Doppelsiegel-MLU
logo
Research Projects
"Our group participated in the EU COST action BM1403 "Native Mass Spectrometry and Related Methods for Structural Biology". "The First Community-Wide, Comparative Cross-linking Mass Spectrometry Study.". Community Paper (PubMed)
Novel MS-Cleavable Cross-Linkers for Protein Structure Characterization

Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross-linked products presents an alternative approach for assessing low-resolution protein structures and for gaining insight into protein interfaces. This project aims to design innovative amine-reactive, N-hydroxy succinimide (NHS) cross-linkers. Our collision-induced dissociative cross-linkers lead to the formation of indicative fragment ions and constant neutral losses in MS/MS spectra, allowing an unambiguous identification of cross-linked peptides. ESI and MALDI mass spectrometry can be used for analyzing the created cross-linked products.

Our MS-cleavable, urea-based linker is shown below, offering the possibility to identify cross-linked products in an efficient and robust manner. This project is conducted in cooperation with Dr. Mathias Schäfer, University of Cologne (DFG project Si 867/15-1 and 15-2).


Our cross-linking reagent is now commercially available at Thermo Fisher Scientific with the name DSBU (disuccinimidyl dibutyric urea):

Thermo Fisher Product Catalog: DSBU
Structure of Full-Length p53 Tumor Suppressor Probed by Chemical Cross-Linking and Mass Spectrometry

The tumor suppressor p53 presents a great challenge for 3D-structural analysis due to its inherent flexibility. In this project, our aim is to gain insights into the structure of full-length wild-type human p53 in solution by chemical cross-linking/mass spectrometry (MS). This approach allows us obtaining structural information of DNA-free p53 in solution without making use of the ultrastable quadruple p53 variant. The cross-links within one p53 monomer are in good agreement with the SAXS-based model of full-length p53. Our cross-linking data between different p53 molecules in the tetramer however indicate a large degree of flexibility in the C-terminal regulatory domain of full-length p53 in the absence of DNA. The cross-links suggest that the C-terminal regulatory domains are much closer to each other, resulting in a more compact arrangement of the p53 tetramer than perceived by the SAXS model.

Structural Insights into Retinal Guanylylcyclase/GCAP-2 Interaction by Cross-Linking and Mass Spectrometry

The retinal guanylylcyclases ROS-GC 1 and 2 are regulated via the intracellular site by guanylylcyclase-activating proteins (GCAPs). The mechanism of how GCAPs activate their target proteins remains elusive as exclusively structures of non-activating calcium-bound GCAP-1 and -2 are available. In this work, we apply a combination of chemical cross-linking with amine-reactive cross-linkers and photo-affinity labeling followed by MS analysis of the created cross-linked products to study the interaction between N-terminally myristoylated GCAP-2 and a peptide derived from the catalytic domain of full length ROS-GC 1. Based on the distance constraints imposed by the cross-links we are creating structural models of the calcium-loaded complex between myristoylated GCAP-2 and the GC peptide.

Investigating Calmodulin/Munc13 Interaction

The efficacy of synaptic transmission between neurons can be transiently altered during neuronal network activity. This phenomenon of short-term plasticity is a key determinant of network properties, is involved in many physiological processes such as motor control, sound localization, or sensory adaptation, and is critically dependent on cytosolic calcium concentration. Due to their essential function in synaptic vesicle priming and in the modulation of synaptic strength, Munc13 proteins are key regulators of presynaptic short-term plasticity. However, the underlying molecular mechanisms and the identity of the calcium sensor/effector complexes involved are unclear. All four Munc13 isoforms share a common domain structure, including a calmodulin (CaM) binding site in their otherwise divergent N-termini. By combining chemical cross-linking, photoaffinity labeling, and mass spectrometry, we show that all neuronal Munc13 isoforms exhibit similar CaM binding modes. Moreover, we demonstrate that the 1-5-8-26 CaM binding motif discovered in Munc13-1 cannot be induced in the classical CaM target skMLCK (skeletal muscle myosin light chain kinase), indicating unique features of the Munc13 CaM binding motif.

This project is conducted in cooperation with Dr. Olaf Jahn, Max-Planck Institute for Experimental Medicine, Göttingen, and was financially supported by the Graduiertenkolleg GRK 1026 (Conformational Transitions in Macromolecular Interactions) at the Martin-Luther University Halle-Wittenberg until 2014.
© 2019 Sinz-Lab