6.3. Non fluorescence based optical methods:

There are a number of label-free optical methods that can report on IDP-membrane binding. Circular dichroism (CD) spectroscopy is a sensitive method to characterize the secondary structure content of the whole protein molecule. Alpha-helices and beta sheets have characteristic CD spectral signatures and this property is usually employed as a first-line tool to quantify secondary structure changes on binding to lipid membranes [131]. In addition, one can measure apparent membrane-binding affinity by analyzing CD spectra in a titration series [132]. In order to study curvature-dependence of membrane binding, proper characterization of vesicle size is essential. Dynamic light scattering (DLS) allows estimation of vesicle size distribution for binding experiments as well as detection of changes in vesicle size or morphology upon IDP binding, which could arise from shape alterations, vesicle clustering, fusion, fission, and rupture [58,133]. However, DLS data is weighted by particle size and careful interpretation is recommended in the presence of even small quantities of large particles [64]. Finally, surface plasmon resonance (SPR) methods for determining protein-lipid interactions are gaining in popularity with the advent of commercially available chips with functionalized gold surfaces that allow preparation of various membrane-mimetic surfaces on them [134]. The advantages for SPR include rapid label-free detection of protein-lipid interactions in real time with low sample concentration requirement. One can determine binding rate constants and apparent equilibrium constants using SPR, which is essentially the change in plasmon resonance frequency at a thin gold-plated measurement tip due to binding of biomolecules, measured as the change in angle of reflection at which an attenuation of a monochromatic totally internally reflected beam occurs [135]. SPR has been used to study membrane interactions of alpha-synuclein variants [136]. In principle, SPR can be used as a powerful screening tool for membrane interaction modulators of IDPs, much like screening for IDP-protein interaction inhibitors [137,138].