This PhD student project is a representative instance of one of the current major challenges in structural biology, namely the determination of membrane protein structure using solution NMR (Nuclear Magnetic Resonance) spectroscopy. The project will be carried out mainly at the very well-equipped Swedish NMR Center (SNC) at Gteborg University located in the Hasselblad Laboratory (http://www.nmr.se). It currently includes one 900, one 800, two 600s and one 500 MHz NMR Varian spectrometers fully equipped for high-resolution triple-resonance experiments on isotope-labeled biomolecules. The student in this PhD project will get ample hands-on experience using these state-of-the-art machines. The student will also get knowledge in standard molecular biology techniques when preparing selected isotope-labeled proteins. Students suitable for this project probably have a MasterŐs degree with a chemistry, physics or biophysics major and have taken one or more advanced courses in physical chemistry or biophysical chemistry or related subjects. Also students with a predominately biological or biochemical education background can also come into consideration if this has been supplemented with several university-level courses in mathematics (analysis, linear algebra) and physical chemistry (thermodynamics, quantum mechanics, spectroscopy).
Membranes, and
hence membrane proteins, are omnipresent in all forms of life but detailed
three-dimensional (3D) structures of membrane proteins are scarce and
determination of such structures is at present considered a challenge also for
the
experienced NMR spectroscopist. Membrane proteins play an essential role in cellular
processes by providing vital communication channels between the cell and its
surroundings. Membrane proteins are already the target
of the vast majority of drugs currently in use and structural information could
greatly improve the efficiency of drug discovery. In fact, many currently available
drugs target membrane proteins whose structures are poorly understood. Gaining
a better understanding of a target proteinŐs structure is an essential step
towards understanding its precise function and can help in the development of
more specific and efficient drugs. Membrane proteins represent 20 to 30 % of
currently sequenced genomes, but less than 1 % of solved structures are
membrane proteins. The reason for this discrepancy is that membrane proteins are
relatively difficult to work with
and difficult to obtain in large quantities (a problem in both NMR and
X-ray studies), when compared to the more conventional (water) soluble proteins.
The properties of the cell membrane environment, with its
water, lipid and protein components, impose certain structural and functional
features on membrane proteins which make them much more difficult to study than
the soluble proteins found elsewhere in the cell. A primary difficulty in dealing with integral membrane proteins
using NMR, is that
integral membrane protein samples are simply too large to
tumble with sufficiently low correlation time to yield narrow and well-resolved
resonance lines, thus effectively preventing structure determination of them in
their natural environment (lipid bilayers). The most common resolution of this
difficulty is to embed integral membrane proteins in micelles of detergents
(such as dihexanoylphospatidylcholine (DHPC)), which enables the use of solution
NMR techniques for their structure determination. However, the method of
embedding integral membrane proteins in micelles invariably leads to fairly
large molecular assemblies, typically beyond 50 kDa even for small membrane
proteins. Conventional NMR spectra of molecular structures in solution larger
than 40 kDa typically display broad lines, effectively resulting in poor
resolution and sensitivity. The TROSY technique suppresses transverse
nuclear
spin relaxation, which is the direct cause of the deterioration of the NMR
spectra of large molecular structures, and is therefore at present the
fundamental enabling technique for the structure determination of integral
membrane proteins, and has been successfully applied for this purpose by an as
yet small, but increasing, number of researchers.
As a starting-point for our investigation of (alpha-helical) membrane
proteins we have
chosen the small
membrane protein CcmD from the inner membrane of E. coli. CcmD will be expressed, purified and biophysically characterized
in order to be able to undertake solution NMR measurements to investigate the
structure and dynamics of the protein. CcmD is a small membrane protein
involved in heme delivery to the heme chaperone CcmE during cytochrome c maturation. CcmD interacts with, in addition to CcmE, with
CcmC, another essential component of the heme delivery system. Subsequent
studies will most likely be NMR investigations of CcmE and CcmC (and possibly
other membrane proteins), in combination with
supporting method- and methodological
development to increase the feasibility and effectiveness of using NMR to study
membrane proteins.