Functional Structure Biology of Biomembranes and Membrane Proteins

In view of recent achievements in sequencing and gene expression projects, it became more evident that the bottleneck in the sequence->protein->structure->function->altered function chain is structural biology. This is especially true for membrane proteins. The delivery of new membrane protein sequences happens at an alarmingly higher rate than the delivery of membrane protein structures, due to the enormous work needed to determine the 3D structure for a membrane protein by conventional structural methods (X-ray and NMR spectroscopy). There is therefore a permanent demand for improved structure prediction algorithms and alternative biophysical techniques to provide structural data on membrane proteins in their native membranous environment. The statement 'membranous environment' should be emphasized, since both X-ray crystallography and NMR spectroscopy are subject to well known difficulties when applied to membrane proteins. These difficulties are often overcome by techniques which disrupt the native protein-lipid interface. Our working strategy is that structural, dynamic and thermodynamic data on native and reconstituted lipid-protein systems are obtained during permanent control of the biological function using a range of biophysical techniques which are then consistently interpreted in detailed molecular models and related to the biological function. This non-conventional approach can be considered as "function-controlled spectroscopy-based structural biology" (see e.g. [Bashtovyy et al., 2001]). Data are obtained with a variety of techniques and their combination. These include membrane protein purification; FTIR, site-specific (spin label) and spin-trapping electron paramagnetic resonance (EPR), polarised attenuated total internal reflection (PATIR) FTIR, UV-VIS and fluorescence spectroscopies; high-sensitivity DSC, theory and computation (mathematical models, spectrum simulations and molecular modelling). We are keen on developing new detection modes and novel spectrum analysis/simulation techniques that best suit the purpose. We were able to assign distinct bands of the FTIR spectra to the trans and gauche segments of the lipid fatty acyl chains in both model and biological membranes [Kota et al., 1999]. Notable is the combination of non-linear EPR techniques with paramagnetic quenching and spin-spin interactions to gain structural data on membrane proteins [Pali and Marsh, 2002].