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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.