Priprava porotvornih proteinov stiholizina I in II ter njunih mutant za vrednotenje z mikroskopijo na atomsko silo

Majnik, Manca (author), Lunder, Mojca (mentor), García Linares, Sara (comentor)

Aktinoporini so skupina majhnih in bazičnih α-porotvornih toksinov, ki jih proizvajajo morske vetrnice. Kot vsi porotvorni proteini lahko tudi stiholizini obstajajo v dveh različnih konformacijah. Nastajajo kot topni monomeri, ki so v odsotnosti tarčnih membran stabilno zviti in vodotopni. Ko pridejo v bližino membrane tarčne celice, pride v njihovi strukturi do sprememb, ki vodijo v oligomerizacijo monomerov in do nastanka pore v membrani celice. To povzroči osmotski šok in neizogibno smrt tarčne celice. Stiholizina I in II (StnI in StnII) sta zelo podobna aktinoporina, ki ju proizvaja morska vetrnica Stichodactyla helianthus. Izkazujeta 93% homologijo zaporedja, a imata kljub temu različno hemolitično aktivnost. Ta razlika je posledica ionske interakcije, ki je prisotna v StnI, ne pa tudi v StnII, zaradi česar je odcepitev N-konca pri StnI otežena in hemolitična aktivnost nižja. Da bi to hipotezo podrobneje preučili, je skupina z Univerze Complutense v Madridu, Fakulteta za kemijske vede, Oddelek za biokemijo in molekularno biologijo pripravila dvojni cisteinski mutanti StnI (StnI I7C/K68C) in II (StnII I6C/K67C). Ti mutanti sta sposobni tvorbe disulfidnega mostička znotraj svoje strukture, kar posnema ionsko interakcijo pri stiholizinu I in oteži oddaljitev α-vijačnice in tvorbo pore. Ti štirje proteini (StnI, StnII, StnI I7C/K68C in StnII, StnII I6C/K67C) so bili funkcionalno okarakterizirani, pri čemer je bilo ugotovljeno, da so se mutanti res vežeta na membrane, ki vsebujejo sfingomielin, vendar ne napredujeta do oblikovanja pore. Naše delo je bilo razdeljeno na tri dele. Prvi in glavni del je bil kloniranje vseh štirih genov, ki kodirajo beljakovine (StnI in StnII divjega tipa ter StnI I7C/K68C in StnII I6C/K67C) v izbrani plazmid, ki omogoča izražanje izražanje rekombinantnih proteinov s peptidnimi dodatki NPS3 tako na N- kot na C- koncu, le-ti pa omogočajo specifično kovalentno vezavo v vezavni sistem s peptidoma NPS2 in NPS4, ki se uporabljata v enomolekulski tehniki mikroskopije na atomsko silo. Drugi del je predstavljalo izražanje in čiščenje rekombinantnega StnII. Tako pripravljen rekombinantni protein se lahko uporablja v enomolekularni tehniki mikroskopije na atomsko silo, pri kateri je preiskovani protein razpet med premičnim elementom in površino. Predpostavlja se, da bo ta tehnika z eno molekulo posnemala mehanske sile, katerim so stiholizini podvrženi med molekularno transformacijo ki je potrebna za njihovo sestavo v transmembransko poro. Tretji del je bil test hemolize z vsemi štirimi proteini v odsotnosti in prisotnosti DTT, sredstva, ki reducira disulfidne vezi. V prisotnosti DTT so vse štiri beljakovine pokazale primerljivo hemolitično aktivnost, medtem ko sta v odsotnosti DTT le proteina divjega tipa pokazala vidno hemolizo.

• Language: Slovenian
• Keywords: stiholizini, mutant, interakcija protein-lipid, mikroskopija na atomsko silo, tvorba pore
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-143481
• Publication date in RUL: 22.12.2022

Secondary language

• Language: English
• Title: Preparation of pore–forming proteins sticholysin I and II and their mutants for atomic force microscopy measurements

Abstract

Actinoporins constitute a group of small and basic α pore-forming toxins produced by sea anemones. In an aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. Sticholysins I and II (StnI and StnII) are two very similar actinoporins produced by the Caribbean Sea anemone Stichodactyla helianthus. They show 93% of sequence homology but, quite surprisingly, different pore-formation activity, when measured as their ability to produce hemolysis. This is due to a salt bridge, present in StnI but not in StnII, which makes the N-terminal detachment in StnI more difficult and thus impairs the hemolysis. To study this hypothesis in deeper detail, the group at Complutense University of Madrid, Faculty of Chemical Sciences, Department of Biochemistry and Molecular Biology has prepared mutants of StnI (StnI I7C/K68C) and II (StnII I6C/K67C) where two Cys residues have been strategically located to covalently bind, employing a disulfide bond, the Nterminal stretch to the β-sandwich core. The four proteins (StnI, StnII, StnI I7C/K68C, and StnII StnII I6C/K67C) have been characterized in functional terms, revealing that the mutants do bind to sphingomyelin-containing membranes but do not progress to form a pore. Our work was divided into three parts. The first and main part was the cloning of all four genes coding for proteins (StnI and StnII wild-type as well as StnI I7C/K68C and StnII I6C/K67C) in the selected plasmid, that enables expression of recombinant proteins within a specific peptides-construct facilitating further research of the proteins with atomic force microscopy. The second part was expression and purification of recombinant StnII. The peptides construct, called NPS3, would allow us to use the proteins in a single-molecule technique of AFM, in which a purified protein of interest is tethered between the tip of an AFM cantilever and a surface that can be retracted with sub-nanometer precision, thanks to piezoelectric actuators. The assumption is that this single-molecule technique will mimic the mechanical perturbations suffered by sticholysins during the molecular metamorphosis needed to assemble them into a transmembrane pore.The third part was a hemolysis assay with all four proteins in the absence and in the presence of DTT, an agent that reduces the disulfide bonds. In the presence of DTT, all four proteins showed comparable hemolytic activity, while in the absence of DTT, only wild-type proteins showed visible hemolysis.

• Keywords: sticholysins, mutants, protein-lipid interaction, atomic force microscopy, pore formation