Evolution, structure and stability studies of bacteriorhodopsin mutants and crystallographic studies of a sugar-binding membrane protein from the hyperthermophile Pyrobaculum aerophilum

Chapter 2 deals with kinks in membrane proteins. The majority of transmembrane helices are bent, primarily at proline residues, but about 40% at nonproline residues. We propose an evolutionary hypothesis in which nonproline kinks originate from proline kinks, which themselves occur by random mutation. I describe evidence supporting this hypothesis, and a method to predict the occurrence of all kinks in transmembrane helices with >90% reliability.; In Chapter 3, I discuss the structure and stability effects of inserting a proline into a transmembrane helix at random locations. Surprisingly, these mutations were not much less favorable than alanine mutations at the same positions, showing that a protein will tolerate a mutation to proline. We solved four of the mutants by x-ray crystallography, and found that three induced significant structural shifts. The other mutant, positioned at a nonproline kinking position, revealed very minor changes.; C-H···O H-bonds (the theme of Chapter 4) are common in protein structures, and though expected to be weaker than traditional H-bonds, some calculations suggest that C alpha-H···O H-bonds could be half the strength of a traditional H-bond. Thus, they have the potential to be highly significant in stabilizing protein structures. Surprisingly, no experimental tests of their energetic contribution to stability have been performed. We probed the strength of a Calpha-H··· O H-bond in the membrane protein bacteriorhodopsin, and found it to be slightly destabilizing.; Chapter 5 describes a naturally highly-expressed single-transmembrane sugar-binding protein we isolated from the hyperthermophile Pyrobaculum aerophilum. We grew crystals and collected 2.8A X-ray diffraction data. Phases could not be obtained from heavy atom experiments. I crystallized the recombinant soluble portion of PA-SBP and collected 2.4A data. We are growing seleno-methionine crystals to solve the structure by multiple anomalous dispersion.