<p>Ssu72 is a protein phosphatase that selectively targets phosphorylated serine residues at the 5th position (pS5) in the heptad repeats of the C-terminal domain (CTD) of RNA polymerase II, in order to regulate the CTD-mediated coupling between eukaryotic transcription and co-transcriptional events. The biological importance of Ssu72 is underscored by (1) the requirement of its activity for viability in yeast, and (2) the numerous phenotypes - affecting all three stages of the transcription cycle - that result from its mutation in yeast. Despite limited homology to the low molecular weight (LMW) subclass of protein tyrosine phosphatases (PTPs), several lines of evidence suggest that Ssu72 represents the founding member of a new class of enzymes, including its unique substrate specificity and an in vivo connection with the activity of proline isomerase Ess1.</p><p>The main focus of this thesis has been to structurally and kinetically characterize Ssu72, in order to define its relation to known enzyme families, to provide biochemical explanations for extant in vivo observations, and to allow future structure-guided investigations of its role in coordinating transcription with co-transcriptional events. To this end, we solved the structure of Ssu72 in complex with its pS5 CTD substrate, revealing an enzyme fold with unique structural features and a surprising substrate conformation with the pS5-P6 motif of the CTD adopting the cis configuration. Together with kinetic assays, the structure provides a new interpretation of the role of proline isomers in regulating the CTD phosphorylation state, with broad implications for CTD biology.</p><p>The second goal of this thesis has been to develop new methods for NMR studies of large proteins, which present unique challenges to conventional methods, including fast signal decay and severe signal degeneracy. The first of these new methods, the `just-in-time' HN(CA)CO, improves the sensitivity of a common backbone assignment experiment. The next two methods, the 4-D diagonal-suppressed TROSY-NOESY-TROSY and the 4-D time-shared NOESY, were designed for use with sparse sampling techniques that allow the acquisition of high-resolution, high-dimensionality datasets. These efforts culminate with global fold calculations for large proteins, including the 23 kDa Ssu72, with accurate and unambiguous automated assignment of NOE crosspeaks. We expect that the methods presented here will be particularly useful as the NMR community continues to push toward higher molecular weight targets.</p> / Dissertation

Die vorliegende Arbeit untersucht mit bioinformatischen Methoden die biologische Bedeutung von Ähnlichkeiten in Kleinstrukturen und peptidischen Sequenzmotiven sowie lokaler und globaler Sequenzähnlichkeit. Der erste Teil der Arbeit behandelt chemische Ähnlichkeiten. Ausgehend von bekannten Inhibitoren der Fehlfaltung des Prionproteins wurde eine Datenbank pharmakologischer Wirkstoffe nach chemisch und strukturell ähnlichen Substanzen durchsucht und 16 Substanzen als neue potentielle Inhibitoren der Fehlfaltung vorgeschlagen. Der nächste Teil untersucht Ähnlichkeiten in Sequenzmotiven, die eine Interaktion mit Pex19, dem Importrezeptor für peroxisomale Membranproteine, vermitteln. In Zusammenarbeit mit einer experimentellen Arbeitsgruppe konnte die Bindestelle charakterisiert und Präferenzen für bestimmte Aminosäuren herausgearbeitet werden. Das Bindemotiv ist eine vermutlich helikale Region mit verzweigtkettigen aliphatischen und basischen Aminosäuren. Aus experimentellen Daten konnte eine positionsabhängige Vorhersagematrix erstellt und validiert werden. Die Beziehung zwischen lokalen Sequenzähnlichkeiten und der Konformation von Prolylbindungen in Proteinen ist Thema des dritten Teils. Die Aminosäurepräferenzen in der Nachbarschaft von cis- und trans-Prolylresten unterscheiden sich, und beide zeigen unterschiedliche Austauschpräferenzen bei Mutationen. Im Gegensatz zu lokaler Sequenzähnlichkeit ist eine globale Sequenzähnlichkeit von nur 20% ein wesentlich besserer Indikator für das Auftreten von cis-Prolylbindungen. Der letzte Teil befaßt sich mit inverser Sequenzähnlichkeit zwischen Proteinen, die wesentlich öfter auftritt als erwartet. Proteine aus einem nichtredundanten Datensatz wurden gleich- und gegenläufig aligniert und strukturelle Ähnlichkeiten zwischen den aufgefundenen Proteinpaaren untersucht. Es konnte gezeigt werden, daß bis auf kurze Sekundärstruktur-Einheiten eine inverse Sequenzähnlichkeit zwischen Proteinen keine strukturelle Ähnlichkeit impliziert. / This work is dealing with the biological impact of similarities between chemical structures, protein sequence motifs and local sequence surrounding as well as global sequence similarity. All four aspects are analyzed by computational methods. The first part is dealing with chemical similarities. Based on a recently published set of prion protein misfolding inhibitors, a data base of approved drugs has been screened for compounds with chemical and structural similarities to these substances. 16 drugs are proposed as new potential inhibitors of prion protein aggregation. The next part addresses similarities of sequence motifs which mediate the interaction with the peroxisomal membrane protein import receptor Pex19. In cooperation with an experimental group, the binding site could be characterized, and amino acid preferences of the different positions of the motif have been determined. The binding motif is a probably helical region of target proteins bearing branched aliphatic and basic residues. A position specific scoring matrix for the prediction of Pex19 binding sites could be generated and validated. The relation between local sequence similarity and prolyl bond conformation is examined in the third part. Amino acid preferences of neighboring residues differ between cis and trans prolyl residues, and both species show different amino acid exchange patterns upon mutation. In contrast to local sequence similarity, overall sequence similarity between proteins as low as 20% is a much better indicator for the occurrence of cis prolyl bonds. The last part focuses on inverse sequence similarity between proteins which occurs far more often than expected by chance. Proteins from a nonredundant data set have been aligned in parallel and antiparallel, and structural similarities between the detected protein pairs have been examined. It could be shown that, with the exception of short secondary structural elements, inverse sequence similarity does not imply structural similarity.