Optical Spectroscopy of Membrane
Protein/Amphipol Complexes
8
Summary
Most optical spectroscopy approaches can be applied to membrane protein/amphipol
(MP/APol) complexes, namely UV-visible absorbance spectroscopy, light scattering,
circular dichroism, static and time-resolved fluorescence measurements, fluorescence
quenching and Förster resonance energy transfer studies, surface plasmon resonance
measurements, etc. A notable exception is infrared absorbance studies in the peptide
bond absorbance bands. Indeed, most APols comprise amide bonds, in which case
absorbance spectroscopy studies of APol-trapped MPs at the peptide bond wavelengths have proven intractable. Resonance Raman studies however are possible, as
well as surface-enhanced Raman spectroscopy.
A large number of APols carrying fluorescent labels have been developed,
opening the way to a vast range of applications, from the study of the composition,
organization, and dynamics of MP/APol complexes to topological and conformational
studies of MPs and to imaging of the distribution and elimination of APols in cell
cultures and live animals.
8.1
Introduction
Optical spectroscopy has been used in most studies of amphipol (APol)-trapped membrane proteins
(MPs), usually in the form of UV-visible absorbance spectroscopy, often in circular dichroism (CD)
determination of the secondary structure of the trapped protein and/or dynamic light scattering (DLS)
estimation of particle sizes. Fluorescence studies have also been numerous. As is the case for A8-35,
most APols do not contain conjugated double bonds, making them transparent in the visible and near-UV
regions of the spectrum. A8-35 absorbs only in the far UV (Fig. 8.1A), with a peak at ~219 nm, so that, at
and above 280 nm, it interferes very little with absorbance measurements. Unlabeled A8-35 is readily
detectable in the 220–230-nm region, with an extinction coefficient ε 219 % 1.3 gÁL
À1
Ácm
À1
. At 280 nm, ε
does not exceed 0.03 gÁL
À1
Ácm
À1
. Since APols are seldom used at APol/protein mass ratios exceeding
5:1 (see Chap. 5) and the extinction coefficient of proteins at 280 nm is typically !10 LÁg
À1
Ácm
À1
, the
contribution of A8-35 to the absorbance at 280 nm of a preparation of MP/A8-35 complexes is generally
<2%. The situation is probably similar for most other APols, except for the styrene-maleic acid (SMA)
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_8
381
Protein/Amphipol Complexes
8
Summary
Most optical spectroscopy approaches can be applied to membrane protein/amphipol
(MP/APol) complexes, namely UV-visible absorbance spectroscopy, light scattering,
circular dichroism, static and time-resolved fluorescence measurements, fluorescence
quenching and Förster resonance energy transfer studies, surface plasmon resonance
measurements, etc. A notable exception is infrared absorbance studies in the peptide
bond absorbance bands. Indeed, most APols comprise amide bonds, in which case
absorbance spectroscopy studies of APol-trapped MPs at the peptide bond wavelengths have proven intractable. Resonance Raman studies however are possible, as
well as surface-enhanced Raman spectroscopy.
A large number of APols carrying fluorescent labels have been developed,
opening the way to a vast range of applications, from the study of the composition,
organization, and dynamics of MP/APol complexes to topological and conformational
studies of MPs and to imaging of the distribution and elimination of APols in cell
cultures and live animals.
8.1
Introduction
Optical spectroscopy has been used in most studies of amphipol (APol)-trapped membrane proteins
(MPs), usually in the form of UV-visible absorbance spectroscopy, often in circular dichroism (CD)
determination of the secondary structure of the trapped protein and/or dynamic light scattering (DLS)
estimation of particle sizes. Fluorescence studies have also been numerous. As is the case for A8-35,
most APols do not contain conjugated double bonds, making them transparent in the visible and near-UV
regions of the spectrum. A8-35 absorbs only in the far UV (Fig. 8.1A), with a peak at ~219 nm, so that, at
and above 280 nm, it interferes very little with absorbance measurements. Unlabeled A8-35 is readily
detectable in the 220–230-nm region, with an extinction coefficient ε 219 % 1.3 gÁL
À1
Ácm
À1
. At 280 nm, ε
does not exceed 0.03 gÁL
À1
Ácm
À1
. Since APols are seldom used at APol/protein mass ratios exceeding
5:1 (see Chap. 5) and the extinction coefficient of proteins at 280 nm is typically !10 LÁg
À1
Ácm
À1
, the
contribution of A8-35 to the absorbance at 280 nm of a preparation of MP/A8-35 complexes is generally
<2%. The situation is probably similar for most other APols, except for the styrene-maleic acid (SMA)
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_8
381