Crystallization of apotransferrin derivatives with small chiral molecules

Abstract

Transferin je protein krvne plazme koji se nalazi u skoro svim živim bićima i njegove su glavne funkcije transport i reguliranje količine željeza u krvi. Apotransferin je slobodan oblik proteina, onaj na koji nije vezano željezo. Koristi se u kapilarnoj elektroforezi za razdvajanje enantiomera nekih malih kiralnih molekula no još nije dovoljno istraženo gdje i kako se te molekule vežu na apotransferin. Rentgenskom strukturnom analizom mogu se dobiti takve informacije. U ovom radu opisan je postupak kristalizacije apotransferina s malim kiralnim molekulama metodom difuzije para viseće kapi. Cilj je rada bio dobiti što kvalitetniji kristal različitih kompleksa apotransferin – mala kiralna molekula, a koji bi bili podvrgnuti difrakciji rentgenskih zraka. Na taj bi način dobili uvid u strukturu te mjesto i moguće načine vezanja malih molekula na apotransferin. Uspješno je iz kristalizacijskih otopina kojima su dodane male molekule nastalo nekoliko kristala no nažalost nisu bili zadovoljavajuće kvalitete za strukturno istraživanje difrakcijom rentgenskih zraka.Transferrin is a blood plasma protein found in almost all living organisms and its main functions are to transport and regulate the amount of iron in the blood. Apotransferrin is the free form of the protein, without bound iron. It has been used in capillary electrophoresis to separate enantiomers of some small chiral molecules. There are no data yet about the sites and ways these molecules bind to apotransferrin. X-ray diffraction of such molecules can give us such information. In this thesis the crystallization process of apotransferrin with small chiral molecules by the hanging drop vapour diffusion method is described. The aim of this work was to obtain crystals of different complexes of apotransferrin with small chiral molecules. Crystals would be subjected to X-ray diffraction which would give insight into the structure and possible ways of binding of small molecules to apotransferrin. Several crystals were successfully obtained from crystallization solutions with added small molecules. Unfortunately the resulting crystals were not of satisfactory quality for the X-ray diffraction structure analysis