Computational Investigation of Structural and Thermodynamic Properties of Beta-Barrel Membrane Proteins

Transmembrane (TM) proteins are important as they serve as gateways to permit substance transport or signaling transduction between interior and exterior of cells. Beta barrel membrane proteins (beta MPs) are one major type of TM proteins. They are solely found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplast. Beta MPs serve a multitude of essential cellular functions, including reaction catalysis, protein anchoring, metabolite transportation, and outer-membrane biogenesis. In bacteria, beta MPs are also found to be responsible for the release of virulence factors and are implicated in multidrug resistance. Dysfunctional beta MPs in mitochondria are also related to neurodegenerative diseases. The effective pore formation ability and the high stability in the membrane of beta MPs grant them huge potential in bionanotechnology. Beta MPs have drawn increasing attention in their promising application, including protein profiling, DNA sequencing, and small molecule detection. In order to investigate the roles of beta MPs in biological and pathological processes and to engineer or to design novel beta MPs for biotechnical applications, it is critical for us to understand structural and thermodynamical properties of beta MPs. Despite importance of beta MPs in biology and nanotechnology, limited availability of beta MP structures hinders understanding of their structural properties and structure-function relationship. It was estimated that there exist hundreds of beta MPs in each Gram-negative bacterium genome, while there are only around 60 non-homologous structures deposited in the Protein Data Bank when this study was conducted. This lack is due to the great difficulty of experimental determination of TM protein structures because of their amphipathic nature. It is therefore important to develop accurate and efficient computational structure prediction methods for these proteins. We have developed a method to predict the 3D structures of beta MPs. We predict strand registers and construct 3D structures of TM domains of beta MPs accurately, including proteins for which no prediction has been attempted before. Our method also accurately predicts structures from protein families with a limited number of sequences and proteins with novel folds. An average mainchain RMSD of 3.48A is achieved between predicted and experimentally resolved structures of TM domains, which is a significant improvement over a recent study. For beta MPs with NMR structures, the deviation between predictions and experimentally solved structures is similar to the difference among the NMR structures, indicating excellent prediction accuracy. Moreover, we can now accurately model the extended beta-barrels and loops in non-TM domains, increasing the overall coverage of structure prediction by 30%. In additional to structural properties, it is also important to characterize thermodynamical properties of beta MPs, which is important to understand their folding and stability, and may help in understanding the structure-function relationship. Free energy of transferring amino acid sidechains from aqueous environment into lipid bilayers, known as transfer free energy (TFE), provides important information on the thermodynamic stability of membrane proteins. However, experimental measurement of TFEs of beta MPs is challenging. A recent computational method has been developed to calculate TFEs, the results of which are in excellent agreement with experimentally measured values. However, the method does not scale up, and is limited to small beta MPs. We have improved this method and developed an approximation method, which is comparably accurate but much faster than the original method. The new method enables the calculation of TFEs of all beta MP regardless of the size of the proteins. With this method, we derived a TFE profile named General Transfer Free Energy Profile (GeTFEP) based on computation of the TFEs of 58 beta MPs. The GeTFEP agrees well with experimentally measured and computationally derived TFEs. Analysis based on the GeTFEP shows that residues in different regions of the TM segments of beta MPs have different roles during the membrane insertion process. Results further reveal the importance of the sequence pattern of TM strands in stabilizing beta MPs in the membrane environment. In addition, we show that GeTFEP can be used to predict the positioning and the orientation of beta MPs in the membrane. We also show that GeTFEP can be used to identify structurally or functionally important amino acid residue sites of beta MPs. Furthermore, the TM segments of helical membrane proteins can be accurately predicted with GeTFEP, suggesting that the GeTFEP captures fundamental thermodynamic properties of amino acid residues inside membrane, and is of general applicability in studying membrane protein. The methods reported in this thesis require only sequence information, implying their general applications to genome-wide studies. The structure prediction and the TFE characterization methods provide ways to investigate properties of novel beta MPs without conducting expensive wet lab experiments. They will also be useful in bionanotechnologies such as engineering existing beta MPs and in design novel ones.