Probing Early Events in Ferrous Cytochrome c Folding with Time- Resolved Natural and Magnetic Circular Dichroism Spectroscopies.

Chen, Eefei; Goldbeck, Robert A.; Kliger, David S.

doi:10.2174/138920309789352001

Cited by 15 publications

(13 citation statements)

References 78 publications

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“…The folding and unfolding of cyt c in solution have been explored extensively (Winkler, 2004; Maity et al, 2005; Ensign et al, 2008; Chen et al, 2009; Yu et al, 2012). Hydrogen-deuterium amide exchange experiments have suggested that cyt c consists of five cooperative folding units (foldons) of varying thermodynamic stability that undergo folding and unfolding in a stepwise, sequential mechanism (Roder et al, 1988; Bai et al, 1995; Maity et al, 2004; Krishna et al, 2006).…”

Section: Cytochrome C and Its Conformational Dynamicsmentioning

confidence: 99%

Structural transformations of cytochrome c upon interaction with cardiolipin

Muenzner

Pletneva

2014

Chemistry and Physics of Lipids

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Interactions of cytochrome c (cyt c) with cardiolipin (CL) play a critical role in early stages of apoptosis. Upon binding to CL, cyt c undergoes changes in secondary and tertiary structure that lead to a dramatic increase in its peroxidase activity. Insertion of the protein into membranes, insertion of CL acyl chains into the protein interior, and extensive unfolding of cyt c after adsorption to the membrane have been proposed as possible modes for interaction of cyt c with CL. Dissociation of Met80 is accompanied by opening of the heme crevice and binding of another heme ligand. Fluorescence studies have revealed conformational heterogeneity of the lipid-bound protein ensemble with distinct polypeptide conformations that vary in the degree of protein unfolding. We correlate these recent findings to other biophysical observations and rationalize the role of experimental conditions in defining conformational properties and peroxidase activity of the cyt c ensemble. Latest time-resolved studies propose the trigger and the sequence of cardiolipin-induced structural transitions of cyt c.

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Section: Cytochrome C and Its Conformational Dynamicsmentioning

confidence: 99%

Structural transformations of cytochrome c upon interaction with cardiolipin

Muenzner

Pletneva

2014

Chemistry and Physics of Lipids

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“…3–6 In this work we use mesoporous silica nanoparticles (MSN) as delivery vehicle because they are biocompatible, biodegradable, and generally recognized as safe by the US Food and Drug Administration (FDA). 7, 8 MSN have been developed as delivery vehicles for proteins some years ago and it has been established that they are capable of transporting membrane-impermeable proteins (i.e., Cyt c) into the cytoplasm of target cells including the HeLa cell line used herein by us. 9,10 Previously, we reported on the improvement of the use of MSN as carriers for protein drugs and by covalently immobilizing the model protein carbonic anhydrase on a thiolated surface.…”

Section: Introductionmentioning

confidence: 99%

Delivery of Chemically Glycosylated Cytochrome c Immobilized in Mesoporous Silica Nanoparticles Induces Apoptosis in HeLa Cancer Cells

et al. 2013

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Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7±0.9 using mono-(lactosylamido)-mono-(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 μg/mL) for 24 to 72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 μg/mL and 37.5 μg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.

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“…[36][37][38][39] A large body of kinetic and thermodynamic data suggests the presence of molten globules on the folding path of proteins. [40][41][42][43][44][45][46][47][48] Recently, Chen et al 49 provided experimental support to the classical folding pathway U -MG -N from their kinetic studies of GdmCl-induced and SDS-induced denaturation of cyt-c. These studies suggested that the protein folding is hierarchic.…”

Section: Introductionmentioning

confidence: 99%

Conformation and thermodynamic stability of pre-molten and molten globule states of mammalian cytochromes-c

2011

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Proteins in cells fold via a number of intermediates. These intermediates are quite important as they guide the protein to attain its unique native conformation. To solve the immensely difficult problem of protein folding, it is necessary to characterize intermediates which will unravel the mystery of the steps involved in the proper folding of proteins. Cytochromes-c (cyts-c) have played an important role in studies of the earliest events and intermediates in protein folding. They have always been considered as model proteins for protein folding studies due to their intrinsic properties that can be measured by multiple probes. A large number of different solvent conditions have been employed to obtain equilibrium intermediates of cyts-c. These intermediates show structural heterogeneity which is mainly due to the different solvent conditions used to induce them. In this review we present results of conformational and thermodynamic characterization of equilibrium intermediates (molten globules and pre-molten globules) of the mammalian cyts-c under different solvent conditions.

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Probing Early Events in Ferrous Cytochrome c Folding with Time- Resolved Natural and Magnetic Circular Dichroism Spectroscopies.

Cited by 15 publications

References 78 publications

Structural transformations of cytochrome c upon interaction with cardiolipin

Structural transformations of cytochrome c upon interaction with cardiolipin

Delivery of Chemically Glycosylated Cytochrome c Immobilized in Mesoporous Silica Nanoparticles Induces Apoptosis in HeLa Cancer Cells

Conformation and thermodynamic stability of pre-molten and molten globule states of mammalian cytochromes-c

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