Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

Jordi, W.; Kruijff, Ben de; Marsh, Derek

doi:10.1021/bi00449a007

Cited by 26 publications

(19 citation statements)

References 29 publications

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“…The final level of FPE fluorescence (at 10 s; Figure 5) suggests that only a fraction of the polypeptide inserts into the hydrophobic core of the micelle. This interpretation is consistent with previous studies by Jordi et al (10,36), in which it was shown that the N-and C-terminal helices insert into lipid membranes. Also, a recent study by polarized attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) of apocyt c in lipid membranes (Bryson et al, unpublished results) revealed that the helical domains of apocyt c in lipid membranes have a net orientation perpendicular to the membrane, which is compatible with a partial lipid-inserted conformation of apocyt c. It is important to note that the insertion events monitored by the FPE probe (kinetic phases of decreasing fluorescence) are typically associated with a movement of the polypeptide from the membrane surface to a deeper location in the lipid membrane, which may or may not coincide with a transmembrane topology of the inserted R-helix (29)(30)(31)(32).…”

Section: Discussionsupporting

confidence: 94%

Folding of Apocytochrome c in Lipid Micelles: Formation of α-Helix Precedes Membrane Insertion

et al. 1999

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Apocytochrome c, which in aqueous solution is largely unstructured, acquires a highly alpha-helical structure upon interaction with lipid. The alpha-helix content induced in apocytochrome c depends on the lipid system, and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The folding kinetic mechanism of apocytochrome c induced by zwitterionic micelles of lysophosphatidylcholine (L-PC), predominantly driven by hydrophobic lipid-protein interactions, was investigated by fluorescence stopped-flow measurements of Trp 59 and fluorescein-phosphatidylethanolamine-(FPE) labeled micelles, in combination with stopped-flow far-UV circular dichroism. It was found that formation of the alpha-helical structure of apocytochrome c precedes membrane insertion. The unfolded state in solution (U(W)) binds to the micelle surface in a helical conformation (I(S)) and is followed by insertion into the lipid micelle, i.e., formation of the final helical state H(L). Binding of apocytochrome c to the lipid micelle (U(W) --> I(S)) is concurrent with formation of a large fraction (75-100%, depending on lipid concentration) of the alpha-helical structure of the final lipid-inserted state H(L). The highly helical intermediate I(S) is formed on the time scale of 3-12 ms, depending on lipid concentration, and inserts into the lipid micelle (I(S) --> H(L)) in the time range of approximately 200 ms to >1 s, depending on lipid-to-protein ratio. The final lipid-inserted helical state H(L) in L-PC micelles has an alpha-helix content approximately 65% of that of cytochrome c in solution and has no compact stable tertiary structure as revealed by circular dichroism results.

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Section: Discussionsupporting

confidence: 94%

Folding of Apocytochrome c in Lipid Micelles: Formation of α-Helix Precedes Membrane Insertion

et al. 1999

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“…The observed importance of the N-terminal region of apocytochrome c with respect to its association with mitochondria, as compared to the relative insignificance of the C-terminal region, correlates well with the observations ofDe Kruijff and collaborators (16)(17)(18) on the insertion of apocytochrome c into artificial lipid vesicles, even though the preliminary evidence (8) seems to indicate that for the system employed here the portal for transmembrane passage of the apocytochrome c is likely to be a pore.…”

Section: Discussionsupporting

confidence: 89%

“…By contrast, it is known that apocytochrome c can penetrate into artificial lipid vesicles (13)(14)(15) and N-terminal segments containing 38-65 residues are translocated nearly as well as the full-length apoprotein, whereas C-terminal fragments of 65-23 residues penetrate into, but not through, the lipid bilayer (16)(17)(18).…”

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confidence: 99%

Amino acid sequence requirements for the association of apocytochrome c with mitochondria.

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Hakvoort

Koshy

et al. 1990

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

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To examine the amino acid sequence requirements for the biphasic association of Drosophila melanogaster apocytochrome c with mouse liver mitochondria in vitro, recombinant constructs of the protein were prepared. Removal of the C-terminal sequence to residue 58 had little influence, but truncation to residue 50 decreased the association to low levels and removal to residue 36 was even more effective. However, a mutant missing the segment between residues 35 and 66 was fully functional, but, when the C-terminal segment from residue 36 was replaced with a noncytochrome c sequence, the high-affinity phase of the association was lost. A mutant in which residues 90, 91, 92, 96, and 100 were replaced by lysine, leucine, proline, proline, and proline, respectively, to prevent the possible formation of the C-terminal a-helix and another mutant in which the C-terminal segment from residue 90 to residue 120 was a noncytochrome c sequence had normal association. In contrast, replacing lysine-5, -7, and -8 by glutamine, glutamic acid, and asparagne, respectively, resulted in loss of the high-affinity phase. The same mutations in the apoprotein lacking the segment between residues 35 and 66 caused, in addition, a decrease of the low-afinty phase association. Thus, the N-terminal region is most critical for apocytochrome c association, but alternative segments of the central and/or C-terminal region can be utilized, where noncytochrome c sequences are ineffective. These results emphasize the wide disparity between the structural requirements for association with mitochondria and for the production of a functional holoprotein.

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“…Experiments with various fragments of apocytochrome c have localized the major site of penetration to the amino-terminal region [37]. Such results are consistent with the precursor first inserting into the lipid regions of the outer mitochondrial membrane, prior to interaction of the Nterminal portion of the protein with the internallyfacing haem lyase.…”

Section: Protein Penetrationsupporting

confidence: 61%

Lipid‐protein interactions in membranes

Marsh

1990

FEBS Letters

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The interactions of lipids with integral and peripheral proteins can be studied in reconstituted and natural membranes using spin label electron spin resonance (ESR) spectroscopy.The ESR spectra reveal a reduction in mobility of the spin-labelled lipid chains on binding of peripheral proteins to negatively-charged lipid bilayers. A selectivity of interaction has been established for different lipid species. and in certain cases evidence is obtained for a partial penetration of the peripheral proteins into the membrane. The latter may be relevant to the import mechanism of apocytochrome c into mitochondria.Integral proteins induce a more direct motional restriction of the spin-labelled lipid chains, allowing the stoichiometry and specificity of the interaction, and the lipid exchange rate at the protein interface to be determined from the ESR spectra. In this way, a population of very slowly exchanging cardiohpin associated with the mitochondrial ADP-ATP carrier has been identified. The residues involved in the specificity for charged lipids of the myelin proteolipid protein have been localized to the deletion in the DM-20 mutant, and the difference in lipid-protein interactions with the .&sheet and x-helical conformations of the M-13 coat protein, has been characterized.

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Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

Cited by 26 publications

References 29 publications

Folding of Apocytochrome c in Lipid Micelles: Formation of α-Helix Precedes Membrane Insertion

Folding of Apocytochrome c in Lipid Micelles: Formation of α-Helix Precedes Membrane Insertion

Amino acid sequence requirements for the association of apocytochrome c with mitochondria.

Lipid‐protein interactions in membranes

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