Deconvolution of the circular dichroism spectra of the proteins: The circular dichroism spectra of antiparallel β-sheet
A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure β-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:-669–679, 1991), was improved by the addition of proteins with high β-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (α-helix, antiparallel β-pleated sheet, β-turns, and unordered conformation), as well as a curve attributed to the “aromatic contribution” in the wavelength range of 195–240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the α-helical structure, but upon analysis yielded a considerable amount of β-sheet in agreement with the X-ray structure. © 1992 Wiley-Liss, Inc.
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- "The far-UV CD spectrum of E1 341 E2 661 showed a minimum at 208 nm and a shoulder at 223 nm (Fig. 5). Deconvolution of this spectrum using the program Convex Constraint Analysis (CCA)  yielded the following percentages of secondary structure elements: 13% a-helix, 48% b-sheet, and 39% non-ordered structure. "
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ABSTRACT: Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which are involved in viral attachment and entry into target cells. We have obtained in insect cells infected by recombinant baculovirus a chimeric secreted recombinant protein, E1(341)E2(661,) containing the ectodomains of E1 and E2. The described procedure allows the purification of approximately 2mg of protein from 1L of culture media. Sedimentation velocity experiments and SDS-PAGE in the absence of reducing agents indicate that the protein has a high tendency to self-associate, the dimer being the main species observed. All the oligomeric forms observed maintain a conformation which is recognized by the conformation-dependent monoclonal antibody H53 directed against the E2 ectodomain. The spectroscopic properties of E1(341)E2(661) are those of a three-dimensionally structured protein. Moreover, the chimeric protein is able to bind to human antibodies present in HCV-positive human sera. Accordingly, this chimeric soluble polypeptide chain may be a valuable tool to study the structure-function relationship of HCV envelope proteins.
Protein Expression and Purification 02/2010; 71(2):123-31. DOI:10.1016/j.pep.2010.02.012 · 1.70 Impact Factor
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- "The spectra were calculated by using 110 as the mean residue molecular mass and the results are expressed in terms of residue molar ellipticity in deg cm 2 dmol − 1 . The secondary structure of the protein was evaluated by computer fit of the dichroism spectra according to Convex Constraint Analysis (CCA) . This method relies on an algorithm that calculates the contribution of the secondary structure elements that give rise to the original spectral curve without referring to spectra from model systems. "
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ABSTRACT: The role of preS domains of the hepatitis B virus (HBV) envelope proteins in the first steps of viral infection has been restricted to their implication in virus attachment to a putative hepatocyte receptor. In order to explore a fusion activity in these regions, we used recombinant preS domains to characterize their interaction with liposomes. Binding experiments carried out with NBD-labeled proteins indicated that preS were able to interact in a monomeric way with acidic phospholipid vesicles, being the partition coefficient similar to that described for peptides which can insert deeply into bilayers. Fluorescence depolarization of DPH-labeled vesicles confirmed the specificity for negative charged phospholipids. Upon interaction the proteins induced aggregation, lipid mixing and release of internal contents of acidic vesicles at both acid and neutral pH in a concentration-dependent manner. Taken together, all these data indicate that preS domains are able to insert into the hydrophobic core of the bilayer. Moreover, the insertion resulted in a protein conformational change which increased the helical content. Therefore all these results suggest that, besides their participation in the recognition of a cellular receptor, the preS domains could be involved in the fusion mechanism of HBV with the plasma membrane of target cells.
Biochimica et Biophysica Acta 12/2008; 1788(2):417-24. DOI:10.1016/j.bbamem.2008.10.014 · 4.66 Impact Factor
Available from: Kyusung Park
- "The LINCOMB method (Perczel et al., 1992a) was used for the analysis of the CD spectra, derived from 25 globular proteins by the CCA method (Perczel et al., 1992b). The CCA utilizes three constraints. "
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ABSTRACT: The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222–245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83–93). The result of the CD analysis of Hsc70 (15% -helix, 24% β-sheet, 24% β-turn, and 38% remainder) was very similar to the predicted secondary structure for the β-sheet (24%) and the β-turn (29%). However, there is disagreement between the -helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of β-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of β-sheet conformation (–15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.
Protein Science 03/2008; 2(3):325 - 330. DOI:10.1002/pro.5560020304 · 2.85 Impact Factor
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