KLINKE Sebastian
congresos y reuniones científicas
The role of the flanking regions of the LOV domain from the Brucella photoreceptor LOV-histidine kinase (Comunicación Oral)
Congreso; III Reunión de Fotobiólogos Moleculares Argentinos "GRAFOB del Bicentenario"; 2016
Institución organizadora:
Grupo Argentino de Fotobiología GRAFOB
Light modulates the virulence of the bacteriumBrucella abortus through a histidine kinase (HK) containing a light-oxygen-voltage(LOV) domain [1]. LOV domains consist of an alpha/beta core where the cofactor FMN isbound. Light absorption causes the formation of a covalent bond between aconserved cysteine residue and the C(4a) atom from the FMN ring. While thecovalent bond formation is fast (in microseconds) in all LOV proteinscharacterized, the recovery to the dark state varies among differentLOV-containing proteins, ranging from 20-30 seconds in phototropins to hours insome prokaryotic photoreceptors. According to secondary structure predictions andbased on other models, the LOV domain from Brucella LOV-HK is formed by aconserved alpha/beta fold (core) which is flanked by an N-terminal helix (N-helix) anda C-terminal helix (J-helix). Previous work in our laboratory was performedusing a construct from the LOV domain that encompasses the core region, whichis an unstable dimer in solution. Its structure shows that it adopts thetypical alpha/beta PAS domain fold, consisting of a beta-sheet and alpha-helical connectorelements. NMR studies point out that the beta-scaffold is a key element in thelight activation [2]. Also, we reported that upon illumination the recovery tothe dark state takes several hours (half-life of 5.8 hours at pH 7.0 and 35C)[2]. In the present work we aimed to study the role ofthe helices flanking the LOV core region. For that purpose, we cloned,expressed, and purified different LOV constructs containing one or both of theterminal helices. The analysis of their oligomeric state through static anddynamic light scattering indicates that the N-helix is essential for theformation of a stable dimer. We also studied dark recovery kinetics by UV-VISspectroscopy, and comparing the results from different constructs it can bededuced that the presence of the N-helix increases dramatically the half-lifeof the lit state. Finally, we purified a construct of the LOV domain which includesboth the N- and J-helices (LOV 15-155) and a mutant version insensitive tolight, in which the reactive cysteine residue was replaced by serine (LOV15-155 C69S). These proteins were submitted to a screening of crystallizationsolutions, obtaining different conditions in which crystals were obtained.These conditions were further optimized and the crystals will be used indiffraction experiments that are under way.