Abstract - Stief
Sensitivity-enhanced 15N NMR relaxation experiments with improved water-suppression at high magnetic fields provide novel insights into the structural dynamics of the SNARE protein SNAP25a
Chemical information transfer between neurons in our brain is driven by an unidirectional flow of neurotransmitters between neurons. Neurotransmitters are packaged in vesicles located in the axon terminal of the neuron. To release neurotransmitters, the vesicle membrane fuses with the presynaptic plasma membrane. After membrane fusion, the neurotransmitters pass the synaptic cleft to dedicated receptors in the postsynaptic neuron's membrane. This process of neurotransmitter release is known as neuronal exocytosis. SNARE (soluble N-ethylmaleimide-sensitive-factor attachment receptor) proteins are central for driving synaptic vesicle fusion at the neuronal synapse. The assembly of the SNARE complex provides the energy necessary for membrane fusion. The structural transitions of the SNARE proteins and their membrane interactions are, however, not well understood at the molecular level. We studied SNAP25a (synaptosomal-associated protein of 25kDa), one of the three SNARE proteins, by NMR spectroscopy to obtain new structural insights in the pre-fusion state: SNAP25a is mostly intrinsically disordered and shows high internal flexibility. Two α-helices form the N-terminal part of the first SNARE motif of SNAP25a, but the remainder of the protein is intrinsically disordered, including the second SNARE motif. We hypothesize that the SNAP25a N-erminus may act as a nucleation site for initiating SNARE zippering. [1] To study the backbone dynamics of SNAP25a and other intrinsically disordered proteins (IDPs), we optimized a set of sensitivityenhanced backbone relaxation experiments (R1, R1ρ) for improved water-suppression, yielding high signal-to-noise (SNR) for intrinsically disordered proteins (IDPs) and small proteins. We tested these sequences on protonated and deuterated samples and found a strong correlation.
[2] [1] Stief T, Gremer L., Pribicevic S., Espinueva D.F., Vormann K., Biehl R., Jahn R., Pérez-Lara Á., Lakomek N.A., J Mol Biol. 2023 Mar 30;435(10):168069. [2] Stief T., Vormann K., Lakomek N.A., Methods 2024, 223,1-15