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- Christova, P, et al.
(författare)
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Fluorescence studies on denaturation and stability of recombinant human interferon-gamma
- 2003
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Ingår i: Zeitschrift fur Naturforschung. C, Journal of biosciences. - : Walter de Gruyter GmbH. - 0939-5075. ; 58:3-4, s. 288-294
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Tidskriftsartikel (refereegranskat)abstract
- Unfolding/folding transitions of recombinant human interferon-gamma (hIFNγ) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 m and ΔG0 = 13.4 kJ/mol) than urea (C* = 2.8 m and ΔG0 = 11.7 kJ/mol). The close ΔG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNγ is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNγ remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNγ, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNγ.
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- Gruber, CEM, et al.
(författare)
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Geographical Variability Affects CCHFV Detection by RT-PCR: A Tool for In-Silico Evaluation of Molecular Assays
- 2019
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 11:10
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Tidskriftsartikel (refereegranskat)abstract
- The Crimean–Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient’s travel history and geographic distribution of the different CCHFV strains.
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- Knapp, S, et al.
(författare)
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Thermal unfolding of small proteins with SH3 domain folding pattern
- 1998
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 31:3, s. 309-319
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Tidskriftsartikel (refereegranskat)abstract
- The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy, The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec), At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. (C) 1998 Wiley-Liss, Inc.
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