Probing electrostatic interactions in cytochrome c using site-directed chemical modification

2002 ◽  
Vol 80 (2) ◽  
pp. 197-203 ◽  
Author(s):  
Christian Blouin ◽  
J Guy Guillemette ◽  
Carmichael JA Wallace
Keyword(s):  
Cytochrome C ◽  
Chemical Modification ◽  
Redox Potential ◽  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Heme Iron ◽  
Dielectric Constants ◽  
Protein Protein Interactions ◽  
Potential Control ◽  
Electrostatic Probe

This communication reports the generation of an electrostatic probe using chemical modification of methionine side chains. The alkylation of methionine by iodoacetamide was achieved in a set of Saccharomyces cerevisiae iso-1-cytochrome c mutants, introducing the nontitratable, nondelocalized positive charge of a carboxyamidomethylmethionine sulfonium (CAMMS) ion at five surface and one buried site in the protein. Changes in redox potential and its variation with temperature were used to calculate microscopic effective dielectric constants operating between the probe and the heme iron. Dielectric constants (ε) derived from ΔΔG values were not useful due to entropic effects, but εΔΔH gave results that supported the theory. The effect on biological activity of surface derivatization was interpreted in terms of protein–protein interactions. The introduction of an electrostatic probe in cytochrome c often resulted in marked effects on activity with one of two physiological partners: cytochrome c reductase, especially if introduced at position 65, and cytochrome c oxidase, if at position 28.Key words: protein engineering, chemical modification, cytochrome c, electron transport, protein electrostatics, redox potential control.

scholarly journals ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1646
Author(s):  
Jordan Bye ◽  
Kiah Murray ◽  
Robin Curtis
Keyword(s):  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Colloidal Stability ◽  
Conformational Stability ◽  
Kinetic Studies ◽  
Structural Factors ◽  
Protein Protein Interactions ◽  
Protein Aggregate ◽  
Aggregate Growth

A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to high colloidal stability, but often has the undesirable effect of lowering conformational stability. We show this trade-off can be overcome by using small multivalent polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (TPP) as excipients. These ions are equally effective at suppressing aggregation of ovalbumin and bovine serum albumin (BSA) upon thermal stress as monitored by dynamic and static light scattering. Monomer loss kinetic studies, combined with measurements of native state protein–protein interactions and ζ-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), α-chymotrypsinogen (α-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and α-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely packed cellular environments. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.

scholarly journals Colloidal stability of the living cell

2020 ◽  
Vol 117 (19) ◽  
pp. 10113-10121 ◽  
Author(s):  
Håkan Wennerström ◽  
Eloy Vallina Estrada ◽  
Jens Danielsson ◽  
Mikael Oliveberg
Keyword(s):  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Colloidal Stability ◽  
Surface Heterogeneity ◽  
Specific Protein ◽  
Cellular Systems ◽  
Dispersion Force ◽  
Protein Protein Interactions ◽  
Protein Surfaces ◽  
Dense Crowd

Cellular function is generally depicted at the level of functional pathways and detailed structural mechanisms, based on the identification of specific protein–protein interactions. For an individual protein searching for its partner, however, the perspective is quite different: The functional task is challenged by a dense crowd of nonpartners obstructing the way. Adding to the challenge, there is little information about how to navigate the search, since the encountered surrounding is composed of protein surfaces that are predominantly “nonconserved” or, at least, highly variable across organisms. In this study, we demonstrate from a colloidal standpoint that such a blindfolded intracellular search is indeed favored and has more fundamental impact on the cellular organization than previously anticipated. Basically, the unique polyion composition of cellular systems renders the electrostatic interactions different from those in physiological buffer, leading to a situation where the protein net-charge density balances the attractive dispersion force and surface heterogeneity at close range. Inspection of naturally occurring proteomes and in-cell NMR data show further that the “nonconserved” protein surfaces are by no means passive but chemically biased to varying degree of net-negative repulsion across organisms. Finally, this electrostatic control explains how protein crowding is spontaneously maintained at a constant level through the intracellular osmotic pressure and leads to the prediction that the “extreme” in halophilic adaptation is not the ionic-liquid conditions per se but the evolutionary barrier of crossing its physicochemical boundaries.

Cytochrome P-450 and NADPH-Cytochrome c (P-450) Reductase in Reconstituted Phospholipid Vesicles as Demonstrated by Negative Staining Techniques

1981 ◽  
Vol 39 ◽  
pp. 558-559
Author(s):  
Jane H. Dees ◽  
Linda K. Parkhill ◽  
L. Dean Coe ◽  
Betty Ann Germany ◽  
R. T. Okita ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Protein Interactions ◽  
Membrane Vesicles ◽  
Protein Protein Interactions ◽  
Ultrastructural Studies ◽  
Nadph Cytochrome ◽  
Hydrophobic Tail ◽  
Staining Techniques ◽  
Cytochrome P 450 ◽  
Structural Aspects

Artificial membrane vesicles or liposomes have become an accepted model for the study of enzyme-membrane interactions under controlled conditions.The development of reconstituted vesicle models for the microsomal mixed function oxidase enzymes has provided a system in which to study not only the biochemistry of these enzymes in a near physiologic milieu, but also structural aspects of the protein-phospholipid and protein-protein interactions. Ultrastructural studies on liposomes reconstituted with NADPH- cytochrome c (P-450) reductase (an amphipathic protein, 78,000 daltons, thought to be partially embedded in the microsomal membrane by its hydrophobic tail), and cytochrome P-450 (a hydrophobic protein, about 50,000 daltons, thought to be embedded within the lipid bilayer) either singly or together, should reveal information on whether these proteins are randomly arranged in the membrane or arranged in an ordered manner, an issue currently debated among cytochrome P-450 researchers.

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation

1995 ◽  
Vol 1252 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Janice L Theodorakis ◽  
Eric A.E Garber ◽  
John McCracken ◽  
Jack Peisach ◽  
Abel Schejter ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Chemical Modification ◽  
Heme Iron

scholarly journals Identification of β-Strand Mediated Protein-Protein Interaction Inhibitors Using Ligand-Directed Fragment Ligation

2020 ◽  
Author(s):  
Zsófia Hegedüs Zsófia Hegedüs ◽  
Fruzsina Hobor ◽  
Deborah K. Shoemark ◽  
sergio Celis ◽  
Lu-Yun Lian ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Significant Proportion ◽  
Selective Inhibition ◽  
Peptide Sequence ◽  
Protein Protein Interactions ◽  
X Ray Crystallography ◽  
Protein Protein Interaction ◽  
Ligand Discovery ◽  
Dynamics Simulations

β-Strand mediated protein-protein interactions (PPIs) represent underexploited targets for chemical probe development despite representing a significant proportion of known and therapeutically relevant PPI targets. β-strand mimicry is challenging given that both amino acid side-chains and backbone hydrogen-bonds are typically required for molecular recognition, yet these are oriented along perpendicular vectors. This paper describes an alternative approach using GKAP/SHANK1 PDZ as a model and dynamic ligation screening to identify small-molecule replacements for tranches of peptide sequence. A peptide truncation of GKAP functionalized at the N- and C-termini with acylhydrazone groups was used as an anchor. Reversible acylhydrazone bond exchange with a library of aldehyde fragments in the presence of the protein as template and <i>in situ</i> screening using a fluorescence anisotropy (FA) assay identified peptide hybrid hits with comparable affinity to the GKAP peptide binding sequence. Identified hits were validated using FA, ITC, NMR and X-ray crystallography to confirm selective inhibition of the target PDZ-mediated PPI and mode of binding. These analyses together with molecular dynamics simulations demonstrated the ligands make transient interactions with an unoccupied basic patch through electrostatic interactions, establishing proof-of-concept that this unbiased approach to ligand discovery represents a powerful addition to the armory of tools that can be used to identify PPI modulators.

Lysine methylation modulates the protein-protein interactions of yeast cytochrome C Cyc1p

PROTEOMICS ◽  
2015 ◽  
Vol 15 (13) ◽  
pp. 2166-2176 ◽  
Author(s):  
Daniel L. Winter ◽  
Dhanushi Abeygunawardena ◽  
Gene Hart-Smith ◽  
Melissa A. Erce ◽  
Marc R. Wilkins
Keyword(s):  
Cytochrome C ◽  
Protein Interactions ◽  
Lysine Methylation ◽  
Protein Protein Interactions

scholarly journals Regulation of COX Assembly and Function by Twin CX9C Proteins—Implications for Human Disease

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 197
Author(s):  
Stephanie Gladyck ◽  
Siddhesh Aras ◽  
Maik Hüttemann ◽  
Lawrence I. Grossman
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Atp Synthase ◽  
Protein Interactions ◽  
Human Disease ◽  
Electron Transport Chain ◽  
Intermembrane Space ◽  
Protein Protein Interactions ◽  
Inner Mitochondrial Membrane ◽  
Transport Chain

Oxidative phosphorylation is a tightly regulated process in mammals that takes place in and across the inner mitochondrial membrane and consists of the electron transport chain and ATP synthase. Complex IV, or cytochrome c oxidase (COX), is the terminal enzyme of the electron transport chain, responsible for accepting electrons from cytochrome c, pumping protons to contribute to the gradient utilized by ATP synthase to produce ATP, and reducing oxygen to water. As such, COX is tightly regulated through numerous mechanisms including protein–protein interactions. The twin CX9C family of proteins has recently been shown to be involved in COX regulation by assisting with complex assembly, biogenesis, and activity. The twin CX9C motif allows for the import of these proteins into the intermembrane space of the mitochondria using the redox import machinery of Mia40/CHCHD4. Studies have shown that knockdown of the proteins discussed in this review results in decreased or completely deficient aerobic respiration in experimental models ranging from yeast to human cells, as the proteins are conserved across species. This article highlights and discusses the importance of COX regulation by twin CX9C proteins in the mitochondria via COX assembly and control of its activity through protein–protein interactions, which is further modulated by cell signaling pathways. Interestingly, select members of the CX9C protein family, including MNRR1 and CHCHD10, show a novel feature in that they not only localize to the mitochondria but also to the nucleus, where they mediate oxygen- and stress-induced transcriptional regulation, opening a new view of mitochondrial-nuclear crosstalk and its involvement in human disease.

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