A Study of the Influence of the Hydrophobic Core Residues of Yeast Iso-2-cytochrome c on Phosphate Binding: A Probe of the Hydrophobic Core-Surface Charge Interactions

Taniuchi, Hiroshi; Shi, Ying; Miguel, Gloria I. San; Ferretti, James A.; Mack, James W.; Fisher, Alice; Shah, Rashed; Schechter, Alan N.; Shiloach, Joseph

doi:10.1023/a:1010906929793

Cited by 4 publications

(5 citation statements)

References 78 publications

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“…In the case of equine and bovine cyt c , binding of phosphate or chloride anions to the protein has been reported to lower the heme iron reduction potential, , with a shift of ≈6 mV seen for 0.1 M chloride relative to 0.1 M acetate. Although yeast cyt c has also been found to interact with phosphate, − our observations suggest that the influence of anion binding on potential is much smaller for WT* than for mammalian cyt c . Further, it appears that the interaction of Lys79 with HP6 in WT* is not in competition with specific anion binding.…”

Section: Resultsmentioning

confidence: 67%

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochromec

et al. 2018

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The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (pKa) of the protein. However, the reduction potential of the heme iron decreases; further, the pKH of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively-charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c.

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Section: Resultsmentioning

confidence: 67%

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochromec

et al. 2018

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“…The characterization of the dependence of protein stability on pH and salt concentration indicates that the core substitutions at residue 30 do not appear to lead to significant modifications of surface electrostatic interactions, and changes in stability can be attributed to modifications of the core packing and/or hydrophobicity. This is not always the case, and in some proteins substitutions of core aliphatic residues have been shown to induce changes in surface electrostatics ( , ).…”

Section: Discussionmentioning

confidence: 99%

Thermodynamics of Core Hydrophobicity and Packing in the Hyperthermophile Proteins Sac7d and Sso7d

2004

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The influence of core hydrophobicity and packing on the structure and stability of the hyperthermophile proteins Sac7d and Sso7d have been studied by calorimetry, circular dichroism, and NMR. Valine 30 is positioned in Sac7d to allow a cavity-filling Val --> Ile substitution which occurs naturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has been characterized and compared to the reciprocal Ile --> Val mutation in Sso7d. A detailed analysis of the stability of the proteins was obtained by globally fitting the variation of DSC parameters and circular dichroism intensities as a function of temperature (0-100 degrees C), salt (0-0.3 M), and pH (0-8). A global analysis over such a range of conditions permitted an unusually precise measure of the thermodynamic parameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from the linked effects of protonation and chloride binding associated with acid-induced folding. The results indicate differences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysis of DSC data alone using conventional methods. The sign and magnitude of the changes in DeltaG, DeltaH, TDeltaS, and DeltaC(P) of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent with differences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions. The benefit of increased hydrophobicity of the core increased with temperature, with maximal effect around 116 degrees C. Increased hydrophobicity of the core achieved not only an increase in the free energy of unfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.

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“…The residues found in the major hydrophobic cores of staphylococcal nuclease and its homologues are summarized in Table 2, which include the residues equivalent to the wildtype staphylococcal nuclease positions 14,17,23,25,34,36,39,62,65,66,72,74,92, 99, and 103. These 15 residues represent a broad definition of the major hydrophobic core in nuclease.…”

Section: Occurrence Of Various Amino Acid Types In Thementioning

confidence: 99%

“…For other sets of residues, these interactions can include other factors; for example, the interaction of two charged residues would certainly include electrostatic interactions. A further caution is necessary since it has recently been shown by the elegant work of Taniuchi et al (25) that alterations in the core of yeast iso-2-cytochrome c altered stability in part by altering interactions between charged residues on the surface of the protein. Nevertheless, it seems reasonable to make the assumption to a first approximation that among these buried aliphatic residues the principal interaction is packing, which is to say optimizing van der Waals interactions and minimizing cavities.…”

Section: Correlation Of Consensus Sequence With Favorable Interaction...mentioning

confidence: 99%

“…Staphylococcal Nuclease Mutants with the Consensus Core Sequences. Positions 23,25,72, and 92 in staphylococcal nuclease were all substituted simultaneously with the side chains found in the consensus core sequence (23I/25V/72V/ 92V). Not every possible variant of this set of four residues was constructed as we have detailed elsewhere (10,11) for other residue sets, but 11 variants were made.…”

Section: Correlation Of Consensus Sequence With Favorable Interaction...mentioning

confidence: 99%

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Packing Is a Key Selection Factor in the Evolution of Protein Hydrophobic Cores

Chen

Stites

2001

Biochemistry

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The energy derived from optimized van der Waals interactions in closely packed, folded proteins has been proposed to be of similar energetic magnitude to hydrophobicity in stabilizing the native state. If packing is this energetically important, it should influence the evolution of protein core sequences. To test this hypothesis, the occurrence of various amino acid side chains in the major hydrophobic core of staphylococcal nuclease and 42 homologous proteins was determined. Most such positions in this protein family are usually isoleucine, leucine, or valine. Previously we have constructed and measured the stabilities of 12 single, 44 double, 64 triple, and 32 quadruple mutants, representing all possible permutations of these three side chains at two overlapping sets of four positions in the core of staphylococcal nuclease. The stabilities and interaction energies of those mutants with various combinations of the most common, or consensus, sequence were compared to the stabilities of all other mutants. Mutants which had the consensus side-chain combinations were not necessarily the most stable, but usually were found to have the best interaction energies. In other words, these proteins were far more stable than would be predicted from simply summing the observed energetic effects of the component single mutations, apparently reflecting particularly favorable packing interactions that are possible for the most common side chains. An additional 12 mutants which tested possible alternate explanations of the results were constructed. The stabilities and interaction energies of these mutants also support the conclusion that packing is a crucial determinant guiding the sequence evolution of protein cores.

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A Study of the Influence of the Hydrophobic Core Residues of Yeast Iso-2-cytochrome c on Phosphate Binding: A Probe of the Hydrophobic Core-Surface Charge Interactions

Cited by 4 publications

References 78 publications

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochromec

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochromec

Thermodynamics of Core Hydrophobicity and Packing in the Hyperthermophile Proteins Sac7d and Sso7d

Packing Is a Key Selection Factor in the Evolution of Protein Hydrophobic Cores

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