ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P193. A Unique Synchrotron-Radiation Flow Linear Dichroism
Spectroscopy Facility for the Study of Oriented Macromolecules
P.W. Thulstrup a , S.V. Hoffmann b
a
Department of Natural Sciences, LIFE, University of Copenhagen, Thorvaldsensvej 40, DK-1871
Frederiksberg, Denmark, e-mail: pwt@life.ku.dk
b
Institute for Storage Ring Facilities, University of Aarhus, Ny Munkegade, Bldg. 1520, DK-8000 Århus,
Denmark.
Spectroscopic studies of ordered samples constitute an underutilized, yet highly useful range of techniques,
particularly for studies of biological systems, which often possess an inherent orientation. One prominent
technique is Linear Dichroism (LD) spectroscopy, where a sample with a partial molecular orientation is probed
with linearly polarized light. The LD signal is defined as the difference in absorption between the polarization
oriented parallel and perpendicular to the sample orientation axis: LD = ∆A = A⊥ - A||
Naturally oriented samples include crystals, fibres and membranes, but sample orientation may also be induced,
as for example in Flow LD (see e.g. ref. [1]). This technique (illustrated in the figure) can be applied to any rigid,
elongated sample molecule. The most notable examples are DNA and fibrous proteins. They are oriented in a
laminar flow due to their rigidity and their extended molecular shape. Since the sample is in aqueous solution the
structural properties can be studied as a function of temperature and chemical composition (e.g. salts, denaturing
agents, and other small molecules), where intra- and intermolecular reactions can be studied and physical
changes can be monitored.
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Figure 1: Long molecules like e.g. DNA can
be oriented in a flow field of a Couette cell.
Small molecules (e.g. a potential drug) remain
randomly oriented until they bind to the DNA.
The plane polarized light passes through the
central part of the cell and thus only probes the
molecules aligned perpendicular to the direction
of the light.
Small molecules are not oriented by the laminar flow gradient in the sample. Flow-LD studies can, therefore,
reveal information both on molecular structure of the oriented sample (i.e. changes in molecular shape / protein
secondary structure for fibrous proteins) as well as information on the binding of small molecules to the oriented
sample. In the latter case information can both be obtained with regard to kinetics, equilibrium binding constants
as well as structural information. Thus, linear dichroism spectroscopy can provide many different types of
information; both with regard to the sample constitution and with regard to molecular shape, symmetry and
structure.
The flow LD facility has been designed specifically for the study of:
• The interaction between membranes and membrane-binding peptides and proteins.
• The interaction between DNA and DNA-binding molecules, including proteins and coordination compounds.
• The structure and structural dynamics of fibre-forming peptides and proteins.
The use of flow oriented LD spectroscopy has until very recently only been realised on commercial CD
instruments modified into LD spectrometers. This despite the fact that the same advantages in spectral range and
intensity can be realised with synchrotron radiation based LD (SRLD) as has been obtained for SRCD. A
Couette Flow LD facility has been implemented at the existing SRCD facilities on the UV1 and CD1 beamlines
at the synchrotron radiation source ASTRID at Aarhus University in Denmark. This SRLD facility is the first of
its kind world wide. The implementation has been very successful, and it has shown that SRLD is a drastically
improvement over the commercial LD spectrometers: The dynamic range of molecular concentrations is higher,
the spectral quality is far better, and lower wavelengths can be measured enabling the studies of protein-lipid
membrane interactions/insertions.
Acknowledgement: A grant from The Danish Natural Science Research Council is gratefully acknowledged
(no. 272-07-0240)
References: [1] R. Marrington, T.R. Dafforn, D.J. Halsall, J.I. MacDonald, M. Hicks, A. Rodger, Analyst, 130,
1608 (2005).