BEGIN:VCALENDAR VERSION:2.0 PRODID:-//CERN//INDICO//EN BEGIN:VEVENT SUMMARY:Contr. talk - Production of Bcl-2 proteins involved in regulation of mitochondrial apoptosis DTSTART;VALUE=DATE-TIME:20210526T080000Z DTEND;VALUE=DATE-TIME:20210526T082000Z DTSTAMP;VALUE=DATE-TIME:20260721T193242Z UID:indico-contribution-185-1138@lindico453.srv.lu.se DESCRIPTION:Speakers: Jörgen Ådén (Umeå University)\nProgrammed cell d eath (apoptosis) is essential for human life. In its intrinsic pathway\, t he Bcl-2 (B-cell lymphoma 2) protein family regulates cell life and death by controlling permeability of the mitochondrial outer membrane (MOM). How ever\, the molecular basis of cell protection by its anti-apoptotic Bcl-2 members remains elusive due to the lack of detailed structural insight int o their action at the MOM to ensure its integrity. To provide atomic-level insight into their functioning\, we will use the founding member of this family\, the human antiapoptotic Bcl-2 protein itself\, whose involvment i n p53 regulation and its overexpression\nplays a notorious role in many ca ncers and their treatment resistance. However\, for a long time\, obtainin g sufficient protein was cumbersome due to its insolubility as a membrane\ nprotein and low yields. Therefore\, we establishes an expression and puri fication protocol [1] to produce routinely mg amounts of the fully functio nal full-length human isoform 2 of Bcl-2\n(Bcl-2(2)) which can also be iso topically labelled as 15N/13C/2H versions\, ideally suited for neutron and NMR studies. The protocol even allows to generate residue specific Bcl-2\ nmutants and various constructs [2].\n\n1. Aden\, A.U. Mushtaq\, A. Dingel dein\, M. Wallgren\, G. Gröbner. A novel recombinant expression and purif ication approach for the full-length anti-apoptotic membrane protein Bcl-2 . Protein expression and purification. 172 (2020) 105628.\n2. A. Ul Mushta q\, J. Aden\, T. Sparrman\, M. Hedenstrom\, G. Gröbner. Insight into Func tional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein-A Promis ing Cancer Drug Target. Molecules 26 (2021) 1467\, 1-14\n\nhttps://lindico 453.srv.lu.se/event/219/contributions/1138/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1138/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - Towards Neutron crystallography of membrane proteins : Insights into production of deuterium-labelled OmpF DTSTART;VALUE=DATE-TIME:20210526T074000Z DTEND;VALUE=DATE-TIME:20210526T080000Z DTSTAMP;VALUE=DATE-TIME:20260721T193242Z UID:indico-contribution-185-1137@lindico453.srv.lu.se DESCRIPTION:Speakers: Swati Aggarwal (European Spallation Source)\nHydroge n bonds play a crucial role for protein function and involved in almost ev ery mechanism from foundation of protein structure to enzyme catalysis. Hy drogen (1H) atoms form the basis of hydrogen bond that is not scattered by X-ray crystallography due to its poor scattering power. Neutron protein c rystallography (NPX) is a powerful tool that is capable of locating hydrog ens and study the significance of hydrogen bonding interactions in biomacr omolecules. However\, due to\nthe requirement of large crystals very few n eutron structures have been deposited in PDB with no membrane protein stru cture determined yet. Additionally\, 1H has a negative scattering length a nd large incoherent cross-section giving rise to a significant background noise in neutron data collection.\nThis effect can be minimized by isotopi c substitution of 1H with its heavier isotope deuterium (2H or D) leading to less ambiguous data analysis and better structure interpretation. Overa ll ˜25% H atoms in a protein are solvent exchangeable and can be exchange d by dissolving in heavy water. However\, complete deuterium labelling (pe rdeuteration) is required for the remaining 75% H atoms. In this work\, an optimized methodology for large scale production of perdeuterated bacteri al outer membrane protein F (OmpF) has been designed. OmpF was produced in deuterated minimal medium with different carbon sources. Mass spectrometr y and thermal stability experiments\nverified the purity and level of deut eration of OmpF protein. Perdeuterated OmpF crystals also diffracted X-ray s to 9 Å resolution emphasising the need of fine tuning of perdeuterated crystallisation conditions.\n\nhttps://lindico453.srv.lu.se/event/219/cont ributions/1137/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1137/ END:VEVENT BEGIN:VEVENT SUMMARY:Keynote talk - Membrane protein purification and application in dr ug discovery DTSTART;VALUE=DATE-TIME:20210526T070000Z DTEND;VALUE=DATE-TIME:20210526T074000Z DTSTAMP;VALUE=DATE-TIME:20260721T193242Z UID:indico-contribution-185-1136@lindico453.srv.lu.se DESCRIPTION:Speakers: Arjan Snijder (Astra Zeneca)\nIn the presentation I will discuss our capabilities\, processes and methods for membrane protein expression and purification\, including our recently developed teabag pur ification method. The teabag method allows rapid purification of membrane proteins with reduced hands-on and processing time. Application of membran e proteins in drug discovery will be illustrated by our work on the protea se-activated receptor 2\, where extensive protein engineering allowed DNA- encoded library screening on purified receptor\, establishment of biophysi cal assays\, and structure assisted drug design.\n\nhttps://lindico453.srv .lu.se/event/219/contributions/1136/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1136/ END:VEVENT END:VCALENDAR