Part A: Structural studies of bacterial methyl-accepting chemotaxis proteins. Part B: Water polygons in protein crystals

In Part A, I describe the structural studies of bacterial chemotaxis receptors. Bacterial chemotaxis is one of the best understood biological signaling pathways. Almost all motile eubacteria, proteobacteria, and archaea use the chemotaxis to direct their movement toward a more favorable environment and away from unfavorable environment. The chemotaxis signaling system is a member of the bacterial two-component system, which is divided into a sensor and response-regulator. The major component of the sensor is a group of transmembrane receptors known as methyl-accepting chemotaxis proteins (MCP). An MCP is a ∼60 kDa protein with two transmembrane alpha helices. Although all attempts so far to solve the atomic structure of an intact MCP have been unsuccessful, the X-ray crystal structures of the domains have been solved. The structures of the periplasmic sensor domain and cytoplasmic signaling domain in both the apo and signal-activated state showed little conformational difference on ligand binding obscuring the intra MCP signal transduction mechanism.;Solving the crystal structure of an intact MCP in the apo and activated form will give us a better understanding of the intra signal transduction mechanism through the transmembrane, as well as, the inter signal transduction mechanism between MCPs that contributes to the signal amplification and wide dynamic range properties of the bacterial chemotaxis system. Knowing that membrane protein structural biology is renowned for its difficulty, we decided to follow the structural genomics approach to overcome this problem.;As presented in Chapter 2, I screened 37 MCP genes from 10 prokaryotes using a high-throughput (HTP) method to successfully crystallize the E. coli ribose-galactose chemotaxis receptor (Trg). However, the crystals diffracted to 20 A, and could not be improved by changing purification and crystallization conditions. Therefore, as presented in Chapter 3, we concentrated on the three best targets to generate rationally designed mutants to improve the crystallization. Although I was able to successfully crystallize two of the three targets to medium X-ray diffraction resolution of 3 A, we realized that it was a crystal of a contaminant membrane protein AcrB, which will be presented in Chapter 4.;Since MCP is a transmembrane protein, some HTP methods could not be directly applied to this project. Therefore, I tried to modify and improve existing HTP techniques. As presented in Chapter 5, I fixed the low binding affinity (to metal chelating columns) problem of His-tagged MCPs by making a series of HTP ligation-independent cloning vectors that allow recombinant protein expression with different C-terminal His-tag lengths without expressing any unwanted cloning sequence. As presented in Chapter 6, I improved the quality of purified MCPs by exploiting the high ionic strength tolerance of detergent-solubilized membrane proteins. I also modified an existing I-ITP purification robot to automate the detergent exchange screening in a preparatory scale that is more suitable for structural genomics, which will be presented in Chapter 7.;In Part B, I present experimental evidence that suggests the presence of water polygons in liquid water. Despite the fact that water is the most important molecule for all living organisms on Earth, many properties of liquid water are not well understood. The oligomeric state of water molecules in liquid water has been debated as one of the factors important for understanding the properties of liquid water. To find experimental evidence, I have analyzed the interstitial water (ISW) structures of 1,500 high resolution protein crystal structures. I observed varieties of water polygons: trigons, tetragons, as well as expected pentagons, hexagons, higher polygons, partial dodecahedrons, and disordered networks. This investigation sets a basis for a water polygon population study in different temperatures, which might provide new experimental data to correlate with various water properties that are not yet explained including the density of water being highest at 4 C°.