Circular Dichroism Spectroscopy and X-ray Crystallography: A Dynamic Duo
B. A. Wallace
Centre for Membrane Structure and Dynamics, Daresbury Laboratory
and
Dept. of Crystallography, Birkbeck College, University of London
At the recent Daresbury PX Users meeting, the complementary natures of circular dichroism (CD) spectroscopy and X-ray crystallography were discussed. While it is clear that CD is no substitute for X-ray data, it can provide both dynamic and static structural information and information that can be of assistance in producing crystals.
What you can learn from CD Spectroscopy (while you are waiting for crystals to grow):
CD spectroscopy is a useful technique for examining protein structures in solution under different conditions and for examining both their dynamics and folding pathways. The former types of experiments can be important for establishing the "physiological relevance" of the conditions used, say, for crystallisation. The latter can be important for examining the conformational effects of ligand binding or complex formation with another macromolecule. In addition, the processes associated with protein folding can be monitored on a fast time scale.
Secondary and tertiary structures can be monitored by the peptide transitions in the far UV (~190-220 nm) and by the aromatic side chain transitions in the near UV (~270-290 nm), respectively. Other chromophores in the visible region (for coloured proteins) may be valuable for establishing the fidelity of tertiary and quaternary folding.
In advance of obtaining a crystal structure, CD can be useful in conjunction with modelling efforts: it can provide a good test of the secondary structural content of any model produced, and can be used to determine the class of molecule. For crystallography, it may be used as an assay of whether a particular structure will be a reasonable model for molecular replacement.
CD can also be used to examine conformational changes associated with ligand or drug binding, thereby aiding in elucidating the mechanism of action of enzymes and receptors. These changes can be quantitated by CD since shifts in conformation involving as few as 10 amino acids may be readily detectable.
How CD Spectroscopy can help you to grow (or improve) crystals:
CD spectroscopy may be useful for establishing protein integrity and conditions for complex formation:
Ligand binding studies can allow titration of complex formation, thereby assuring a single species under the crystallisation conditions (as opposed to a mixture of proteins with and without ligand bound). It can also be useful for monitoring complex formation with other macromolecules.
Examination of the effects of different pHs, detergents, solvents or other additives can be a guide to the integrity of the protein and "physiological relevance" of the crystallisation conditions.
The secondary structures and thermal stability of molecular biology constructs created to produce domain structures for crystallisation can be assayed, as can the fidelity of refolding of proteins initially expressed as inclusion bodies or in other denatured forms.
Finally, thermal denaturation studies by CD (melting curves) can be used to establish means of enhancing the stability of the protein by addition of various types of additives to the crystallisation mixtures.
The Centre for Protein and Membrane Structure and Dynamics (CPMSD):
Earlier this year the BBSRC created the CPMSD at Daresbury as one of six Structural Biology Centres throughout the U.K. This centre is focused on CD spectroscopy, both conventional and synchrotron-based. As well as providing a state-of-the-art facility for SRCD data collection, the Centre will be a "virtual centre" for CD and its interpretation, where "visitors" will have access to an Expert Panel of advisors, a user-friendly website for data analyses, an interactive bulletin-board, and BioMOO-type virtual meetings to discuss current techniques and methods for analysis of data. The SRCD at the Centre will be a unique facility world-wide with capabilities for low wavelength, high resolution and very fast stopped-flow measurements.
The annual U.K. CD Spectroscopy Meeting will be held on 21 October 1999 at the Daresbury Laboratory. Registration is free and open to any interested person. If you wish to attend, please contact: p.b.broadhurst@dl.ac.uk prior to 15 October.
More information on CD spectroscopy and the CPMSD can be obtained at: http://www.dl.ac.uk/SRS/VUV-IR/CD/cpmsd.html