Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength
| Autor: | Elodie Monsellier, Hugues Bedouelle |
|---|---|
| Přispěvatelé: | Prévention et thérapie moléculaires des maladies humaines, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2005 |
| Předmět: |
Protein Denaturation
Protein Folding cooperativity MESH: Protein Folding Immunoglobulin Variable Region Analytical chemistry envelope glycoprotein MESH: Immunoglobulin Variable Region Thermodynamics Bioengineering Cooperativity MESH: Dengue Virus Curvature Biochemistry Stability (probability) Fluorescence Spectral line free-energy MESH: Protein Structure Tertiary 03 medical and health sciences Viral Envelope Proteins Methods Emission spectrum MESH: Methods Molecular Biology unfolding MESH: Tryptophan 030304 developmental biology Envelope (waves) Quantitative Biology::Biomolecules 0303 health sciences dengue virus Chemistry scFv antibody fragment MESH: Fluorescence 030302 biochemistry & molecular biology Tryptophan Protein Structure Tertiary [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biophysics Wavelength Spectrometry Fluorescence denaturant MESH: Viral Envelope Proteins MESH: Muramidase Muramidase MESH: Protein Denaturation MESH: Spectrometry Fluorescence Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection Protein Engineering, Design and Selection, Oxford University Press (OUP), 2005, 18 (9), pp.445-56. ⟨10.1093/protein/gzi046⟩ Protein Engineering, Design and Selection, 2005, 18 (9), pp.445-56. ⟨10.1093/protein/gzi046⟩ |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzi046 |
| Popis: | International audience; The fluorescence of tryptophan is used as a signal to monitor the unfolding of proteins, in particular the intensity of fluorescence and the wavelength of its maximum lambda(max). The law of the signal is linear with respect to the concentrations of the reactants for the intensity but not for lambda(max). Consequently, the stability of a protein and its variation upon mutation cannot be deduced directly from measurements made with lambda(max). Here, we established a rigorous law of the signal for lambda(max). We then compared the stability DeltaG(H(2)O) and coefficient of cooperativity m for a two-state equilibrium of unfolding, monitored with lambda(max), when the rigorous and empirical linear laws of the signal are applied. The corrective terms involve the curvature of the emission spectra at their lambda(max) and can be determined experimentally. The rigorous and empirical values of the cooperativity coefficient m are equal within the experimental error for this parameter. In contrast, the rigorous and empirical values of the stability DeltaG(H(2)O) generally differ. However, they are equal within the experimental error if the curvatures of the spectra for the native and unfolded states are identical. We validated this analysis experimentally using domain 3 of the envelope glycoprotein of the dengue virus and the single-chain variable fragment (scFv) of antibody mAbD1.3, directed against lysozyme. |
| Databáze: | OpenAIRE |
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