How to study DNA and proteins by linear dichroism spectroscopy.(Report)

ABSTRACT

The technique of linear dichroism (LD) is a simple absorbance technique that uses two polarised light beams. Since only oriented molecules show different absorbances for different polarisations, LD detects only oriented molecules.

In aqueous solutions, flow orientation is an attractive orientation methodology as it selects long molecules or molecular assemblies. LD thus is selective for molecules that are particularly challenging to study by more standard biophysical techniques. In this article, a brief review of the application of LD to DNA, DNA drug systems, DNA--protein enzymatic complexes, fibrous proteins and membrane peptides and proteins is given.

Keywords: linear dichroism, DNA, proteins

Introduction

This article is predicated on the assumption that if we know about the structure of complicated molecules in biological systems then we can begin to work out how they function. With some molecules we can get them to crystallise into regular lattices and determine the structure using X-ray crystallography. However, we are not always sure that the structure the molecule adopts when it is in a regular lattice is the same as when it is in solution interacting with lots of different molecules. In addition, for some classes of molecules crystallography is simply not possible. Such molecules include long DNAs and assemblies of molecules into fibres as well as membrane-bound molecules including membrane proteins. The purpose of this article is to illustrate the advantages and disadvantages of solution-phase flow linear dichroism (LD) spectroscopy for structural characterization of solutions of long biomacromolecular molecules. LD can also be used for any long not completely flexible molecules; some carbon nanotube work is reported in references (1,2). The key to the success of the recent work outlined below was the invention in 2003 of LD cells that only required (on a good day) 25 [micro]l of sample (3); 50 [micro]l makes the experiment easy to do. This was an order of magnitude improvement in sample requirement for LD and opened up a wide range of DNA and protein experiments that had been previously impossible. A convenient side-effect of the new cells was that they also reduced artefacts, gave better quality data, and are significantly easier to clean than our previous generations of cells.

Samples for LD need to be oriented so the concept of Couette flow is introduced below, followed by a description of linear dichroism and some recent applications, which suggest where the developments will go.