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

Welfle, Heinz; Misselwitz, Rolf; Fabian, Heinz; Damerau, W.; Hoelzer, W.; Gerlach, Dieter; Kalnin, N.N.; Venyaminov, Sergei Yu.

doi:10.1016/s0141-8130(05)80013-3

Cited by 20 publications

(20 citation statements)

References 30 publications

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“…IF). This is consistent with previous studies that used differential scanning calorimetry (Radek & Castellino, 1989;Welfle et al, 1992). Figure 8 shows the same process monitored by NMR (see also Teuten et al, 1993).…”

Section: Thermal Unfolding Of Three-domain Intact Streptokinasesupporting

confidence: 91%

“…The origin of these relatively downfield resonances is unclear, although it is likely that they correspond to residual /3 structure within the SK chain. This is consistent with the presence of significant ellipticity around 220 nm in the far-UV CD spectrum of intact SK and SK fragments at high temperatures observed by us and by others (Radek & Castellino, 1989;Welfle et al, 1992). Additionally, the majority of resonances in the NMR spectrum of SK at 65 "C are very broad.…”

Section: Thermal Unfolding Of Three-domain Intact Streptokinasesupporting

confidence: 90%

“…Several fragments and modified forms of SK have been isolated previously (Brockway & Castellino, 1974;Siefring & Castellino, 1976;Jackson et al, 1986;Malke et al, 1987;Misselwitz et al, 1992;Rodriguez et al, 1994Rodriguez et al, , 1995Shi et al, 1994;Reed et al, 1995;Young et al, 1995). A number of SK fragments remain folded when isolated in solution and a minority maintain an ability to cause Plg activation, although, in most cases, this occurs at substantially reduced rates when compared with native SK.…”

mentioning

confidence: 99%

“…A variety of techniques has been used to investigate these processes, including differential scanning calorimetry, CD, fluorescence spectroscopy, and NMR spectrometry (Novokhatny et al, 1991;Teuten et al, 1991Teuten et al, , 1993Vysotchin et al, 1993;Nowak et al, 1994;Kurochkin et al, 1995). A previous DSC study with intact SK reported two major thermal transitions occurring at very similar temperatures under several conditions (Welfle et al, 1992). A number of proteolytic SK fragments were later studied by DSC (Misselwitz et al, 1992), although complete assignment of the unfolding transitions to specific regions of SK was not possible because of the lack of fragments representing all regions of the molecule.…”

mentioning

confidence: 99%

“…A previous DSC study with intact SK reported two major thermal transitions occurring at very similar temperatures under several conditions (Welfle et al, 1992). A number of proteolytic SK fragments were later studied by DSC (Misselwitz et al, 1992), although complete assignment of the unfolding transitions to specific regions of SK was not possible because of the lack of fragments representing all regions of the molecule. Unfolding by temperature and guanidine hydrochloride of the intact protein followed by NMR showed that three regions of SK experience independent unfolding (Teuten et al, 1993), but the results could not be correlated to the domain organization of the protein.…”

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confidence: 99%

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Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

et al. 1996

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Streptococcus equisirnilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al ., 1996, Protein Sci 5693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46°C and its stability is largely independent of the presence of the other domains. The hightemperatwe transition in intact SK (at approximately 63 "C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (AI and A2) adopt unstructured conformations when separated. AI binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.

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Section: Thermal Unfolding Of Three-domain Intact Streptokinasesupporting

confidence: 91%

Section: Thermal Unfolding Of Three-domain Intact Streptokinasesupporting

confidence: 90%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

et al. 1996

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Conformation and stability of streptokinases from nephritogenic and nonnephritogenic strains of streptococci

et al. 1997

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Conformation and stability of three Sks from Streptococcus equisimilis strain H46A, Streptococcus pyogenes strain A374, and Streptococcus pyogenes strain AT27 were compared by limited proteolysis, CD, and fluorescence measurements and by DSC. The general similarity of the peptide CD spectra in the spectral region 185 to 260 nm indicates the same type of folding for the three proteins. Fluorescence and aromatic CD spectra are consistent with a predominant surface localization of the aromatic amino acids and a low rigidity of their surroundings. A major difference among the three Sks is shown by deconvolution of their excessive heat capacity functions. Deconvolution reveals two energetic folding units in Sk H46A but three energetic folding units in Sk A374 and Sk AT27. Digestion of the Sks with trypsin indicates a reduced sensitivity of the C-terminal region of Sk A374 and Sk AT27 in comparison to Sk H46A. This suggests that amino acids of the C-terminal region participate in the formation of the third folding unit of Sk A374 and Sk AT27.

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Solubilization of inclusion body proteins using low and very low concentrations of chemicals: implications of novel combined chemical treatment designs in enhancement of post‐solubilization target protein purity and biological activity

Mohammadian

Kaghazian

Kavianpour

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND Solubilization of inclusion body (IB) proteins by conventional methods (i.e. high concentrations of denaturants such as chaotropes) is a challenging process due to denaturation of the native‐like secondary structures and subsequently low recovery into bioactive forms. The main objective of this work was to study the effect of a range of chemicals at low and very low concentrations on the solubilization of IB proteins produced in Escherichia coli and the enhancement of the target protein biological activity subsequent to refolding. RESULTS Performance of chemical combinations at low and very low concentrations through the solubilization process of recombinant streptokinase (rSK) from IBs isolated from E. coli was appraised based on values pertinent to three parameters including target protein solubilization yield, purity and biological activity. In comparison with the conventional IBs' solubilization method (i.e. 4 mol L‐1 urea), combinations of 0.5–1 mol L‐1 urea with very low to low concentrations (0.05–1%) of detergents resulted in considerable target protein solubilization (by 100%), very high post‐solubilization target protein purities (up to 100%) and biological activities (up by 360%). CONCLUSION Owing to the improvements in the abovementioned integral parameters, these chemical treatments are good candidates to be considered for more efficient and cost‐effective recovery of recombinant target proteins from IBs. © 2017 Society of Chemical Industry

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

Cited by 20 publications

References 30 publications

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

Conformation and stability of streptokinases from nephritogenic and nonnephritogenic strains of streptococci

Solubilization of inclusion body proteins using low and very low concentrations of chemicals: implications of novel combined chemical treatment designs in enhancement of post‐solubilization target protein purity and biological activity

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