Semisynthesis of horse heart cytochrome c analogues from two or three fragments.

Kortenaar, Paul B. W. Ten; Adams, P. J.; Tesser, G. I.

doi:10.1073/pnas.82.24.8279

Cited by 13 publications

(2 citation statements)

References 14 publications

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“…S2) that has been suggested to play a role in closing up the heme crevice in cyt c (52-54). Previous modifications to Val, Asn, and α-aminobutyric acid at this site have destabilized the protein and altered the heme reduction potential (52,55). However, introduction of a Cys for ligand interactions with the heme instead increases the stability of ferric variants compared with the wild-type WT* protein (Table 2).…”

Section: Discussionmentioning

confidence: 99%

Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins

Zhong

Lisi

Collins

et al. 2014

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

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Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys-heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.metalloenzyme | folding | electron transfer

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Section: Discussionmentioning

confidence: 99%

Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins

Zhong

Lisi

Collins

et al. 2014

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

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show abstract

“…Some in the latter category must be essential not only for the functional properties of cytochrome c studied in isolation but also for its essential interactions with other components of the respiratory chain such as its redox partner, cytochrome c peroxidase. The 'fast flipping' Phe-82 of cytochromes c, a residue near the surface and some 0.5 nm from the haem, has been replaced in the yeast iso-I protein by Ser, Gly or Tyr by oligonucleotide mismatch mutagenesis (Pielak et al, 1985) and by Leu by using chemical semisynthesis (ten Kortenaar et al, 1985). All of the protein variants are stable and the reduction potentials of the Tyr-82 and Leu-82 cytochromes are those of the wild-type.…”

Section: Al-antitrypsinmentioning

confidence: 99%

Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw

1987

Biochemical Journal

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Sequence simplification and the intra‐ and intermolecular self‐recognition properties of vasopressin/neurophysin biosynthetic precursor

Fassina

Chaiken

1989

J of Molecular Recognition

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The self-assembly properties of the arginine 8-vasopressin/bovine neurophysin II (AVP/BNPII) biosynthetic precursor were studied using glycopeptide-deleted and sequence-redesigned semisynthetic derivatives. Semisynthetic precursors were prepared by chemically coupling synthetic vasopressinyl sequence domains and native protein-derived neurophysin II domain. Measurement of precursor-protein association by the extent of affinity chromatographic retardation on agarose-immobilized BNPII verified that the semisynthetic precursor with native AVP sequence has an enhanced self-association propensity similar to that predicted for native precursor. Here, the stabilizing contacts between hormone and neurophysin domains, mainly the positively charged protonated alpha-amino group and tyrosyl 2 side chain of the hormone, are retained. Semisynthetic precursor variants in which the hormone domain is sequence-simplified by introducing alanyl residues in positions not considered important for neurophysin recognition show non-reduced association to BNPII. In contrast, removal of one of the main contact elements between hormone and neurophysin by acetylation of the hormone alpha-amino group abolishes potentiation of precursor self-association. The results show that the presence of the C-terminal glycopeptide sequence domain of native vasopressin precursor is not required to promote self-assembly of the precursor. The data verify the view proposed for the oxytocinyl precursor that intramolecular domain interaction is the triggering event which promotes the increase in affinity of precursor self-association (intermolecular self-recognition). The data also define some of the intramolecular self-recognition elements in the folded precursor required for the high affinity intermolecular self-recognition.

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Semisynthesis of horse heart cytochrome c analogues from two or three fragments.

Abstract: Horse heart cytochrome c was cleaved with cyanogen bromide.

Cited by 13 publications

References 14 publications

Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins

Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins

Protein engineering. The design, synthesis and characterization of factitious proteins

Sequence simplification and the intra‐ and intermolecular self‐recognition properties of vasopressin/neurophysin biosynthetic precursor

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