Andrew H. A. Clayton

Spectral Properties of Fluorescein in Solvent‐Water Mixtures: Applications as a Probe of Hydrogen Bonding Environments in Biological Systems

Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by Martin (Chem. Phys. Lett. 35, 105–111, 1975), that the absorption maximum of fluorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this work by studying the spectral properties of the dianion form of the probe in solvent–water mixtures. We show that the magnitude of the shift correlates with the α and β parameters of Kamlet and Taft (J. Am. Chem. Soc. 98, 377–383; 2886–2894, 1976), which provide a scale of the hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent–water mixtures, these shifts reflect general effects of the solvents on the hydrogen bonding environment of the fluorescein through water–solvent hydrogen bonding and specific effects due to fluorescein–solvent hydrogen bonding. Indeed, both the absorption and fluorescence properties appear to be dominated by these effects indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein–antifluorescein antibody complex where it appears that antibodies selected during the immune response possess binding sites that are increasingly dehydrated and hydrophobic.

Tryptophan Rotamer Distributions in Amphipathic Peptides at a Lipid Surface

The fluorescence decay of tryptophan is a sensitive indicator of its local environment within a peptide or protein. We describe the use of frequency domain fluorescence spectroscopy to determine the conformational and environmental changes associated with the interaction of single tryptophan amphipathic peptides with a phospholipid surface. The five 18-residue peptides studied are based on a class A amphipathic peptide known to associate with lipid bilayers. The peptides contain a single tryptophan located at positions 2, 3, 7, 12, or 14 in the sequence. In aqueous solution, the peptides are unstructured and a triple-exponential function is required to fit the decay data. Association of the peptides with small unilamellar vesicles composed of egg phosphatidylcholine reduces the complexity of the fluorescence decays to a double exponential function, with a reduced dependence of the preexponential amplitude on peptide sequence. The data are interpreted in terms of a rotamer model in which the modality and relative proportions of the lifetime components are related to the population distribution of tryptophan chi1 rotamers about the Calpha-Cbeta bond. Peptide secondary structure and the disposition of the tryptophan residue relative to the lipid and aqueous phases in the peptide-lipid complex affect the local environment of tryptophan and influence the distribution of side-chain rotamers. The results show that measurement of the temporal decay of tryptophan emission provides a useful adjunct to other biophysical techniques for investigating peptide-lipid and protein-membrane interactions.

On the rate of radiationless intermolecular energy transfer

The effects of higher multipole interactions (up to electric quadrupole–quadrupole) and small exchange contributions on the rate of intermolecular energy transfer are examined. Second‐order Coulombic interactions are described within a molecular quantum electrodynamical framework. A correction due to a small first‐order exchange mechanism is then proposed. It is concluded that use of the multipole expansion in the interaction Hamiltonian is not always a good approximation at interchromophore separations of less than about 10 A. This is attributed to a combination of large molecular dimensions compared to intermolecular separation, and wave function overlap effects. For larger separations, the interaction is described well by the usual dipolar coupling formalism. The inclusion of small exchange effects in a simplistic model at small to intermediate separations demonstrates the likelihood of a substantially greater rate of energy transfer than that predicted by either a Forster‐type (dipole–dipole) or a pur...

The structure and orientation of class-A amphipathic peptides on a phospholipid bilayer surface.

Abstract The amphipathic α-helix is a recognised structural motif that is shared by membrane-associating proteins and peptides of diverse function. The aim of this paper is to determine the orientation of an α-helical amphipathic peptide on the bilayer surface. We use five amphipathic 18-residue peptide analogues of a class A amphipathic peptide that is known to associate with a bilayer surface. Tyrosine and tryptophan are used as spectroscopic probes to sense local environments in the peptide in solution and when bound to the surface of unilamellar phosphatidylcholine vesicles. In a series of peptides, tryptophan is moved progressively along the sequence from the nonpolar face (positions 3, 7, 4) to the polar face of the peptide (positions 2, 12). The local environment of the tryptophan residue at each position is determined using fluorescence spectroscopy employing quantum yield, and the wavelength of the emission maximum as indicators of micropolarity. The exposure of the tryptophan residues at each site is assessed by acrylamide quenching. On association with vesicles, the tryptophan residues at positions 3, 7 and 14 are in nonpolar water-shielded environments, and the tryptophan at position 12 is in an exposed polar environment. The tryptophan at position 2, which is located near the bilayer-water interface, exhibits intermediate behaviour. Analysis of the second-derivative absorption spectrum confirmed that the tyrosine residue at position 7 is in a nonpolar water-shielded environment in the peptide-lipid complex. We conclude that these class A amphipathic peptides lie parallel to the lipid surface and penetrate no deeper than the ester linkages of the phospholipids.

Site-Specific Tryptophan Dynamics in Class A Amphipathic Helical Peptides at a Phospholipid Bilayer Interface

The amphipathic helix plays a key role in many membrane-associating peptides and proteins. The dynamics of helices on membrane surfaces might be of importance to their function. The fluorescence anisotropy decay of tryptophan is a sensitive indicator of local, segmental, and global dynamics within a peptide or protein. We describe the use of frequency domain dynamic depolarization measurements to determine the site-specific tryptophan dynamics of single tryptophan amphipathic peptides bound to a phospholipid surface. The five 18-residue peptides studied are based on a class A amphipathic peptide that is known to associate at the interface of phospholipid bilayers. The peptides contain a single tryptophan located at positions 2, 3, 7, 12, or 14 in the sequence. Association of the peptides with egg phosphatidylcholine vesicles results in complex behavior of both the tryptophan intensity decay and the anisotropy decay. The anisotropy decays were biphasic and were fitted to an associated model where each lifetime component in the intensity decay is associated with a particular rotational correlation time from the anisotropy decay. In contrast, an unassociated model where all components of the intensity decay share common rotational modes was unable to provide an adequate fit to the data. Two correlation times were resolved from the associated analysis: one whose contribution to the anisotropy decay was dependent on the exposure of the tryptophan to the aqueous phase, and the other whose contribution reflected the position of the tryptophan in the sequence. The results are compared with existing x-ray structural data and molecular dynamics simulations of membrane-incorporated peptides.

Oriented circular dichroism of a class A amphipathic helix in aligned phospholipid multilayers.

The effect of lipid phase state on the orientation and conformation of a class A alpha-helical peptide on aligned lipid multilayers was examined using oriented circular dichroism spectroscopy. A comparison of oriented spectra in aligned peptide-lipid multilayers with CD spectra of unaligned peptide lipid vesicle complexes is consistent with a preferential alignment of helices parallel to the membrane surface at temperatures above and below the main acyl-chain melting transition temperature of the phospholipid. Changes are observed in the oriented CD spectra with lipid phase state which are attributed to a subtle conformational change of the peptide on the lipid surface. The results are compared with available experimental data on membrane-active lytic and antimicrobial helical peptides.

Photoinduced electron transfer in rigidly linked dimethoxynapthalene-N-methylpyridinium donor-acceptor molecules

Abstract Photoinduced electron transfer (ET) is studied in a series of novel molecules containing a dimethoxynaphthalene (DMN) donor and either a pyridine (P) or N-methylpyridinium (P-Me + ) acceptor covalently linked via a rigid nonbornalogous bridge ( n sigma bonds in length). ET rates of the order of 10 10 s −1 were measured for the DMN- n -P-Me + series ( n = 4, 6), while no appreciable ET was observed for the DMN- n -P compounds. Electronic and nuclear factors are discussed and the results rationalized in terms of Marcus—Hush and non-adiabatic ET theories.

Electron and singlet energy transfer in rigid supramolecular systems

Abstract Fluorescence spectroscopic measurements are reported on the N,N-dimethylaniline-{polynorbornyl (4, σ-bonds)}-dimethoxynaphthalene (DMA[4]DMN[2]) dyad and the H-isomer of the trichromophore, N,N-dimethylaniline-{polynorbornyl (4, σ-bonds)}-dimethoxynaphthalene-{polynorbornyl (8, σ-bonds)}-dicyanovinyl (DMA[4]DMN[8]-DCV) to probe electron transfer (ET) and singlet energy transfer (EnT) in these novel systems. In acetonitrile, the DMA[4]DMN[2] dyad is shown to undergo rapid and complete photoinduced ET following excitation of DMA and DMN chromophores. In n-hexane, very little ET is apparent for DMA[4]DMN[2] and this allows the observation of a very efficient singlet EnT process from locally excited DMA to the lowest DMN singlet excited state. A mode of vectorial excitation EnT from locally excited DMA to DMN followed by ET from DMN to DCV is observed for DMA[4]DMN[8]DCV in n-hexane.