Stability and structural characterization of the FlgD protein from Helicobacter pylori

Abstract

Protein FlgD bakterije Helicobacter pylori (HpFlgD) šaperon je koji igra ulogu tijekom sastavljanja kuke biča. Kuka biča dio je aparata za pokretanje koji je ujedno i faktor virulencije u ove bakterije. Bakterije koje su mutanti i nemaju tog proteina daju fenotipski nepokretne bakterije što govori da je njegova funkcija vrlo bitna prilikom sinteze kuke biča. Ovaj rad opisuje transformaciju bakterija Escherichia coli vektorom s ugrađenim genom proteina HpFlgDHis6, prekomjernu ekspresiju, pročišćavanje afinitetnom kromatografijom i gel filtracijom, postavljanje kristalizacije s čistim uzorkom proteina i rješavanje kristalne strukture proteina. Tijekom kristalizacije korišteni su uvjeti u kojima je variran udio (w/v) PEG 1500, pH pufera SPG, temperatura (4 °C i 16 °C) i koncentracija proteina. Podatci dobiveni rentgenskom strukturnom analizom kristala proteina HpFlgD dali su strukturni model do rezolucije od 2,86 Å. Riješena struktura je pokazala da je protein građen iz dvije domene, Tudor i Fn III, koje su sastavljene od β ploča i petlji. Struktura je slična dvijema poznatim strukturama proteina, FlgD iz bakterija Xanthomonas campestris i Pseudomonas aeruginosa. Ključna razlika između već poznatih struktura i ove jest zarotirani položaj domene Tudor.FlgD protein from Helicobacter pylori (HpFlgD) is a chaperon, which plays a role during hook assembly. The hook is a part of the motility apparatus and is also a virulence factor. The lack of the flgD gene makes bacteria immotile and that fact highlights the importance of the FlgD protein during synthesis of flagella. This thesis describes the transformation of Escherichia coli with the vector with sequence coding for HpFlgD His6, over-expresion of protein, isolation by affinity chromatography and gel-filtration methods, and crystallization trials with a pure protein sample. During the crystallization trials the following conditions were varied: concentration of PEG 1500 (w/v), pH of the SPG buffer, temperature (4 °C and 16 °C), and concentration of the protein. The data obtained by the X-ray structure analysis of FlgD protein enabled us to make a structural model up to 2,86 Å resolution. The solved structure of the HpFlgD protein is similar to the two already known structures of FlgD proteins from Xanthomonas campestris and Pseudomonas aeruginosa. The protein has two domains, Tudor and Fn III, which are built up of β sheets and loops. The main difference between the two known structures and this one is the rotated position of the Tudor domain