Chapter 21
Flow Linear Dichroism of Protein–Membrane Systems
Matthew R. Hicks, Sarah R. Dennison, Adewale Olamoyesan,
and Alison Rodger
Abstract
Linear dichroism (LD) is the differential absorbance of light polarized parallel and perpendicular to an
orientation direction. Any oriented sample will show a signal in its electronic as well as vibrational
transitions. Model membrane small unilamellar vesicles or liposomes provide an oriented system when
they are subject to shear flow in a Couette or other type of flow cell. Anything, including peptides and
proteins, that is bound to the liposome also gives an LD signal whereas unbound analytes are invisible. Flow
LD is the ideal technique for determining the orientation of different chromophores with respect to the
membrane normal. To illustrate the power of the method, data for diphenyl hexatriene, fluorene, antimicrobial peptides (aurein 2.5 and gramicidin), are considered as well as another common chromophore,
fluorene, often used to increase the hydrophobicity and hence membrane binding of peptides. How LD can
be used both for geometry, structure analysis and probing kinetic processes is considered. Kinetic analysis
usually involves identifying binding (appearance of an LD signal), insertion (sign change), often followed by
loss of signal, if the inserted protein or peptide disrupts the membrane.
Key words Linear dichroism, Membrane bilayers, Liposomes, Electronic spectroscopy, Infrared
spectroscopy
1 Introduction
Although there were 1522 membrane protein structures (though
not independent proteins) in the Protein Data Bank (PDB) archive
at the end of 2018, this is little more than 0.1% of all PDB structures. Even if we have a crystal or NMR structure of a membrane
protein, the data do not necessarily provide information about the
protein in an environment of interest. This is most obviously true
for membrane-inserting peptides which are often unfolded in aqueous solution and adopt a helical structure in a membrane. A typical
example is shown by the circular dichroism (CD) spectra in Fig. 1,
where the spectrum of aurein 2.5 in water has a small negative CD
signal at 200 nm characteristic of an unfolded peptide whereas the
spectrum in a lipid environment (above the gel to fluid phase
transition) has large negative signals at 222 nm and 208 nm and a
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_21, © Springer Science+Business Media, LLC, part of Springer Nature 2021
449
Flow Linear Dichroism of Protein–Membrane Systems
Matthew R. Hicks, Sarah R. Dennison, Adewale Olamoyesan,
and Alison Rodger
Abstract
Linear dichroism (LD) is the differential absorbance of light polarized parallel and perpendicular to an
orientation direction. Any oriented sample will show a signal in its electronic as well as vibrational
transitions. Model membrane small unilamellar vesicles or liposomes provide an oriented system when
they are subject to shear flow in a Couette or other type of flow cell. Anything, including peptides and
proteins, that is bound to the liposome also gives an LD signal whereas unbound analytes are invisible. Flow
LD is the ideal technique for determining the orientation of different chromophores with respect to the
membrane normal. To illustrate the power of the method, data for diphenyl hexatriene, fluorene, antimicrobial peptides (aurein 2.5 and gramicidin), are considered as well as another common chromophore,
fluorene, often used to increase the hydrophobicity and hence membrane binding of peptides. How LD can
be used both for geometry, structure analysis and probing kinetic processes is considered. Kinetic analysis
usually involves identifying binding (appearance of an LD signal), insertion (sign change), often followed by
loss of signal, if the inserted protein or peptide disrupts the membrane.
Key words Linear dichroism, Membrane bilayers, Liposomes, Electronic spectroscopy, Infrared
spectroscopy
1 Introduction
Although there were 1522 membrane protein structures (though
not independent proteins) in the Protein Data Bank (PDB) archive
at the end of 2018, this is little more than 0.1% of all PDB structures. Even if we have a crystal or NMR structure of a membrane
protein, the data do not necessarily provide information about the
protein in an environment of interest. This is most obviously true
for membrane-inserting peptides which are often unfolded in aqueous solution and adopt a helical structure in a membrane. A typical
example is shown by the circular dichroism (CD) spectra in Fig. 1,
where the spectrum of aurein 2.5 in water has a small negative CD
signal at 200 nm characteristic of an unfolded peptide whereas the
spectrum in a lipid environment (above the gel to fluid phase
transition) has large negative signals at 222 nm and 208 nm and a
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_21, © Springer Science+Business Media, LLC, part of Springer Nature 2021
449