Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger

1998 ◽  
Vol 76 (5) ◽  
pp. 837-842 ◽  
Author(s):  
Daniel Gebreselassie ◽  
Krishna Rajarathnam ◽  
Larry Fliegel
Keyword(s):  
Circular Dichroism ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Cytoplasmic Domain ◽  
Random Coil ◽  
Glutathione S Transferase ◽  
Carboxyl Terminal ◽  
Circular Dichroïsm

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.Key words: Na+/H+ exchanger, pH regulation, membrane protein, circular dichroism.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

Circular Dichroism of C-Phycoerythrin: A Conformational Analysis

1980 ◽  
Vol 35 (5-6) ◽  
pp. 367-375 ◽  
Author(s):  
Elisabeth Langer ◽  
Harald Lehner ◽  
Wolfhart Rüdiger ◽  
Barbara Zickendraht-Wendelstadt
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Spectral Region ◽  
Protein Unfolding ◽  
Extensive Study ◽  
Random Coil ◽  
Linear Superposition ◽  
Thermally Induced ◽  
Circular Dichroïsm

Abstract An extensive study of the chiroptical properties of C-phycoerythrin and the α-and β-subunits in the spectral region from 700 -200 nm is presented. Based on the VIS-circular dichroism inherently chiral conform ations are proposed for the co­ valently linked chromophores. By means of mean residue ellipticities and the experimental circular dichroism spectra in the region of the n → π* peptide transition the a-helix contents of the apoproteins of the ac-and ß-subunits are estimated to amount to 60% and 40%, respectively. The circular dichroism spectrum of native C-phycoerythrin is congruent with a linear superposition of the α-and β-subspectra, in the whole spectral region studied. Since a-and β-subunits are associated in native C-phycoery-thrin as revealed by sedim entation analysis the interactions between the subunits in the native chromoprotein are not accom panied by substantial conform ational changes. In the temperature range 0°-40°C the thermally induced changes of the chrom ophores in native C-phycoerythrin are not associated with changes of the secondary structure of the apoprotein. Unfolding occurs at 60°-70°C but slowly leads to irreversible denaturation. Protein unfolding starts at 3 M urea. The random coil secondary structure of the apoproteins is reached at 8 M urea. At this concentration the absorbance and the optical activity of the chrom o­ phores are reduced by a factor 3 and 10, respectively. The conformational changes in the peptide with increasing denaturant concentration are not synchronous with those induced in the Chromo­ phore indicating that a m ultistep process is operative during unfolding. The C D results on dena­ turation are supplem ented by absorption and em ission spectroscopy.

scholarly journals Theoretical study of two-photon circular dichroism on molecular structures simulating aromatic amino acid residues in proteins with secondary structures

RSC Advances ◽  
2014 ◽  
Vol 4 (105) ◽  
pp. 60974-60986 ◽  
Author(s):  
Yuly Vesga ◽  
Carlos Diaz ◽  
Florencio E. Hernandez
Keyword(s):  
Circular Dichroism ◽  
Comparative Analysis ◽  
Theoretical Study ◽  
Secondary Structures ◽  
Molecular Structures ◽  
Random Coil ◽  
Amino Acid Residues ◽  
Two Photon ◽  
Α Helix ◽  
Circular Dichroïsm

Calculation and comparative analysis of the theoretical two-photon circular dichroism (TPCD) spectra of l-His, l-Phe, and l-Tyr simulating residues in proteins with secondary structures (α-helix, β-strand and random coil), down to the far-UV region (FUV).

The Secondary Structure of Proteins in the Thylakoid Membrane

1973 ◽  
Vol 28 (3-4) ◽  
pp. 128-130 ◽  
Author(s):  
Wilhelm Menke ◽  
Rolf-Dieter Hirtz
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Thylakoid Membrane ◽  
Circular Dichroism Spectrum ◽  
Random Coil ◽  
Reference Substance ◽  
Secondary Structure Of Proteins ◽  
Circular Dichroïsm ◽  
Structure Of Proteins

With reference spectra derived from proteins of known structure (CHEN, YANG, and MARTINEZ, Biochemistry 11, 4120 [1972]) a better approximation of the circular dichroism spectrum of fragments of the thylakoid membrane is achieved, than by the use of polylysine as reference substance. Most probably the protein in the thylakoid membrane consists of 40 per cent helix, 42 per cent random coil and 18 per cent β-structure.

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