Naslov
Temperature-dependent structural changes in Cas3 protein in Escherichia coli

Autori
Markulin, Dora ; Peharec Štefanić, Petra ; Čulo, Anja ; Pandžić, Marta ; Matković, Marija ; Ivančić Baće, Ivana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
FEBS Open Bio, 9 / - , 2019, 271-271

Skup
44th FEBS Congress ; From Molecules to Living Systems

Mjesto i datum
Kraków, Poljska, 06.07.2019. - 11.07.2019

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
CRISPR-Cas ; Cas3 ; circular dichroism ; intrinsic tryptophan fluorescence ; E. coli

Sažetak
The CRISPR-Cas system is a significant mechanism of bacteria and archaea that provide adaptive immunity against viruses and plasmids. It consists of DNA repeats separated by spacers of foreign origin (CRISPR locus), and cas genes responsible for various stages of defense. In E. coli, Cas3 protein is involved in a degradation of invader DNA as a last stage of defense. Recent studies showed that Cas3 activity is temperature-dependent and is lost at 37°C, unless the protein is present in abundance. In this work, we wanted to investigate the mechanism of Cas3 activity loss by determining conformational changes of purified protein at different temperatures in vitro. We monitored structural changes by measuring a change of ellipticity using a circular dichroism method (CD) and by measuring intrinsic tryptophan fluorescence (ITF) using a fluorescence spectrometry method. Employing CD method, we observed a conformational change in helical region at 35°C what was confirmed by ITF method. The obtained results are in an agreement with protein activity change in vivo. Furthermore, we introduced point mutations in three different α-helices in order to investigate which helical region is responsible for temperature-dependent activity of Cas3. Based on our in vitro and in vivo data, our results indicate a possible role for one α-helix that we discuss and propose a model of regulation. Overall, the results of this research will contribute to a better understanding of the regulation of Cas3 activity as well as to the progress of the CRISPR-Cas field.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA