Entropic Stabilization of Cytochrome c upon Reduction

Cohen, David S.; Pielak, Gary J.

doi:10.1021/ja00111a001

Cited by 68 publications

(59 citation statements)

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“…The higher positive charge carried by the oxidized heme alters the solvation of the protein which may result in the larger ⌬S D . It has been reported that the stability differences between the two redox forms of wild type iso-1-cytochrome c result from differences in the transitional entropies (12). The authors proposed that the folded oxidized wild type protein may bind in excess of 6 additional surface water molecules relative to the reduced form of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the functional properties of cytochrome c are dependent on the oxidation state of the protein, and knowledge of the energetics of protein stability with respect to oxidation state is central to our understanding of function (11). For example, the addition of an electron to ferricytochrome c results in modified functional properties including a significant increase in stability (12).…”

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confidence: 99%

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The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

Lett¹,

Berghuis

Frey

et al. 1996

Journal of Biological Chemistry

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Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule ( Water in and around proteins is recognized as being important to protein structure, function and stability (4 -6). Surface and bound water molecules have been identified in protein structures using crystallographic methods and NMR spectroscopy. Eukaryotic cytochromes c, the paradigms of electron transfer proteins, are ideally suited for investigating the structural and functional purposes of water-protein interactions (7). Crystallographic studies performed on the oxidized and reduced states of both tuna and yeast iso-1-cytochrome c proteins have indicated that a conserved and internally bound water molecule (Wat166), 1 along with the surrounding hydrogen bond network are central to the structural transition of cytochrome c between oxidation states (8, 9). The water molecule is adjacent to the heme and the hydrogen bonding network which is composed of conserved residues Asn 52 , Tyr 67 , and Thr 78 , which are also hydrogen bonded in ferrocytochrome c to the Met 80 sulfur which is one of the two heme iron ligands. The oxidation-reduction or redox potential that determines the direction of electron flow between electron transfer proteins is dependent upon the heme ligands and the surrounding peptide (10). In addition, the functional properties of cytochrome c are dependent on the oxidation state of the protein, and knowledge of the energetics of protein stability with respect to oxidation state is central to our understanding of function (11). For example, the addition of an electron to ferricytochrome c results in modified functional properties including a significant increase in stability (12).Several studies using classical genetic procedures or site directed mutagenesis have shown that the hydrogen bond network and Wat166 modulate redox potential and the stability of the protein (13-20). The high resolution three dimensional structures of the reduced and oxidized states of yeast iso-1-cytochromes c carrying mutations at position 52 and/or 67 have been recently reported (1-3). When compared to the wild type protein structure, these mutants show significant changes in their hydrogen bonding networks adjacent to the heme as well as either the displacement of the conserved internally bound Wat166 or the addition of a second internally bound water molecule.Recent investigations have shown that depending on the amino acid substitutions, varying degrees of change in the free energy of unfolding are observed for the two redox states of cyto...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

Lett¹,

Berghuis

Frey

et al. 1996

Journal of Biological Chemistry

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“…Interestingly, the final global unfolding is stabilized by an additional 2 kcal͞mol. This finding suggests that the last unfolding step finally exposes the buried heme iron (14), which carries a destabilizing charge in oxidized Cyt c but is neutral in the reduced form. Fig.…”

Section: Native-state Hx (Nhx)mentioning

confidence: 93%

Protein folding: The stepwise assembly of foldon units

Maity

Mallela

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

291

320

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Equilibrium and kinetic hydrogen exchange experiments show that cytochrome c is composed of five foldon units that continually unfold and refold even under native conditions. Folding proceeds by the stepwise assembly of the foldon units rather than one amino acid at a time. The folding pathway is determined by a sequential stabilization process; previously formed foldons guide and stabilize subsequent foldons to progressively build the native protein. Four other proteins have been found to show similar behavior. These results support stepwise protein folding pathways through discrete intermediates.cytochrome c ͉ hydrogen exchange ͉ stability labeling T o understand how proteins fold to their native state, it will be necessary to define the structure of the intermediate forms that they pass through, as for any other biochemical mechanism. This effort has proven to be exceptionally difficult. Partially folded kinetic intermediates live for Ͻ1 s, and they cannot be isolated and studied by the usual structural methods. Spectroscopic observations widely used to detect fast kinetic folding events provide very little structural detail and can be misleading in respect to structure formation. Theoretical methods are not yet able to simulate the folding of sizeable proteins.These problems can be avoided by studying proteins under native conditions. Under these conditions proteins predominantly occupy their lowest free energy state (1) but they must repeatedly unfold and refold, thermodynamically cycling through all possible higherenergy forms. The high-energy forms generally exist only at minuscule levels and therefore are invisible to most methods, which perceive only the overwhelmingly populated native state. These high-energy forms can be detected by hydrogen exchange (HX) methods because the predominant native structure makes no contribution to the HX rates that one measures. Hydrogens that are protected in the native protein can exchange with solvent only when their protecting H bonds are transiently broken as the protein searches through its higher-energy forms.HX measurements provide amino acid-resolved information that can in favorable cases define the structure of partially folded intermediate forms, their equilibrium and kinetic parameters, and their interconversions. Results obtained for cytochrome c (Cyt c) show that it is composed of five subglobally cooperative unfolding͞ refolding units, called foldons, color-coded in Fig. 1. This article integrates our research and prior information for Cyt c and other proteins. All of these results consistently show that proteins at equilibrium are composed of foldon building blocks and that they fold kinetically by the stepwise assembly of their foldon units, guided by the same factors that determine the native state. Materials and MethodsWT equine Cyt c (type VI from Sigma) was further purified as required. The pseudo-WT (pWT) protein and its mutants were expressed in Escherichia coli and purified as described (2). The pWT protein has no N-terminal acetylation; also, tw...

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“…The green unfolding, which includes the 60s helix and the green loop, is similarly suppressed (3.3 kcal͞mol), showing that in the measured green-open state the red, yellow, and green segments are open together (rygB). The blue unfolding, known to represent the final global unfolding to U (rygb) (48,86,87), is suppressed by 3.2 kcal plus an additional 2-kcal increment, caused apparently by neutralization of the destabilizing buried charge when Cyt c is reduced (88).…”

Section: An Entire Pathwaymentioning

confidence: 99%

An amino acid code for protein folding

Rumbley

Hoang

Mayne

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

136

140

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Direct structural information obtained for many proteins supports the following conclusions. The amino acid sequences of proteins can stabilize not only the final native state but also a small set of discrete partially folded native-like intermediates. Intermediates are formed in steps that use as units the cooperative secondary structural elements of the native protein. Earlier intermediates guide the addition of subsequent units in a process of sequential stabilization mediated by native-like tertiary interactions. The resulting stepwise self-assembly process automatically constructs a folding pathway, whether linear or branched. These conclusions are drawn mainly from hydrogen exchange-based methods, which can depict the structure of infinitesimally populated folding intermediates at equilibrium and kinetic intermediates with subsecond lifetimes. Other kinetic studies show that the polypeptide chain enters the folding pathway after an initial free-energy-uphill conformational search. The search culminates by finding a nativelike topology that can support forward (native-like) folding in a free-energy-downhill manner. This condition automatically defines an initial transition state, the search for which sets the maximum possible (two-state) folding rate. It also extends the sequential stabilization strategy, which depends on a native-like context, to the first step in the folding process. Thus the native structure naturally generates its own folding pathway. The same amino acid code that translates into the final equilibrium native structure-by virtue of propensities, patterning, secondary structural cueing, and tertiary context-also produces its kinetic accessibility.

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Entropic Stabilization of Cytochrome c upon Reduction

Cited by 68 publications

References 4 publications

The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

Protein folding: The stepwise assembly of foldon units

An amino acid code for protein folding

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