SCRD: a structural biology tool for the pharmaceutical industry
Dr Bonnie Wallace, Director of the Centre for Protein and Membrane
Structure and Dynamics, one of BBSRC's six structural biology
centres of excellence, describes how the Centre is encouraging
and developing new applications for the pharmaceutical industry.
The UK is a world leader in the technique of Synchrotron Radiation
Circular Dichroism (SRCD) spectroscopy and the Centre, which is
developing a dedicated beamline for steady state and stopped flow
measurements, is internationally renowned for applications in
biological research.
There is growing interest in using SRCD in structural biology
because the high intensity of the SR light source ensures enhanced
measurements compared with those from conventional lab-based instruments.
As a result, measurements can be made to include lower wavelengths
[Figure 1] (and thus contain more information on protein secondary
structures), have a higher signal-to-noise (and thus smaller amounts
of material can be used), be done in a speedier manner (due to
the requirement for less signal averaging due to the stronger
signal), and be done in the presence of buffers and absorbing
components (which better mimic "physiological" conditions). SRCD
has many potential uses in the pharmaceutical industry.
Figure 1: SCRD (red) vs CD (blue spectrum of myoglobin
Drug binding:
CD is an important technique for monitoring conformational changes
in proteins that occur upon drug binding. It can be used to determine
binding constants, to quantitate the number of amino acids involved
in the binding site, and in some cases can also be used to pinpoint
the type of structure involved in the binding site. For example,
we, in collaboration with colleagues at Queen Mary and Westfield
College and in the USA, have used CD to show that the Bcl-2 apoptosis
protein binds the drug Taxol, and that the binding involves ~10-12
amino acids in a loop region expected to be distinct from the
homologous regions of Bcl-XL protein. This has led to exploration
of new related targets for rational drug design.
Figure 2: CD spectra of Taxol binding to Bcl-2
Monitoring protein refolding:
CD can be especially valuable for studies of protein expression
and refolding. In particular, it can be used to follow the refolding
of proteins expressed in large quantities in insoluble forms into
their native conformations. For example, we have expressed a G-protein
coupled receptor in large quantities in inclusion bodies and have
used CD to monitor conditions necessary for it to be refolded
into its native, membrane-bound conformation. This provides a
valuable tool for large scale production of proteins (especially
membrane proteins) for pharmacologic and structural purposes.
Examining the process of protein folding:
As well as monitoring the kinetics of folding, CD can be used
to obtain information on the order of secondary structure formation,
and thus on the mechanism of protein folding in vitro. In the
laboratory of Dr. Gareth Jones, Associate Director of the CPMSD
and Head of Life Sciences at Daresbury, the folding of beta-lactoglobulin
was followed over a wide wavelength range and as a function of
time. This has led to an understanding of the rate and order of
development of the helical and sheet components of the protein.
Such studies employ stopped-flow techniques and are providing
important basic information on the fast processes associated with
protein folding. Nanosecond temperature jump CD will be available
shortly. [Figure 3].
Figure 3: 2-D plot of beta-lactoglobulin folding.
[Acknowledgement: G. Jones and colleagues]
An adjunct to crystallography:
SRCD not only provides complementary information on features
of proteins relative to those determined by crystallography, but
also, since it examines proteins in solution, can be used to determine
environmental effects on structure. It can help in determining
suitable conditions for crystallisation, especially of macromolecular
complexes.
Structural genomics:
CD has the potential to play an important new role in structural
genomics where a key goal is to identify all unique protein folds
that exist and then to use this information to improve the prediction
of protein structures from sequences. Since the CD spectrum of
a protein is a combination of characteristic spectra of known
secondary structures and folding motifs, if the spectrum of a
protein with an as yet unknown structure is not well-fit by the
existing reference databases, then it is likely to contain a new
fold or motif. Because SRCD uses ~1/100 the amount of protein
used by crystallography and takes ~1/100 the time, it promises
to be an efficient method for identifying proteins which are good
candidates for having new folds. Proteins thus identified could
then be targeted for full crystallographic structure determination.
This would make searching and fold recognition far more efficient,
with SRCD acting as a valuable adjunct to bioinformatics in predicting
new folds from sequences.
Membrane protein structures:
An important class of proteins which is greatly under-represented
in the databases of protein structures is that of integral membrane
proteins. It has been estimated that nearly 1/3 of all proteins
in the human genome may be membrane-associated. Until now, it
has not been possible to determine the secondary structures of
membrane proteins very accurately from CD spectra because all
of the reference databases used for these empirical determinations
were derived from soluble proteins, which have very different
spectral characteristics. At the Centre we are undertaking a project
to create a new SRCD reference database derived from membrane
proteins whose crystal structures are known. When completed, this
will be made freely available via the Centre website to aid in
the determination of secondary structures of new membrane proteins.
The Centre for Protein and Membrane Structure and Dynamics
The Centre, which is based at the Daresbury Laboratory, focuses
on the technique of circular dichroism (CD) spectroscopy, especially
using synchrotron radiation as a light source. It is accessible
to any UK investigator using CD spectroscopy, from either academic
or industrial labs. As well as its CD facilities, the Centre also
acts as a bioinformatics resource for the entire CD community
in the UK (both those using conventional, lab-based instruments
and those using the SRCD for data collection). "Members" can access
this via the Centre website, which contains all sorts of information
on methods of analysis, instrumentation, references, etc. and
will soon contain a interactive site (DICHROWEB) for user-friendly
calculations of protein secondary structures.
Information on the Centre and on access to SRCD beamtime can
be found on the Centre website at: http://www.srs.dl.ac.uk/VUV/CD/cpmsd.html
Contacts:
B.A. Wallace, School of Crystallography, Birkbeck College,
Univ. of London and Director, Centre for Protein and Membrane
Structure and Dynamics, Daresbury Lab
ubcga@mail.cryst.bbk.ac.uk
Gareth Jones, Head, Life Sciences Department and Associate
Director, Centre for Protein and Membrane Structure and Dynamics,
Daresbury Lab
g.r.jones@dl.ac.uk
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