NMR Spectroscopy Reveals Cytochrome <i>c</i>–Poly(ethylene glycol) Interactions

Crowley, Peter B.; Brett, Keith; Muldoon, Jimmy

doi:10.1002/cbic.200700603

Cited by 71 publications

(77 citation statements)

References 29 publications

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“…Supporting the non-accidental nature of these interactions, PEG has been shown to bind to cyt c at a hydrophobic patch near the heme. 68 However, we are unable to definitively determine the source of this observed electron density and therefore chose not to include PEG, or another molecule, in our final structural model.…”

Section: Discussionmentioning

confidence: 99%

A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch

et al. 2015

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It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate. Herein we report a high-resolution crystal structure of a Lys73-ligated cyt c conformation that reveals intricate change in the heme environment upon this switch in the heme iron ligation. The structure is surprisingly compact, and the heme coordination loop refolds into a β-hairpin with a turn formed by the highly conserved residues Pro76 and Gly77. Repositioning of residue 78 modifies the intraprotein hydrogen-bonding network and, together with adjustments of residues 52 and 74, increases the volume of the heme pocket to allow for insertion of one of the CL acyl moieties next to Asn52. Derivatization of Cys78 with maleimide creates a solution mimic of the Lys-ligated cyt c that has enhanced peroxidase activity, adding support for a role of the Lys-ligated cyt c in the apoptotic mechanism. Experiments with the heme peptide microperoxidase-8 and engineered model proteins provide a thermodynamic rationale for the switch to Lys ligation upon perturbations in the protein scaffold.

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Section: Discussionmentioning

confidence: 99%

A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch

et al. 2015

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“…To minimize this risk, we had deliberately excluded proteins as crowders since they seem to be particularly prone to interactions1429. However, the ‘neutrality' of various synthetic crowders, after initial enthusiasm, has also been challenged to different degrees30313233.…”

Section: Resultsmentioning

confidence: 99%

An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states

et al. 2017

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Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder's size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalov's interpretation of cold denaturation.

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“…protein crystallization) that I think that there are good reasons for attempting to better understand its behavior anyway. Interesting new information on the nature of PEG-protein interactions has recently become available thanks to the work of the Crowley group who have performed NMR studies to explore the interaction between PEG and the small model protein cytochrome c [66•]. Notably, the largest chemical shift changes induced by the addition of PEG map to a region of the protein’s surface implicated in interactions with other proteins.…”

Section: The Status Of the Fieldmentioning

confidence: 99%

Models of macromolecular crowding effects and the need for quantitative comparisons with experiment

Elcock¹

2010

Current Opinion in Structural Biology

268

228

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SummaryIn recent years significant effort has been devoted to exploring the potential effects of macromolecular crowding on protein folding and association phenomena. Theoretical calculations and molecular simulations have, in particular, been exploited to describe aspects of protein behavior in crowded and confined conditions and many aspects of the simulated behavior have reflected, at least at a qualitative level, the behavior observed in experiments. One major and immediate challenge for the theorists is to now produce models capable of making quantitatively accurate predictions of in vitro behavior. A second challenge is to derive models that explain results obtained from experiments performed in vivo, the results of which appear to call into question the assumed dominance of excluded volume effects in vivo.

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NMR Spectroscopy Reveals Cytochrome c–Poly(ethylene glycol) Interactions

Cited by 71 publications

References 29 publications

A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch

A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch

An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states

Models of macromolecular crowding effects and the need for quantitative comparisons with experiment

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