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pH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence.
Biochemistry. 1997 Nov 25; 36(47):14345-52.B

Abstract

Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) and in triple distilled water (pH 5.5-6.0) was examined by differential scanning calorimetry, UV and CD spectroscopy and fluorescence emission spectroscopy of the added hydrophobic fluorescent probe ANS. At pH 5.5-6.0 and at temperatures below 60 degrees C, the protein exists in a native state characterized by a pronounced tertiary structure, a beta-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (delta H degree)VH = (delta H degree)DSC, into an unfolded state, which is characterized by a complete collapse of its tertiary structure and an incomplete denaturation of its secondary structure. At acidic pH, the EqTxII temperature-induced conformational transition appears at lower temperatures as non-two-state transition accompanied by the formation of an intermediate state which shows characteristics of molten globules, i.e., absence of defined tertiary structure, increase in alpha-rich secondary structure, and high affinity for ANS. At pH 2.0, the low-temperature initial state of EqTxII is already partially denatured; the tertiary structure is partially disrupted, and a pronounced inequality (delta H degree)VH > (delta H degree)DSC is observed. At pH value of 1.1 and below 60 degrees C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 degrees C is not completely denatured. According to numerous studies on the pore forming toxins, such acid-denatured compact states may contribute to the protein's ability to penetrate into biological membranes.

Authors+Show Affiliations

Department of Chemistry, University of Ljubljana, Slovenia.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

9398152

Citation

Poklar, N, et al. "PH and Temperature-induced Molten Globule-like Denatured States of Equinatoxin II: a Study By UV-melting, DSC, Far- and near-UV CD Spectroscopy, and ANS Fluorescence." Biochemistry, vol. 36, no. 47, 1997, pp. 14345-52.
Poklar N, Lah J, Salobir M, et al. PH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence. Biochemistry. 1997;36(47):14345-52.
Poklar, N., Lah, J., Salobir, M., Macek, P., & Vesnaver, G. (1997). PH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence. Biochemistry, 36(47), 14345-52.
Poklar N, et al. PH and Temperature-induced Molten Globule-like Denatured States of Equinatoxin II: a Study By UV-melting, DSC, Far- and near-UV CD Spectroscopy, and ANS Fluorescence. Biochemistry. 1997 Nov 25;36(47):14345-52. PubMed PMID: 9398152.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - pH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence. AU - Poklar,N, AU - Lah,J, AU - Salobir,M, AU - Macek,P, AU - Vesnaver,G, PY - 1997/12/16/pubmed PY - 1997/12/16/medline PY - 1997/12/16/entrez SP - 14345 EP - 52 JF - Biochemistry JO - Biochemistry VL - 36 IS - 47 N2 - Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) and in triple distilled water (pH 5.5-6.0) was examined by differential scanning calorimetry, UV and CD spectroscopy and fluorescence emission spectroscopy of the added hydrophobic fluorescent probe ANS. At pH 5.5-6.0 and at temperatures below 60 degrees C, the protein exists in a native state characterized by a pronounced tertiary structure, a beta-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (delta H degree)VH = (delta H degree)DSC, into an unfolded state, which is characterized by a complete collapse of its tertiary structure and an incomplete denaturation of its secondary structure. At acidic pH, the EqTxII temperature-induced conformational transition appears at lower temperatures as non-two-state transition accompanied by the formation of an intermediate state which shows characteristics of molten globules, i.e., absence of defined tertiary structure, increase in alpha-rich secondary structure, and high affinity for ANS. At pH 2.0, the low-temperature initial state of EqTxII is already partially denatured; the tertiary structure is partially disrupted, and a pronounced inequality (delta H degree)VH > (delta H degree)DSC is observed. At pH value of 1.1 and below 60 degrees C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 degrees C is not completely denatured. According to numerous studies on the pore forming toxins, such acid-denatured compact states may contribute to the protein's ability to penetrate into biological membranes. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9398152/pH_and_temperature_induced_molten_globule_like_denatured_states_of_equinatoxin_II:_a_study_by_UV_melting_DSC_far__and_near_UV_CD_spectroscopy_and_ANS_fluorescence_ DB - PRIME DP - Unbound Medicine ER -