Multiple challenges in protein structure determination by X-ray crystallography : Four ventures, one structure solved

Abstract

Beside DNA and RNA proteins display the basic modules of all cells in nature. Within an organism they take multiple tasks. Hence, they not only confer stability and structure to the cells, but are maschines on molecular level. For example, they mediate transport, catalysis, defense and the recognition of signal substances. However, proteins can also cause extensive damage as toxins, pathogenic structures or by alterations of their genetic code. As a result devastating dysfunctions like spontaneous or inherited diseases can occur that affect the whole organism. This is the case for the neuronal cell adhesion molecule L1. Mutations in the genetic code lead to the so-called CRASH syndrome, that largely influences the lives of persons concerned. One project of this work was to identify a suitable expression system for the first four Ig domains of E587-antigen of the gold fish, that is orthologous to L1, to express the protein fragment for later x-ray structure determination. Another example for a serious malfunction on protein level is the auto-immune disease Myasthenia gravis. Here point of action is the nicotinic acetylcholine receptor that has an important role in signal transduction. The receptor is recognized by auto-antibodies and thus flagged for degradation. In order to improve the crystallization of the transmembrane receptor, monoclonal antibodies were generated. The latter should bind selectively to the extracellular hydrophilic part of the receptor, augment this part to amend the crystal contacts between the receptor molecules.Another two ventures of this dissertation deal from the pathogenic character of two bacterial proteins. One of them is the lysin-N6-hydroxylase of E.coli, that contributes to the biosynthesis of the siderophore receptor aerobactin. Siderophores serve lots of microorganisms for iron uptake, that is essential for their metabolism. Within this work, large surface exposed amino acid side chains were mutated to small side chains to facilitate crystallization. The second protein, the beta-N-acetylglucosaminidase BsNag3A, stems from the gram-positive organism B.subtilis. BsNag3A is engaged in cell wall degradation and recycling. It cleaves the beta-1,4-glycosidic linkage between GlcNAc and MurNAc of the murein. Within the current work the protein could be crystallized in two different ways, with and without its effective inhibitor PUGNAc. Both structures give insight into the active site and the mechanism of the protein.