| 1. |
- Gräslund, Astrid, et al.
(författare)
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Testing membrane interactions of CPPs
- 2011
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Ingår i: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1940-6029. ; 683, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- The chapter deals with some biophysical methods used for investigating CPP-induced changes in membrane properties by spectroscopy methods such as fluorescence or NMR and methods used for probing CPP-induced leakage in membranes. Some useful model systems for biomembranes are described. These include large unilamellar phospholipid vesicles (LUVs) of well-defined size (diameter typically 100 nm). A protocol for the preparation of such vesicles is included. The leakage studies make use of LUVs with entrapped dye molecules. The NMR studies make use of mixed micelles (bicelles) as a membrane mimetic system, which can be oriented in the magnetic field of the spectrometer.
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| 2. |
- Mäler, Lena, et al.
(författare)
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NMR studies of three-dimensional structure and positioning of CPPs in membrane model systems
- 2011
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Ingår i: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1940-6029. ; 683, s. 57-67
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Tidskriftsartikel (refereegranskat)abstract
- CPPs are generally short cationic peptides that have the capability to interact directly with membranes. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes, it is of great importance to investigate CPPs with atomic resolution in a suitable membrane model. Nuclear magnetic resonance (NMR) is an excellent technique both for studying solution structures of peptides as well as for investigating their location within a model bilayer. This chapter outlines protocols for producing model membrane systems for NMR investigations as well as the basic NMR tools for determining the three-dimensional structure of CPPs and for investigating the details in lipid-peptide interactions, i.e., the localization of the CPP in the bilayer.
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| 3. |
- Vukojevic, Vladana, et al.
(författare)
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Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides
- 2011
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Ingår i: Neuropeptides. - Totowa, NJ : Humana Press. - 9781617793097 ; 789, s. 147-70
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Bokkapitel (refereegranskat)abstract
- Neuropeptide plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Trans location across the plasma membrane may represent a previously unknown mechanism by which neuropeptides can signal information to the cell interior. We introduce here two complementary optical methods with single-molecule sensitivity, fluorescence imaging with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), and demonstrate how they may be applied for the analysis of neuropeptide ability to penetrate into live cells in real time. APD imaging enables us to visualize fluorescently labeled neuropeptide molecules at very low, physiologically relevant concentrations, whereas FCS enables us to characterize quantitatively their concentration and diffusion properties in different cellular compartments. Application of these methodologies for the analysis of the endogenous opioid peptide dynorphin A (Dyn A), a ligand for the kappa-opioid receptor (KOP), demonstrated that this neuropeptide may translocate across the plasma membrane of living cells and enter the cellular interior without binding to its cognate receptor.
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