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Interaction of ?-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ¹?F NMR.

ABSTRACT: We show that fluorine NMR can be used to monitor the insertion and change in conformation of a ¹?F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. ?-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of ¹?F resonances of 3-fluoro-l-tyrosine labeled proteins. For ?-synuclein, a decrease in the resonance from position 39 was observed indicating that only the N-terminal third region of the protein interacts with plasma membrane. However, when the fusion construct was incubated with the cells, a decrease in the resonance from the fusion peptide region was noted with no change in the resonances from ?-synuclein region. Longer incubation, studied by using confocal fluorescence microscopy, revealed that the fusion construct translocates into the cells, but ?-synuclein alone did not cross the membrane in significant amounts.

SUBMITTER: Zigoneanu IG

PROVIDER: S-EPMC3319258 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Interaction of α-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ¹⁹F NMR.

Zigoneanu Imola G IG Pielak Gary J GJ

Molecular pharmaceutics 20120313 4

We show that fluorine NMR can be used to monitor the insertion and change in conformation of a ¹⁹F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. α-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of ¹⁹F resonances of 3-fluoro-l-tyrosine labeled proteins. For α-synuclein, a ...[more]

PMID: 22376087

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Project description:Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use (2)H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the ?-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. (2)H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct (2)H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ?40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ?80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.

Dataset Information

Interaction of ?-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ¹?F NMR.

Publications

Interaction of α-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ¹⁹F NMR.

Similar Datasets

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