Conformational properties of streptokinase—secondary structure and localization of aromatic amino acids

• Published in: International journal of biological macromolecules Vol. 14; no. 1; pp. 9 - 18
• Main Authors: Welfle, H., Misselwitz, R., Fabian, H., Damerau, W., Hoelzer, W., Gerlach, D., Kalnin, N.N., Venyaminov, S.Yu
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.02.1992; Elsevier Science
• Subjects: Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Circular Dichroism; e.p.r. spectroscopy; Electron Spin Resonance Spectroscopy - methods; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; i.r. spectroscopy; Protein Conformation; protein structure; Raman spectroscopy; Solvents; Spectrometry, Fluorescence - methods; Spectrophotometry, Ultraviolet - methods; Spectrum Analysis, Raman - methods; Spin Labels; Streptococcus - enzymology; Streptokinase; Streptokinase - chemistry; Transferases; e.p.r. spectroscopy; circular dichroism; i.r. spectroscopy; SL-BrAA; Raman spectroscopy; N-Ac- l-Tyr-OET; N-acetyl- l-tyrosine-ethylester; N-(1-oxy-2,2,5,5-tetramethyl-4-pyrrolidinyl)-bromoacetamide; guanidine hydrochloride; protein structure; Streptokinase; N-acetyl- l-tryptophan-ethylester; N-Ac- l-Trp-OET; Gu.HCl; Streptococcaceae; Temperature; Streptococcus; Enzyme; Denaturation; pH; Bacteria; Micrococcales; Secondary structure; Conformation
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/S0141-8130(05)80013-3

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% α-helices, 38–46% β-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 α + β 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 °C, pH values below 3.0 and urea concentrations above 2 m. At temperatures above 70°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°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°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°C.