Design eines Stabilisierungs- und Schaltmodules für alpha-helikale Proteine

In this work, the concept of a modular approach to stabilize and switch the structure of α-helical proteins and peptides was investigated. To achieve such, we designed a fusion protein which consists of two parts. The first part, an independent protein module, comprises a highly stable helical fold and the possibility to incorporate a light responsive unit for manipulation of the structure. The second part consists of a peptide sequence with a structure-dependent activity. We used the 20 amino acid long Trp-cage miniprotein as basis for the stabilizing and switching module. Its tertiary structure consists of an eight amino acid long N-terminal α-helix, whose fold is induced by encapsulation of the eponymous tryptophan side chain by several C-terminal residues. This results in a very stable tertiary fold of the module. In order to introduce switchability into the miniprotein, we cross-linked the Trp-cage with a photo responsive linker. In this work, we used N-hydroxysuccinimide esters (NHS) as reactive groups for the cross-linking, which are reactive towards primary amines (lysine residues), and azobenzene as the scaffold for a switchable linker. In the second part of this work, one repeat of the α-helical antifreeze protein AFP "Type 1 HPLC6" was fused with the Trp-cage module using a chimera strategy. In order to combine and correlate the secondary structure of the proteins, the sequences were overlapped, resulting in a chimera protein. This protein comprises at the N-terminus the sequence of the antifreeze protein, followed by a chimera region with a combination of both sequences, and is completed by the C-terminal sequence of the Trp-cage. The junction of the sequences is possible in four different ways altering the orientation of the active side of the antifreeze protein relative to the tertiary structure of the Trp-cage. All four variants were spectroscopically characterized in order to ascertain their structure. Furthermore, the activity of the four chimera proteins was determined by observing the ice crystal growth in presence of the proteins.In dieser Arbeit wurde ein modulares Konzept zur Stabilisierung und Schaltung der Struktur α-helikaler Proteine und Peptide untersucht. Zur Realisierung dieses Konzeptes haben wir ein Fusionsprotein entwickelt, welches sich aus zwei Teilen zusammensetzt. Der erste Teil besteht aus einem Protein mit einer stabilen helikalen Faltung und der Möglichkeit, eine photoresponsive Einheit zur Manipulation der Struktur einzuführen. Der zweite Teil wird von einer Peptidsequenz mit strukturabhängiger Aktivität gebildet. Das Schaltmodul basiert auf dem 20 Aminosäuren langen Trp-cage Miniprotein, dessen Struktur eine acht Aminosäuren lange N-terminalen α-Helix enthält. Die Faltung wird durch hydrophobe Wechselwirkungen einer Tryptophan–Seitenkette mit mehreren C-terminalen Aminosäuren induziert, was zu einer hoch stabilen, tertiären Faltung führt. Zur Schaltung der Faltung haben wir in diese Struktur ein photoresponsives Molekül eingeführt. In dieser Arbeit wurde N-Hydroxysuccinimid-Ester als reaktive Gruppe und Azobenzol als Grundgerüst verwendet. Im zweiten Teil der Arbeit wurde eine Wiederholungseinheit des α-helikalen Antigefrier- proteins „Type 1 HPLC6“ mit dem Trp-cage in einem Chimären-Konzept fusioniert. Zur Verknüpfung und Korrelation der Sekundärstrukturen beider Proteine wurden ihre Sequenzen überlappt, was zu einem Chimären-Protein führt. Dieses Protein lässt sich in drei Abschnitte einteilen. Die Sequenz des Antigefrierproteins bildet den N-terminalen Abschnitt, der mittlere Abschnitt enthält Sequenzen beider Proteine, während der C-terminale Abschnitt aus der Trp-cage-Sequenz besteht. Nach diesem Prinzip ergeben sich vier Kombinationsmöglichkeiten unter Berücksichtigung der Orientierung der aktiven Seite des Antigefrierproteins und der Tertiärstruktur des Trp-cages. Alle vier Varianten wurden zur Strukturbestimmung spektroskopisch charakterisiert. Darüber hinaus wurde die Aktivität der vier Chimären-Proteine mittels Beobachtung des Eiskristallwachstums in Gegenwart der Proteine bestimmt

Different Secondary Metabolite Profiles of Phylogenetically almost Identical Streptomyces griseus Strains Originating from Geographically Remote Locations

As Streptomyces have shown an outstanding capacity for drug production, different campaigns in geographically distant locations currently aim to isolate new antibiotic producers. However, many of these newly isolated Streptomyces strains are classified as identical to already described species. Nevertheless, as discrepancies in terms of secondary metabolites and morphology are possible, we compared two Streptomyces strains with identical 16S rRNA gene sequences but geographically distant origins. Chosen were an Easter Island Streptomyces isolate (Streptomyces sp. SN25_8.1) and the next related type strain, which is Streptomyces griseus subsp. griseus DSM 40236T isolated from Russian garden soil. Compared traits included phylogenetic relatedness based on 16S rRNA gene sequences, macro and microscopic morphology, antibiotic activity and secondary metabolite profiles. Both Streptomyces strains shared several common features, such as morphology and core secondary metabolite production. They revealed differences in pigmentation and in the production of accessory secondary metabolites which appear to be strain-specific. In conclusion, despite identical 16S rRNA classification Streptomyces strains can present different secondary metabolite profiles and may well be valuable for consideration in processes for drug discover

Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs.

Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR

A Simple and Convenient Synthesis of Unlabeled and ¹³C-Labeled 3-(3-Hydroxyphenyl)-3-Hydroxypropionic Acid and Its Quantification in Human Urine Samples

An improved method to synthesize the highly abundant and biomedically important urinary metabolite 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) is reported. The modified protocol is based on an indium-mediated sonochemical Reformatsky reaction. The synthesis is a simple two-step route as opposed to a complex four-step route previously reported in the literature that requires specialized equipment, flammable materials, and high-pressure reaction vessels. The described procedure also provides an expedient route to prepare a 13C isotopically labeled HPHPA that can be used as a standard for quantitative LC-MS analysis. This report also illustrates how the synthesized metabolite standard was used to detect and accurately quantify its presence in human urine samples using both NMR and LC-MS techniques

Antitumor Anthraquinones from an Easter Island Sea Anemone: Animal or Bacterial Origin?

The presence of two known anthraquinones, Lupinacidin A and Galvaquinone B, which have antitumor activity, has been identified in the sea anemone (Gyractis sesere) from Easter Island. So far, these anthraquinones have been characterized from terrestrial and marine Actinobacteria only. In order to identify the anthraquinones producer, we isolated Actinobacteria associated with the sea anemone and obtained representatives of seven actinobacterial genera. Studies of cultures of these bacteria by HPLC, NMR, and HRLCMS analyses showed that the producer of Lupinacidin A and Galvaquinone B indeed was one of the isolated Actinobacteria. The producer strain, SN26_14.1, was identified as a representative of the genus Verrucosispora. Genome analysis supported the biosynthetic potential to the production of these compounds by this strain. This study adds Verrucosispora as a new genus to the anthraquinone producers, in addition to well-known species of Streptomyces and Micromonospora. By a cultivation-based approach, the responsibility of symbionts of a marine invertebrate for the production of complex natural products found within the animal’s extracts could be demonstrated. This finding re-opens the debate about the producers of secondary metabolites in sea animals. Finally, it provides valuable information about the chemistry of bacteria harbored in the geographically-isolated and almost unstudied, Easter Island