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Biophysical Studies of Cell-Penetrating Peptides 231 intrinsic binding at the membrane is usually described by an adsorption (mass action) isotherm (e.g., Langmuir) reflecting how the bound molecule (peptide) will become positioned at a site, until the surface becomes fully occupied (saturated). 10.5 BIOPHYSICAL METHODS TO STUDY PEPTIDE–MEMBRANE INTERACTIONS Biomembranes are amphiphilic molecular assemblies able to stabilize peptide conformations upon interaction. When free, short peptides, as distinguished from globular proteins, do not normally have a more hydrophilic “outside” and a more hydrophobic “inside.” Depending on the nature of the residues involved, a peptide will expose both hydrophobic and hydrophilic side chains to the surrounding medium. Peptides with low solubility may self-aggregate and form oligomers. Various spectroscopic methods are used to study the interactions between a peptide and a mixed solvent. Among problems studied are, e.g., distribution of the peptide between the aqueous and amphiphilic phase, location and mobility of the peptide relative to the surface of the membrane mimetic phase, and induction of secondary structure in the peptide by the solvent. These studies are relevant for various other classes of peptides, such as hormones and antibiotic peptides. Other studies concern changes of the membrane induced by the peptide. Such changes range from complete disassembly (lysis) to pore formation or more subtle effects, which may be relevant for the group of peptides under consideration here. The most used spectroscopic techniques are briefly summarized below. 10.5.1 FLUORESCENCE The intrinsic fluorescence of tryptophan (or possibly tyrosine) residue may be investigated in order to determine the polarity of its environment. 29 The tryptophan emission peak wavelength is sensitive to polarity and shifts from ca. 356 nm in an aqueous phase to about 320 nm in a hydrophobic phase. In parallel, the quantum yield becomes enhanced in the hydrophobic phase. Artificial fluorescence probes can be attached to the peptide, cargo, or membrane and may report on interactions or change of state of its host. As a cargo, the green fluorescence protein (GFP) may be used as a natural test molecule. The topology of the peptide and membrane system may be monitored by experiments using quenchers or detecting fluorescence resonance energy transfer (FRET) between two fluorophores (donor–acceptor). Polarization of fluorescence can be used to observe order and dynamics (“fluidity”) of the membrane and perturbations caused by the peptide. Relatively low concentrations and volumes are needed for spectroscopic fluorescence studies: typically a few hundred µl of 1 µ M fluorophore, i.e., about 1 nmol, is enough to give a good fluorescence signal from tryptophan, as well as from any suitable external fluorescence probe. With fluorescence microscopy, in principle, single fluorescent molecules may be monitored by advanced techniques. 31
232 Cell-Penetrating Peptides: Processes and Applications 10.5.2 CIRCULAR DICHROISM Circular dichroism (CD) spectroscopy detects the chirality of molecules as the small difference in absorbtivity for right- and left-hand circularly polarized light. Due to the chiral nature of the peptide bond and the different dihedral angles associated with different types of secondary structure, CD spectra report on secondary structures present in the amino acid chain. The spectral range for this type of peptide or protein study is usually 190 to 300 nm. The use of the CD method is mainly empirical; for analysis of the CD spectra of a protein or a peptide, one compares with standard spectra from proteins with well-known three-dimensional structures. 32 It is possible to estimate percentages of typically five-component spectra: α-helix, antiparallel and parallel β-sheets, β-turn, and random coil. For globular proteins of the same kind used to create the standard set of spectra, this is a reliable evaluation. For short peptides, the results of such an analysis of a new CD spectrum are less clear-cut. However, they may be used as a first estimation of structures present and, particularly, to show directions of change under different conditions. The presence of a clear isodichroic point indicates a two-state situation. CD spectra have similar requirements as to amounts of sample as fluorescence spectroscopy. Light scattering is a severe problem in the region around or below 200 nm when working with concentrated membrane samples or large particles. Oriented CD spectra from a peptide can be measured by use of aligned multibilayers deposited on a quartz plate. 33 10.5.3 FOURIER TRANSFORM INFRARED SPECTROSCOPY Fourier transform infrared (IR) spectroscopy (FTIR) is a vibrational spectroscopy employing an interference technique. Similar to CD, IR spectra carry information about the presence of secondary structures in a peptide under various conditions. 34 The peptide bond has typical amide bands I and II in the IR region from 1680 to 1430 cm –1 , which can be best observed in a D 2O solvent. From these bands it is possible to evaluate secondary structures, which usually requires spectral deconvolution. FTIR sample requirements are typically small amounts of a very concentrated solution (few µl with
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Transportans 69 and still retain ef
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Model Amphipathic Peptides 73 its D
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Model Amphipathic Peptides 75 pmol/
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TABLE 4.1 Internalization of Peptid
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TABLE 4.2 Internalization of Peptid
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Model Amphipathic Peptides 81 relat
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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116 Cell-Penetrating Peptides: Proc
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Interactions of Cell-Penetrating Pe
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Page 202 and 203: Interactions of Cell-Penetrating Pe
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Page 210 and 211: Structure Prediction of CPPs and It
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Kinetics of Uptake of Cell-Penetrat
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Cell-Penetrating Peptide Conjugatio
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FIGURE 15.9 (Color Figure 15.9 foll
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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