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Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles.
Biochemistry. 1997 Oct 21; 36(42):13122-32.B

Abstract

The interaction of cytochrome c with anionic lipid vesicles of DOPS induces an extensive disruption of the native structure of the protein. The kinetics of this lipid-induced unfolding process were investigated in a series of fluorescence- and absorbance-detected stopped-flow measurements. The results show that the tightly packed native structure of cytochrome c is disrupted at a rate of approximately 1.5 s-1 (independent of protein and lipid concentration), leading to the formation of a lipid-inserted denatured state (DL). Comparison with the expected rate of unfolding in solution (approximately 2 x 10(-3) s-1 at pH 5.0 in the absence of denaturant) suggests that the lipid environment dramatically accelerates the structural unfolding process of cytochrome c. We propose that this acceleration is in part due to the low effective pH in the vicinity of the lipid headgroups. This hypothesis was tested by comparative kinetic measurements of acid unfolding of cytochrome c in solution. Our absorbance and fluorescence kinetic data, combined with a well-characterized mechanism for folding/unfolding of cytochrome c in solution, allow us to propose a kinetic mechanism for cytochrome c unfolding at the membrane surface. Binding of native cytochrome c in water (NW) to DOPS vesicles is driven by the electrostatic interaction between positively charged residues in the protein and the negatively charged lipid headgroups on the membrane surface. This binding step occurs within the dead time of the stopped-flow experiments (<2 ms), where a membrane-associated native state (NS) is formed. Unfolding of NS driven by the acidic environment at the membrane surface is proposed to occur via a native-like intermediate lacking Met 80 ligation (MS), as previously observed during unfolding in solution. The overall unfolding process (NS --> DL) is limited by the rate of disruption of the hydrophobic core in MS. Equilibrium spectroscopic measurements by near-IR and Soret absorbance, fluorescence, and circular dichroism showed that DL has native-like helical secondary structure, but shows no evidence for specific tertiary interactions. This lipid-denatured equilibrium state (DL) is clearly more extensively unfolded than the A-state in solution, but is distinct from the unfolded protein in water (UW), which has no stable secondary structure.

Authors+Show Affiliations

Institute for Cancer Research, Fox Chase Cancer Centre, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111, USA. tp@dna.bio.warwick.ac.ukNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

9335575

Citation

Pinheiro, T J., et al. "Structural and Kinetic Description of Cytochrome C Unfolding Induced By the Interaction With Lipid Vesicles." Biochemistry, vol. 36, no. 42, 1997, pp. 13122-32.
Pinheiro TJ, Elöve GA, Watts A, et al. Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles. Biochemistry. 1997;36(42):13122-32.
Pinheiro, T. J., Elöve, G. A., Watts, A., & Roder, H. (1997). Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles. Biochemistry, 36(42), 13122-32.
Pinheiro TJ, et al. Structural and Kinetic Description of Cytochrome C Unfolding Induced By the Interaction With Lipid Vesicles. Biochemistry. 1997 Oct 21;36(42):13122-32. PubMed PMID: 9335575.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles. AU - Pinheiro,T J, AU - Elöve,G A, AU - Watts,A, AU - Roder,H, PY - 1997/10/23/pubmed PY - 1997/10/23/medline PY - 1997/10/23/entrez SP - 13122 EP - 32 JF - Biochemistry JO - Biochemistry VL - 36 IS - 42 N2 - The interaction of cytochrome c with anionic lipid vesicles of DOPS induces an extensive disruption of the native structure of the protein. The kinetics of this lipid-induced unfolding process were investigated in a series of fluorescence- and absorbance-detected stopped-flow measurements. The results show that the tightly packed native structure of cytochrome c is disrupted at a rate of approximately 1.5 s-1 (independent of protein and lipid concentration), leading to the formation of a lipid-inserted denatured state (DL). Comparison with the expected rate of unfolding in solution (approximately 2 x 10(-3) s-1 at pH 5.0 in the absence of denaturant) suggests that the lipid environment dramatically accelerates the structural unfolding process of cytochrome c. We propose that this acceleration is in part due to the low effective pH in the vicinity of the lipid headgroups. This hypothesis was tested by comparative kinetic measurements of acid unfolding of cytochrome c in solution. Our absorbance and fluorescence kinetic data, combined with a well-characterized mechanism for folding/unfolding of cytochrome c in solution, allow us to propose a kinetic mechanism for cytochrome c unfolding at the membrane surface. Binding of native cytochrome c in water (NW) to DOPS vesicles is driven by the electrostatic interaction between positively charged residues in the protein and the negatively charged lipid headgroups on the membrane surface. This binding step occurs within the dead time of the stopped-flow experiments (<2 ms), where a membrane-associated native state (NS) is formed. Unfolding of NS driven by the acidic environment at the membrane surface is proposed to occur via a native-like intermediate lacking Met 80 ligation (MS), as previously observed during unfolding in solution. The overall unfolding process (NS --> DL) is limited by the rate of disruption of the hydrophobic core in MS. Equilibrium spectroscopic measurements by near-IR and Soret absorbance, fluorescence, and circular dichroism showed that DL has native-like helical secondary structure, but shows no evidence for specific tertiary interactions. This lipid-denatured equilibrium state (DL) is clearly more extensively unfolded than the A-state in solution, but is distinct from the unfolded protein in water (UW), which has no stable secondary structure. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9335575/Structural_and_kinetic_description_of_cytochrome_c_unfolding_induced_by_the_interaction_with_lipid_vesicles_ L2 - https://doi.org/10.1021/bi971235z DB - PRIME DP - Unbound Medicine ER -