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Acid-induced molten globule state of a fully active mutant of human interleukin-6.
Biochemistry. 1996 Sep 03; 35(35):11503-11.B

Abstract

Interleukin-6 (IL-6), a four-helix bundle protein, is a multifunctional cytokine which plays an important role in the regulation of the immune system, hematopoiesis, and inflammatory response, as well as in the pathogenesis of multiple myeloma. We have previously shown that a single-disulfide variant of human IL-6, lacking 22 N-terminal amino acids and the disulfide bond connecting Cys-45 and Cys-51 in the 185-residue chain of the wild-type protein, fully retains the conformational, stability, and functional properties of the full-length human IL-6 [Breton et al. (1995) Eur. J. Biochem. 227, 573-581]. In this study, we have investigated the conformational and stability properties of mutant IL-6 at acidic pH (A-state). Using far- and near-ultraviolet (UV) circular dichroism (CD), fluorescence emission, and second-derivative absorption spectroscopy, we have established that mutant IL-6 at pH 2.0 fully retains the helical secondary structure of the native protein at pH 7.5, while the tertiary interactions are much weaker. At variance from the native species, mutant IL-6 in the A-state binds 1-anilinonaphthalene-8-sulfonic acid (ANS), a property considered most typical of a protein in the molten globule state. The pH-induced conformational change from the native to the A-state, monitored either by near-UV CD or by ANS-binding measurements, shows a transition midpoint at pH approximately 4.5, thus indicating that the partial unfolding of the protein is mediated by the titration of glutamic and/or aspartic acid residues. At pH 2.0, the thermal denaturation of mutant IL-6 occurs as a broad process of low cooperativity with a transition at 50-60 degrees C, whereas at pH 7.5 the thermal unfolding is cooperative and characterized by a transition midpoint at 65 degrees C. Of interest, the unfolding of the A-state is not complete even up to approximately 85 degrees C. The urea-mediated unfolding profile of mutant IL-6, measured by far-UV CD, is essentially identical at both pH 7.5 and 2.0, with a midpoint of the cooperative unfolding transition at 5.5 +/- 0.1 M denaturant. Both thermal and urea denaturations of the A-state are complex and cannot fit to a two-state model for unfolding. The unusual stability of mutant IL-6 in acid is also reflected by the resistance to proteolysis at pH 3.6-4.0 by Staphylococcus aureus V8 protease or cathepsin D, an acid protease released by machrophages upon inflammatory stimulation. It is suggested that the molten globule state of IL-6 at acidic pH can play a role in the biological activity of this cytokine, which can exert its activity also at mildly acidic pH, as in inflammation sites.

Authors+Show Affiliations

De Filippis V
CRIBI Biotechnology Centre, University of Padua, Italy.
de Laureto PP
No affiliation info available
Toniutti N
No affiliation info available
Fontana A
No affiliation info available

MeSH

Amino Acid SequenceAnilino NaphthalenesulfonatesCircular DichroismEndopeptidasesFluorescent DyesHumansHydrogen-Ion ConcentrationInterleukin-6Models, MolecularMolecular Sequence DataMutationProtein BindingProtein ConformationProtein DenaturationProtein FoldingProtein Structure, TertiarySequence DeletionSpectrometry, FluorescenceSpectrophotometryTemperatureUrea

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8784206

Citation

De Filippis, V, et al. "Acid-induced Molten Globule State of a Fully Active Mutant of Human Interleukin-6." Biochemistry, vol. 35, no. 35, 1996, pp. 11503-11.
De Filippis V, de Laureto PP, Toniutti N, et al. Acid-induced molten globule state of a fully active mutant of human interleukin-6. Biochemistry. 1996;35(35):11503-11.
De Filippis, V., de Laureto, P. P., Toniutti, N., & Fontana, A. (1996). Acid-induced molten globule state of a fully active mutant of human interleukin-6. Biochemistry, 35(35), 11503-11.
De Filippis V, et al. Acid-induced Molten Globule State of a Fully Active Mutant of Human Interleukin-6. Biochemistry. 1996 Sep 3;35(35):11503-11. PubMed PMID: 8784206.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Acid-induced molten globule state of a fully active mutant of human interleukin-6. AU - De Filippis,V, AU - de Laureto,P P, AU - Toniutti,N, AU - Fontana,A, PY - 1996/9/3/pubmed PY - 1996/9/3/medline PY - 1996/9/3/entrez SP - 11503 EP - 11 JF - Biochemistry JO - Biochemistry VL - 35 IS - 35 N2 - Interleukin-6 (IL-6), a four-helix bundle protein, is a multifunctional cytokine which plays an important role in the regulation of the immune system, hematopoiesis, and inflammatory response, as well as in the pathogenesis of multiple myeloma. We have previously shown that a single-disulfide variant of human IL-6, lacking 22 N-terminal amino acids and the disulfide bond connecting Cys-45 and Cys-51 in the 185-residue chain of the wild-type protein, fully retains the conformational, stability, and functional properties of the full-length human IL-6 [Breton et al. (1995) Eur. J. Biochem. 227, 573-581]. In this study, we have investigated the conformational and stability properties of mutant IL-6 at acidic pH (A-state). Using far- and near-ultraviolet (UV) circular dichroism (CD), fluorescence emission, and second-derivative absorption spectroscopy, we have established that mutant IL-6 at pH 2.0 fully retains the helical secondary structure of the native protein at pH 7.5, while the tertiary interactions are much weaker. At variance from the native species, mutant IL-6 in the A-state binds 1-anilinonaphthalene-8-sulfonic acid (ANS), a property considered most typical of a protein in the molten globule state. The pH-induced conformational change from the native to the A-state, monitored either by near-UV CD or by ANS-binding measurements, shows a transition midpoint at pH approximately 4.5, thus indicating that the partial unfolding of the protein is mediated by the titration of glutamic and/or aspartic acid residues. At pH 2.0, the thermal denaturation of mutant IL-6 occurs as a broad process of low cooperativity with a transition at 50-60 degrees C, whereas at pH 7.5 the thermal unfolding is cooperative and characterized by a transition midpoint at 65 degrees C. Of interest, the unfolding of the A-state is not complete even up to approximately 85 degrees C. The urea-mediated unfolding profile of mutant IL-6, measured by far-UV CD, is essentially identical at both pH 7.5 and 2.0, with a midpoint of the cooperative unfolding transition at 5.5 +/- 0.1 M denaturant. Both thermal and urea denaturations of the A-state are complex and cannot fit to a two-state model for unfolding. The unusual stability of mutant IL-6 in acid is also reflected by the resistance to proteolysis at pH 3.6-4.0 by Staphylococcus aureus V8 protease or cathepsin D, an acid protease released by machrophages upon inflammatory stimulation. It is suggested that the molten globule state of IL-6 at acidic pH can play a role in the biological activity of this cytokine, which can exert its activity also at mildly acidic pH, as in inflammation sites. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8784206/Acid_induced_molten_globule_state_of_a_fully_active_mutant_of_human_interleukin_6_ L2 - https://doi.org/10.1021/bi9604587 DB - PRIME DP - Unbound Medicine ER -