3 results on '"Marika Slivacka"'
Search Results
2. Conformational dynamics of the human serotonin transporter during substrate and drug binding
- Author
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Søren G. F. Rasmussen, Claus J. Loland, Marika Slivacka, Kasper D. Rand, Ulrik Gether, Anne Kathrine Nielsen, and Ingvar R. Möller
- Subjects
0301 basic medicine ,Serotonin ,Science ,General Physics and Astronomy ,02 engineering and technology ,Citalopram ,Molecular Dynamics Simulation ,Neurotransmission ,Ligands ,Serotonergic ,Mass Spectrometry ,Article ,General Biochemistry, Genetics and Molecular Biology ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Cocaine ,Protein Domains ,medicine ,Neurotransmitter ,lcsh:Science ,Serotonin transporter ,Serotonin Plasma Membrane Transport Proteins ,Multidisciplinary ,biology ,Sodium ,Ibogaine ,General Chemistry ,Cations, Monovalent ,021001 nanoscience & nanotechnology ,Recombinant Proteins ,030104 developmental biology ,Structural biology ,chemistry ,Symporter ,Potassium ,biology.protein ,Biophysics ,lcsh:Q ,0210 nano-technology ,Protein Binding ,medicine.drug - Abstract
The serotonin transporter (SERT), a member of the neurotransmitter:sodium symporter family, is responsible for termination of serotonergic signaling by re-uptake of serotonin (5-HT) into the presynaptic neuron. Its key role in synaptic transmission makes it a major drug target, e.g. for the treatment of depression, anxiety and post-traumatic stress. Here, we apply hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs. Our results reveal significant changes in dynamics in regions TM1, EL3, EL4, and TM12 upon binding co-transported ions (Na+/K+) and ligand-mediated changes in TM1, EL3 and EL4 upon binding 5-HT, the drugs S-citalopram, cocaine and ibogaine. Our results provide a comprehensive direct view of the conformational response of SERT upon binding both biologically relevant substrate/ions and ligands of pharmaceutical interest, thus advancing our understanding of the structure-function relationship in SERT., The serotonin transporter (SERT) is responsible for re-uptake of serotonin into the presynaptic neuron and plays a key role in synaptic transmission. Here, the authors use hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs.
- Published
- 2019
3. Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments
- Author
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Kasper D. Rand, Patrick S. Merkle, Ingvar R. Möller, Marika Slivacka, Marek Polák, Růžena Lišková, Alan Kadek, Petr Man, Claus J. Loland, Anne Kathrine Nielsen, Eliska Pospisilova, and Jiří Hausner
- Subjects
Models, Molecular ,Proteases ,Aspartic Acid Proteases ,Swine ,Proteolysis ,Hydrogen Deuterium Exchange-Mass Spectrometry ,010402 general chemistry ,01 natural sciences ,Antiporters ,Analytical Chemistry ,Structure-Activity Relationship ,medicine ,Escherichia coli ,Animals ,Drosophila Proteins ,Humans ,Urea ,Amino Acid Sequence ,Integral membrane protein ,Peptide sequence ,Serotonin Plasma Membrane Transport Proteins ,Dopamine Plasma Membrane Transport Proteins ,medicine.diagnostic_test ,Bacteria ,Chemistry ,Escherichia coli Proteins ,010401 analytical chemistry ,Proteolytic enzymes ,Pepsin A ,Peptide Fragments ,0104 chemical sciences ,Aquifex ,Membrane ,Drosophila melanogaster ,Membrane protein ,Biophysics ,Hydrogen–deuterium exchange - Abstract
Insight into the structure-function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl-/H+ exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MS-compatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence; however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.
- Published
- 2019
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