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Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome.

ABSTRACT: Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E-to-Z isomerization about the C(15) = C(16) double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C(15) methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.

SUBMITTER: Yang X

PROVIDER: S-EPMC3337037 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome.

Yang Xiaojing X Ren Zhong Z Kuk Jane J Moffat Keith K

Nature 20111016 7373

Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses. Several struc ...[more]

PMID: 22002602

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Project description:Local adaptation and its underlying molecular basis has long been a key focus in evolutionary biology. There has recently been increased interest in the evolutionary role of plasticity and the molecular mechanisms underlying local adaptation. Using transcriptome analysis, we assessed differences in gene expression profiles for three brown trout (Salmo trutta) populations, one resident and two anadromous, experiencing different temperature regimes in the wild. The study was based on an F2 generation raised in a common garden setting. A previous study of the F1 generation revealed different reaction norms and significantly higher QST than FST among populations for two early life-history traits. In the present study we investigated if similar reaction norm patterns were present at the transcriptome level. Eggs from the three populations were incubated at two temperatures (5 and 8 degrees C) representing conditions encountered in the local environments. Global gene expression for fry at the stage of first feeding was analysed using a 32k cDNA microarray. The results revealed differences in gene expression between populations and temperatures and population M-CM-^W temperature interactions, the latter indicating locally adapted reaction norms. Moreover, the reaction norms paralleled those observed previously at early life-history traits. We were able to identify 90 cDNA clones among the genes with an interaction effect that were differently expressed between the ecologically divergent populations. These included genes involved in immune- and stress response. We observed less plasticity in the resident as compared to the anadromous populations, possibly reflecting that the degree of environmental heterogeneity encountered by individuals throughout their life cycle will select for variable level of phenotypic plasticity at the transcriptome level. Our study demonstrates the usefulness of transcriptome approaches to identify genes with different temperature reaction norms. The responses observed suggest that populations may vary in their susceptibility to climate change. Brown trout populations from three rivers in Denmark were studied: the Karup (KAR), Norring Moellebaek (NOR) and Lilleaa Rivers (LIL). These rivers experience different temperature regimes during the period lasting from egg incubation until fry emergence. During the autumn of 2004 and the winter of 2004/2005, adults from the three populations were collected by electro fishing. The fish were stripped for milt and eggs to establish an F1 generation. In autumn/winter 2008/2009, mature F1s were used to establish F2 offspring for each of the three populations. For KAR and LIL, 20 full-sib families were established, whereas for NOR, 15 full-sib families were established. Eggs from each full sib family were divided into two pools that were incubated at 5 and 8M-BM-!C in separate hatching troughs. At the time of first feeding (ca. 750-780 day-degrees), 10 individuals from each family and temperature were collected and transferred to separate tubes containing RNAlater (QIAGEN, Hilden, Germany). A 32K cDNA microarray developed for salmonids by cGRASP was used for the analyses. Ten families were randomly selected from each population. From each family two individuals were analyzed for each temperature (5 and 8oC, respectively), amounting to a total of 120 individuals. Of the two individuals from each temperature treatment within a family, one was labeled with CY5 and the other with CY3. For each family there were two comparisons of individuals representing different temperatures, but with dyes swapped between comparisons.

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Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome.

Publications

Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome.

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