Research

We apply and develop various mass spectrometric techniques including cross-linking, native mass spectrometry, proteomics and lipidomics to study protein-lipid complexes. While proteomics and lipidomics are employed to identify the components of the protein-lipid-assemblies, structural mass spectrometric techniques deliver novel insights into protein-lipid interactions. Importantly, the combination of various mass spectrometric techniques delivers insights into the structure and function/regulation of protein-ligand complexes which is difficult to achieve with the individual techniques alone. By implementing biophysical and biochemical approaches, we specifically aim to investigate the architecture of synaptic membranes. We therefore establish membrane mimetics such as liposomes or nanodiscs and combine these with mass spectrometric analyses.

 

SNARE complex assembly and interactions with regulatory proteins

The neuronal SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25, both anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters. While the general sequence of SNARE complex assembly is well-understood, our knowledge on the intermediates that form as well as alternative assembly routes and off-pathway complexes is incomplete. In addition, the interplay of regulators such as Synaptotagmin-1, the Complexins or Munc 13/18 is only sparsely understood. We, therefore, study the assembly of the SNARE complex as well as interactions with various regulatory proteins. Using native mass spectrometry and chemical cross-linking mass spectrometry, we identify the complexes that form, their stoichiometry, ligand binding as well as interaction sites. Combining this information allows us to establish an assembly pathway.
                                         Hesselbarth, Schmidt (2023) Commun Biol 6(1): 198.

 

Unraveling multivalent interactions in the pre-synapse

Signal transmission between neurons takes place at specifically designed contact sites called synapses. Successful transmission of the signal strongly relies on the organisation of the synapse into different functional compartments. In the active zone, synaptic vesicles are tethered to the plasma membrane and maintained in an activated state until an action potential arrives. The active zone assembles from a set of scaffold proteins including RIM, RIM-BP, Munc-13, ELKS and a-Liprins. However, quantitative and mechanistic insights are still missing. In this project, we propose to unravel the architecture of the active zone and to study the phase behaviour of specific scaffold proteins, therefore, highlighting their multivalent interactions. Specifically, by combining experimental and computational approaches, we will generate a coarse-grained model of the active zone and establish phase-diagrams of specific sets of proteins describing their contribution to condensate formation. We will then integrate functional proteins into this model and explore the impact of phase separation on vesicle recruitment. We will thus contribute to the understanding of compartmentalization in the synapse and, in a larger context, the functional role of multivalent interactions in well-orchestrated processes such as signal transmission. The project is part of the "CRC 1551 - Polymer Concepts in Cellular Function" at the Johannes Gutenberg University.

 

Establishing a Protein-Lipid Cross-linking Workflow for Studying Protein-Lipid Interactions in Natural Membranes

Membrane proteins are involved in many fundamental processes of life; however, it became apparent that they do not function alone and rather require a specific lipid environment. The structural analysis of protein-lipid interactions is, therefore, of utmost importance to understand the function and regulation of membrane proteins. Recent developments and improvements made mass spectrometry a powerful tool in structural biology including the analysis of protein-lipid interactions. However, the available approaches do not provide information on the exact interaction sites between proteins and lipids, and accurate models of protein-lipid assemblies are, therefore, difficult to obtain. In this research project, we aim at establishing a complete workflow for protein-lipid cross-linking allowing the analysis of protein-lipid interactions directly from native membranes. We will thus make the next step towards understanding the relevance of protein-lipid contacts in functionally active membrane protein assemblies. The project is funded through a RiseUp! grant of the Boehringer Ingelheim Stiftung.