposed (13,14,16) with the following general features: (i) PLB is an amphipathic peptide with a hydrophilic NH 2 terminus, part of which is predicted to form an ␣-helix whose exact length and position are uncertain, (ii) serine 16 and threonine 17 are the sites for protein phosphorylation by cAMP-dependent protein kinase (PKA) and calcium-calmodulin-dependent protein kinase, respectively, and (iii) the hydrophobic COOH-terminal amino acids (31-52) form a transmembrane ␣-helical segment and are involved in protein oligomerization. The quaternary structure of PLB under nondenaturing conditions is assumed to be pentameric. PLB migrates as a homopentamer with an apparent M r of 28,000 in SDS-PAGE, w...

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Section: Characterization Of Mab 1d11 and Preparation Of The 1d11mentioning

confidence: 99%

“…32 P]ATP and 45 CaCl 2 were obtained from DuPont NEN. The catalytic subunit of cAMP-dependent protein kinase, phosphoenol pyruvate, NADH, and C 12 E 8 were purchased from Sigma.…”

Section: Materials-[␥-mentioning

confidence: 99%

Section: Camentioning

confidence: 99%

Section: Camentioning

confidence: 99%

See 2 more Smart Citations

Biochemical and Biophysical Comparison of Native and Chemically Synthesized Phospholamban and a Monomeric Phospholamban Analog

Mayer

McKenna

Garsky

et al. 1996

Journal of Biological Chemistry

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“…Although glycosylation (Gottschalk, 1972), phosphorylation (Thomas et al, 1980) and N-terminal acetylation (Allen, 1989) are the three major post-translational modifications, many other modified amino acids have been found in naturally occurring proteins such as 5-hydroxylysine, 4-hydroxyproline, sulfotyrosine, y-carboxyglutamic acid, 5-oxoproline, etc. Both phosphorylation and glycosylation affect the secondary structure of proteins and their fragments (Hider et al, 1985;Terzi et al, 1992;Gerken et al, 1989;Urge et al, 1992). For example, both 0-linked and N-linked glycosylations were suggested to form or maintain the secondary/ tertiary structure of proteins (Williamson et al, 1992;Gorman et al, 1991;Sodora et al, 1991).…”

mentioning

confidence: 99%

Aspartate‐Bond Isomerization Affects the Major Conformations of Synthetic Peptides

Szendrei¹,

Fabian

Mantsch

et al. 1994

European Journal of Biochemistry

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The aspartic acid bond changes to an p-aspartate bond frequently as a side-reaction during peptide synthesis and often as a post-translational modification of proteins. The formation of paspartate bonds is reported to play a major role not only in protein metabolism, activation and deactivation, but also in pathological processes such as deposition of the neuritic plaques of Alzheimer's disease. Recently, we reported how conformational changes following the aspartic-acidbond isomerization may help the selective aggregation and retention of the amyloid p peptide in affected brains (Fabian et al., 1994). In the current study we used circular dichroism, Fouriertransform infrared spectroscopy, and molecular modeling to characterize the general effect of the , & aspartate-bond formation on the conformation of five sets of synthetic model peptides. Each of the non-modified, parent peptides has one of the major secondary structures as the dominant spectroscopically determined conformation: a type I p turn, a type I1 p turn, short segments of a or 3,, helices, or extended p strands. We found that both types of turn structures are stabilized by the aspartic acid-bond isomerization. The isomerization at a terminal position did not affect the helix propensity, but placing it in mid-chain broke both the helix and the /?-pleated sheet with the formation of reverse turns. The alteration of the geometry of the lowest energy reverse turn was also supported by molecular dynamics calculations. The tendency of the aspartic acid-bond isomerization to stabilize turns is very similar to the effect of incorporating sugars into synthetic peptides and suggests a common feature of these post-translational modifications in defining the secondary structure of protein fragments.

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Structural Model of the Phospholamban Ion Channel Complex in Phospholipid Membranes

Arkin

Rothman

Ludlam

et al. 1995

Journal of Molecular Biology

122

125

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Evidence for a phosphorylation‐induced conformational change in phospholamban cytoplasmic domain by CD analysis

Abstract: Phospholamban (PLB). an integlal membrane promin of cardiac sarcoplasmi¢ rettculum (SR}. is dcr, cribed as the reilUlator of the C.aZ'.ATPaz¢ pump, via its phosphorylation,,, Show more

Cited by 31 publications

References 18 publications

Biochemical and Biophysical Comparison of Native and Chemically Synthesized Phospholamban and a Monomeric Phospholamban Analog

Biochemical and Biophysical Comparison of Native and Chemically Synthesized Phospholamban and a Monomeric Phospholamban Analog

Aspartate‐Bond Isomerization Affects the Major Conformations of Synthetic Peptides

Structural Model of the Phospholamban Ion Channel Complex in Phospholipid Membranes

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