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- Persson, Daniel, 1972, et al.
(författare)
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Vesicle size-dependent translocation of penetratin analogs across lipid membranes
- 2004
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1665:1-2, s. 142-155
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Tidskriftsartikel (refereegranskat)abstract
- The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles ( > 1 μm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration. © 2004 Elsevier B.V. All rights reserved.
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| 4. |
- Thoren, Per, 1972, et al.
(författare)
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Membrane binding and translocation of cell-penetrating peptides
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 43:12, s. 3471-3489
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R 7 W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of ∼10-11 Å from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.
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- Ardhammar, Malin, 1970, et al.
(författare)
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Absolute configuration and electronic state properties of light-switch complex [Ru(phen)2dppz]2+ deduced from oriented circular dichroism in a lamellar liquid crystal host
- 2002
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Ingår i: Chemical Physics Letters. - 0009-2614. ; 354:1-2, s. 44-50
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Tidskriftsartikel (refereegranskat)abstract
- Circular dichroism (CD) of enantiomers of [Ru(phen)(2)dppz](2+) oriented in a octanoate-decanol-water lamellar liquid crystal has been measured parallel to the orientation axis. where the sample does not exhibit linear dichroism (LD). At an inclined incidence, the emerging LD shows that the chromophores have an along-chain orientation in the liquid crystal. The changes in the CD spectrum compared to an isotropic sample, in conjunction with CD calculations, allow us to assess the absolute configuration of the enantiomers and assign the CD bands to specific electronic transitions.
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| 6. |
- Coates, C. G., et al.
(författare)
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Picosecond time-resolved resonance Raman probing of the light-switch states of Ru(Phen)(2)dppz (2+)
- 2001
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 105:50, s. 12653-12664
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Tidskriftsartikel (refereegranskat)abstract
- Picosecond time-resolved resonance Raman (picosecond-TR3) spectroscopy has been used to conduct an extensive photophysical characterization of the light- switch complex [Ru(phen)(2)dppz](2+) as a function of environment, in which studies have been carried out in aqueous and nonaqueous media and in DNA. The results are considered in rotation to a previous report describing environment-sensitive lowest triplet MLCT states. Vibrational marker features and enhancement patterns were used to determine the rapid progression (< 20 ps) between two triplet MLCT states in aqueous environment, followed by subnanosecond, nonradiative deactivation to the ground state. In nonaqueous environment, the long-lived, emissive triplet MLCT state is spectrally identified as the short-lived first triplet MLCT state observed in water, in agreement with the earlier proposed mechanism. The present data are shown to correlate well with previous nanosecond RR findings for the complex in each environment. Interestingly, a precursor state has been identified upon excitation in both nonaqueous solvent and in DNA, which precedes the triplet MLCT state, and the lifetime of which appears to be environment dependent. Observation of this state is discussed in relation to other recent femtosecond spectroscopic studies on this complex.
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| 7. |
- Engman, Cecilia, 1974, et al.
(författare)
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DNA adopts normal B-form upon incorporation of highly fluorescent DNA base analogue tC: NMR structure and UV-Vis spectroscopy characterization
- 2004
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 32:17, s. 5087-95
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Tidskriftsartikel (refereegranskat)abstract
- The influence of the highly fluorescent tricyclic cytosine base analogue (tC) on duplex DNA conformation is investigated. The duplex properties are characterized by absorbance and circular dichroism (CD) for all combinations of neighbouring bases to tC, and an NMR structure is determined for one tC-containing sequence. For the oligonucleotides with one tC incorporated instead of cytosine, the melting temperature is increased on average by 2.7 degrees C above that for the unmodified ones. CD spectra are practically identical for modified and unmodified sequences, indicating an unperturbed B-DNA conformation. The NMR structure determination of the self-complementary sequence 5'-CTC(tC)ACGTGGAG shows a DNA conformation consistent with B-form for the whole duplex. The root-mean-square distance for the nucleotides of the eight central base pairs between the 10 structures with lowest CYANA target functions and a mean structure is 0.45 +/- 0.17 A. The NMR data confirm correct base pairing for tC by the observation of both intrastrand and interstrand imino proton NOEs. Altogether, this suggests that tC works well as a cytosine analogue, i.e. it is situated in the base stack, forming hydrogen bonds with G in the complementary strand, without distorting the DNA backbone conformation. This first example of an artificial, highly fluorescent DNA base that does not perturb the DNA conformation could have valuable applications for the study of the structure and dynamics of nucleic acid systems.
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| 8. |
- Gisselfält, Katrin, 1969, et al.
(författare)
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Interactions of tris(phenanthroline)ruthenium(II) enantiomers with DNA: Effects on helix flexibility studied by the electrophoretic behavior of reptating DNA in agarose gel
- 2000
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Ingår i: Journal of Physical Chemistry B Materials. - : American Chemical Society (ACS). - 1089-5647 .- 1520-6106 .- 1520-5207. ; 104:15, s. 3651-3659
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Tidskriftsartikel (refereegranskat)abstract
- A combination of measurement of mobility and orientational dynamics of long reptating DNA in agarose gel has been used to reveal how the binding of Delta and Lambda enantiomers of the tris(phenanthroline)ruthenium(II) ion affects the flexibility of the DNA helix. The mobility data, and data over the step length and period time of the reptation cycle, obtained in the presence of the respective enantiomer, are compared with those of free DNA and with data obtained earlier on DNAs with known variations in helix flexibility. The results suggest that the Delta form induces kinks in the DNA helix while the Lambda form gives rise to a local stiffening of the helix.
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| 10. |
- Nordén, Bengt, 1945, et al.
(författare)
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Ambivalent Intercalators for DNA: L-Shaped Platinum(II) Complexes
- 2004
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Ingår i: Inorganic Chemistry. - : American Chemical Society (ACS). - 0020-1669 .- 1520-510X. ; 43:7, s. 2416-2421
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Tidskriftsartikel (refereegranskat)abstract
- Novel platinum(II) square planar coordination complexes, in which two heteroaromatic ligands are held by the metal in an unusual L-shaped geometry orthogonal to each other, have been synthesized, and their interaction with DNA was investigated with absorption and linear dichroism spectroscopy. As a rule, the ligand that is coplanar with the coordination square of Pt is found to be oriented perpendicular relative to the DNA helix axis when bound, suggestive of its intercalation between the base pairs of DNA. However, when this coplanar ligand is replaced by two pyridines, the opposite ligand, orthogonal to the coordination square, is instead preferentially intercalated. This behavior shows that these new complexes do indeed show some properties of true ambintercalators, i.e., compounds that can bind by intercalation of either of two distinct aromatic moieties.
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