Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.
The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.
Welfle
H
H
Central Institute of Molecular Biology, Berlin-Buch, Germany.
Misselwitz
R
R
Fabian
H
H
Damerau
W
W
Hoelzer
W
W
Gerlach
D
D
Kalnin
N N
NN
Venyaminov
S Y
SY
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D012997:Solvents; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods; D013113:Spin Labels; D013291:Streptococcus / Q000201:enzymology; D013300:Streptokinase / Q000737:chemistry*
D000596:Amino Acids; D012997:Solvents; D013113:Spin Labels; D013300:Streptokinase