Solution structure and biological activity of recombinant salmon calcitonin S-sulfonated analog.
ABSTRACT Salmon calcitonin S-sulfonated analog (abbreviated as [S-SO(3)(-)]rsCT) was prepared by introducing two sulfonic groups into the side chains of Cys1 and Cys7 of recombinant salmon calcitonin. The hypocalcemic potency of this open-chain analog is 5500IU/mg, which is about 30% higher than that (4500IU/mg) of the wild type. The solution conformation of [S-SO(3)(-)]rsCT was studied in aqueous trifluoroethanol solution by CD, 2D-NMR spectroscopy, and distance geometry calculations. In the mixture of 60% TFE and 40% water, the peptide assumes an amphipathic alpha-helix in the region of residues 4-22, which is one turn longer than that of the native sCT. The structural feature analysis of the peptide revealed the presence of hydrophobic surface composed of five hydrophobic side chains of residues Leu4, Leu9, Leu12, Leu16, and Leu19, and a network of salt-bridges that consisted of a tetrad of oppositely charged side chains (Cys7-SO(3)(-)-Lys11(+)-Glu15(-)-Lys18(+)). The multiple salt bridges resulted in the stabilization of the longer amphipathic alpha-helix. Meanwhile, the higher hypocalcemic potency of the peptide could be attributed to the array of hydrophobic side chains of five leucine residues of the amphipathic alpha-helix.
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ABSTRACT: To separate and characterize the different positional isomers of mono-PEGylated salmon calcitonins (mono-PEG-sCTs) and to evaluate the effects of the PEGylation site on the stability of different mono-PEG-sCTs in rat kidney homogenate. Mono-PEG-sCTs were prepared using succinimidyl carbonate monomethoxy polyethylene glycol (5,000 Da) and separated by gel-filtration HPLC followed by reversed-phase HPLC. To characterize PEGylated sCTs, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and reversed-phase HPLC of the trypsin digested samples were performed. Mono-PEG-sCTs and sCT in rat kidney homogenates were measured by column-switching reversed-phase HPLC with on-line detection of the radioiodinated samples using a flow-through radioisotope detector. Three different mono-PEGylated sCTs were separated by reversed-phase gradient HPLC. From the MALDI-TOF MS analysis, the average molecular weight of mono-PEG-sCTs was confirmed as around 8650 Da. The presence of PEG moiety in the mono-PEG-sCTs was also manifested by the fact that the distance between two adjacent mass spectum lines was 44 Da which corresponds to PEG monomer unit. Tryptic digestion analysis demonstrated that these mono-PEG-sCTs are 3 positional isomers of N-terminus, Lys18- and Lys11-residue modified mono-PEGylated sCTs. The degradation half-life of these 3 positional isomers in rat kidney homogenates significantly increased in order of the N-terminus (125.5 min), Lys11- (157.3 min), and Lys18 residue modified mono-PEGylated sCT (281.5 min) over the native sCT (4.8 min). Three positional isomers of mono-PEGylated sCTs were purified and characterized. Of these, the resistance to proteolytic degradation was highest for the Lys18-residue modified mono-PEG-sCT. These studies demonstrate that the in vivo stability of PEGylated sCTs is highly dependent on the site of PEG molecule attachment.Pharmaceutical Research 07/1999; 16(6):813-8. · 4.74 Impact Factor
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ABSTRACT: The use of proton-proton nuclear Overhauser enhancement (NOE) distance information for identification of polypeptide secondary structures in non-crystalline proteins was investigated by stereochemical studies of standard secondary structures and by statistical analyses of the secondary structures in the crystal conformations of a group of globular proteins. Both regular helix and beta-sheet secondary structures were found to contain a dense network of short 1H-1H distances. The results obtained imply that the combined information on all these distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra is sufficient for determination of the helical and beta-sheet secondary structures in small globular proteins. Furthermore, cis peptide bonds can be identified from unique, short sequential proton-proton distances. Limitations of this empirical approach are that the exact start or end of a helix may be difficult to define when the adjoining residues form a tight turn, and that unambiguous identification of tight turns can usually be obtained only in the hairpins of antiparallel beta-structures. The short distances between protons in pentapeptide segments of the different secondary structures have been tabulated to provide a generally applicable guide for the analysis of NOESY spectra of proteins.Journal of Molecular Biology 01/1985; 180(3):715-40. · 3.91 Impact Factor
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ABSTRACT: A new procedure based on the statistical method of "variable selection" is used to predict the secondary structure of proteins from circular dichroism spectra. Variable selection adds the flexibility found in the Provencher and Glöckner method (S. W. Provencher and J. Glöckner, 1981, Biochemistry 20, 33-37) to the method of Hennessey and Johnson (J. P. Hennessey and W. C. Johnson, 1981, Biochemistry 20, 1085-1094). Two analytical methods are presented for choosing a solution from the series generated by the Provencher and Glöckner method, and this improves the technique. All three methods are compared and it is shown that both the variable selection method and the improved Provencher and Glöckner methods have equivalent reliability superior to the original Hennessey and Johnson method. For the new variable selection method, correlation coefficients calculated between X-ray structure and predicted secondary structures for data measured to 178 nm are: 0.97 for alpha-helix, 0.75 for beta-sheet, 0.50 for beta-turn, and 0.89 for other structures. Although the variable selection method improves the analysis of circular dichroism data truncated at 190 nm, data measured to 178 nm gives superior results. It is shown that improving the fit to the measured CD beyond the accuracy of the data can result in poorer analyses.Analytical Biochemistry 12/1987; 167(1):76-85. · 2.58 Impact Factor