Disorder Influence on Linear Dichroism Analyses of Smectic Phases

Abstract

Linear dichroism, the unequal absorption of parallel and perpendicular linear polarized light, is often used to determine the anisotropic ordering of rodlike polymers in a smectic phase, such as helices in a lipid bilayer. It is a measure of two properties of the sample: 1), orientation of the chromophore transition dipole moment (TDM) and 2), disorder. Since it is the orientation of the chromophore TDM that is needed for high resolution structural studies, it is imperative to either deconvolve sample disorder, or at a minimum, estimate its effect upon the calculated TDM orientation. Herein, a rigorous analysis of the effects of disorder is undertaken based on the recently developed Gaussian disorder model implemented in linear dichroism data. The calculation of both the rod tilt and rotational pitch angles as a function of the disorder and dichroism, yield the following conclusions: Disorders smaller than 5 degrees have a vanishingly small effect on the calculated polymer orientation, whereas values smaller than 10 degrees have a negligible effect on the calculated parameters. Disorders larger than 10 degrees have an appreciable effect on the calculated orientational parameters and as such must be estimated before any structural characterization. Finally the theory is tested on the HIV vpu transmembrane domain, employing experimental mosaicity measurements from x-ray reflectivity rocking scans and linear dichroism.

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

“…One limitation complicating the use of polarized IR spectroscopy on oriented samples to derive structural information is the sample disorder, which results from the fact that without further corrections the experimentally obtained order parameter is defined with respect to the window normal and not with respect to the membrane normal. As a result, the experimental order parameter needs to be corrected for the second-order parameter of the membrane normal with respect to the window normal, known as the mosaic spread . In addition, the order parameter can only be converted into an angle value by ignoring potential heterogeneities (e.g., mixture of conformations) as well as dynamical aspects (e.g., thermal fluctuations in the angle).…”

Gating Mechanism of the Influenza A M2 Channel Revealed by 1D and 2D IR Spectroscopies

“…Thus, henceforth for simplicity we shall refer to our measurements as yielding the angle between the bilayer normal and the C=O bond. The precise procedures for deriving the aforementioned angle is according to protocols that we have established in the past (Manor et al, 2005) and are outlined in the Supplemental Information Methods Section. Briefly, the process consists of: (i) reconstituting the labelled channels in hydrated vesicles, (ii) removal of bulk solvent whilst depositing the membranes on a solid support to create aligned bilayers, (iii) measuring the dichroism using ATR-FTIR spectroscopy, (iv) assessing the bilayer disorder by X-ray scattering, and finally (v) converting the measured dichroisms to the angles of the carbonyl groups with respect to the membrane normal.…”

“…Similarly, the ATR-FTIR spectroscopy and the X-ray Scattering bilayer mosaicity estimation were undertaken as described elsewhere (Manor et al, 2005; Torres et al, 2000b), and specified in detail in the Supplemental Information Section.…”

Conformation of the synaptobrevin transmembrane domain

“…Reported S L values for DMPC in the literature are in the range of 0.5-0.8 (28), which is similar to the observed S L of 0.7. Explicit analysis of sample disorder in similar DMPC͞peptide samples concluded that sample disorder was not significant (44), but this analysis may not be directly comparable. In any case, our data would not be compatible with a helix perpendicular to the membrane.…”