20.500.12556/RUL-127649 In vitro evolucija holesterol-vezavne domene z uporabo lipidnih membran In vitro evolution of a cholesterol-binding domain using lipid membranes S spremembami genskega materiala so skozi proces evolucije nastale značilne interakcijske površin bioloških molekul, ki z medsebojnim delovanjem usmerjajo fiziološke procese. Veliko interakcijsko platformo številnih proteinov predstavljajo biološke membrane. Periferni membranski proteini lahko specifično prepoznajo membranske lipide in so pogosto vpleteni v mehanizme napada in obrambe. Veliko skupino virulentnih dejavnikov po Gramu pozitivnih bakterij predstavljajo toksini iz družine od holesterola odvisnih citolizinov, katerih arhetipski predstavnik je erfringolizin O iz bakterije Clostridium perfringens. Začetni kontakt vodotopnih monomerov toksina omogočajo aminokisline v zankah vezavne domene, ki delujejo kot determinanta holesterolne specifičnosti, vendar točen mehanizem prepoznave membranskega holesterola še ni pojasnjen. Tudi možna biokemijska raznolikost interakcijeske površine še ni bila opisana. S pristopom in vitro evolucije s tehniko prikaza na ribosomih ob uporabi lipidnih veziklov različne sestave, smo proučevali biokemijsko in biofizikalno raznolikost stične površine proteina perfringolizina O. Po afinitetni selekciji na vezikle z visoko vsebnostjo holesterola se predvsem obogatijo aminokisline, ki so enake ali kemijsko podobne evolucijsko ohranjenim aminokislinam, in tudi redke različice, ki predstavljajo alternativno evolucijsko pot. Po afinitetni selekciji na vezikle brez holesterola nismo zaznali jasne aminokislinske obogatitve kar kaže, da je vezavna domena toksina evolucijsko izoblikovana za specifično prepoznavo membranskega holesterola in da z vsesplošnimi zamenjavami aminokislin ni možno spremeniti specifičnosti vezave. Characteristic interaction surfaces of biological molecules have evolved through modifications of genetic material to control physiological processes through biomolecular interactions. Biological membranes represent a large interaction platform for many proteins. Peripheral membrane proteins can specifically recognize membrane lipids and are often involved in attack and defense mechanisms. Toxins from the family of cholesterol-dependent cytolysins represent a large group of virulence factors of Gram-positive bacteria, the archetypal representative of which is perfringolizine O from the bacterium Clostridium perfringens. The initial contact of the water-soluble toxin monomers is enabled by amino acids in the loops of the binding domain, which act as determinants of cholesterol specificity, although the exact mechanism of membrane cholesterol recognition remains to be elucidated. The biochemical diversity of the interaction sites has also not yet been described. Using the in vitro evolution approach with ribosome display and lipid vesicles of different composition, we investigated the biochemical and biophysical diversity of the perfringolysin O protein interaction surface. Affinity selection for cholesterol-rich vesicles mainly enriched amino acids identical or chemically similar to naturally conserved amino acids. We also identified rare variants representing an alternative evolutionary pathway. No unique amino acid enrichment was detected after affinity selection for cholesterol-free vesicles, suggesting that the binding domain of the toxin is evolutionarily adapted to specifically recognize membrane cholesterol and that amino acid substitutions in the contact surface cannot alter the specificity of the protein. In vitro evolucija ribosomski prikaz od holesterola odvisni citolizini perfringolizin O holesterol-vezavna domena interakcije proteinov in membranskih lipidov genske knjižnice evolucijske poti In vitro evolution ribosomal display cholesterol dependent cytolysins perfringolysin O cholesterol-binding domain protein-membrane lipid interactions gene libraries evolutionary pathways true false false [A. Šakanović] Slovenski jezik Angleški jezik Doktorsko delo/naloga 2021-06-18 07:15:19 2021-06-18 07:15:24 2022-09-06 03:52:25 0000-00-00 00:00:00 2021 0 Ljubljana 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 601.4:575.224.4:602.6:557.087/.88(043.3) 191255 67891459 15214.pdf 15214.pdf 1 247BD8CC27B3221C44EEE197EF34AA43 0c308dd146ebd8500bcd54ec7341b6c716c868db67b8b32bdcf0b80134f77e1e 15a45754-cff4-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=144120 15216.pdf 15216.pdf 1 A90C32033528F39857611AAD7812C9D1 fd92db1e38d8b06dc3831dcc727d5bb520f2c57d8351fd29c5f85428f6ba99c7 15a648c3-cff4-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=144121 15217.pdf 15217.pdf 1 F4BFA75E3E627B0BAECCE80ED921E9D9 f48818ae5ba4dfa29a066b78213dd776462b3a4bc6d100fbded9c385ae65c54a 15a69c00-cff4-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=144122 15218.pdf 15218.pdf 1 0FD4017FA0643B32990DAE7E43F9B1D9 13fba73863f8ec7b35791dff989d4aa66ad420244691ff733422f44c6bec4456 15a6d4d6-cff4-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=144123 Biotehniška fakulteta 0 0 0