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Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift.

ABSTRACT: A protein molecule is an intricate system whose function is highly sensitive to small external perturbations. However, no examples that correlate protein function with progressive subangstrom structural perturbations have thus far been presented. To elucidate this relationship, we have investigated a fluorescent protein, citrine, as a model system under high-pressure perturbation. The protein has been compressed to produce deformations of its chromophore by applying a high-pressure cryocooling technique. A closely spaced series of x-ray crystallographic structures reveals that the chromophore undergoes a progressive deformation of up to 0.8 A at an applied pressure of 500 MPa. It is experimentally demonstrated that the structural motion is directly correlated with the progressive fluorescence shift of citrine from yellow to green under these conditions. This protein is therefore highly sensitive to subangstrom deformations and its function must be understood at the subangstrom level. These results have significant implications for protein function prediction and biomolecule design and engineering, because they suggest methods to tune protein function by modification of the protein scaffold.

SUBMITTER: Barstow B

PROVIDER: S-EPMC2533195 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift.

Barstow Buz B Ando Nozomi N Kim Chae Un CU Gruner Sol M SM

Proceedings of the National Academy of Sciences of the United States of America 20080903 36

A protein molecule is an intricate system whose function is highly sensitive to small external perturbations. However, no examples that correlate protein function with progressive subangstrom structural perturbations have thus far been presented. To elucidate this relationship, we have investigated a fluorescent protein, citrine, as a model system under high-pressure perturbation. The protein has been compressed to produce deformations of its chromophore by applying a high-pressure cryocooling t ...[more]

PMID: 18768811

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Project description:The data in this series forms the basis for the analysis presented in the above named paper. In this study, we used cDNA microarrays to ask if changes in gene expression are observed in response to a dietary shift in Drosophila melanogaster, a dietary generalist. Methods: Flies used in this study were derived from a multifemale collection at Iraklion, Crete, Greece, in August 2002 and reared in the laboratory for several generations. Three replicates were performed. At the start of each replicate, we collected 300 newly eclosed adults, separated them by sex, and stored them at low density in shell vials with standard cornmeal media for 3 days. Sexually mature males and females were placed together and allowed to mate. Mated females were allowed to oviposit overnight in chambers containing cornmeal medium filled Petri dishes (http://flyfood.arl.arizona.edu/cornmeal.php3). Hatching first instar larvae were reared for 36 hours on cornmeal medium in Petri dishes and then transferred to one of two conditions: 1) a control Petri dish consisting of cornmeal medium, or 2) an experimental Petri dish containing pure mashed banana. After 24 hours, larvae were plucked from these dishes, washed, transferred to microcentrifuge tubes in groups of 20, and flash frozen in liquid nitrogen. All samples were stored at -80C until RNA extraction. RNA was extracted from each tube using a Qiagen RNeasy kit (Qiagen, Inc., standard protocol). We used cDNA microarrays printed by the Genetic Analysis and Technology Core Custom Microarray Facility at the University of Arizona to assess expression levels. Printing material was amplified from BDGP Unigene library v1.0 containing 6K ESTs. Products were purified using Millipore Multiscreen filtration plates and re-suspended in 50%DMSO solution. Each product was printed in triplicate on Corning GAPSII aminosilane slides using a Virtek Chipwriter PRO microarray spotter. Between 10-15 micrograms of RNA were used for each reaction. RNA was reverse transcribed and labeled with one of two fluorescent dyes, Cy3 or Cy5 (details of protocol at http://gatc.arl.arizona.edu/Services/Microarray/aminoallyllabelling.html). Hybridization was completed on a Genomic Solutions Gentac automated hybridization station. Labeled material was allowed to hybridize to the array for 16 hours at 60°C. Wash conditions were: 1) low stringency wash twice at 50°C for 40seconds (1X SSC/ 0.2% SDS), 2) medium stringency wash twice at 42°C for 40 seconds ( 0.1X SSC/ 0.2% SDS), 3) high stringency wash twice at 42°C for 40 seconds (0.1X SSC). Hybridized arrays were scanned using the Applied Precision ArrayworxE slide scanner. We performed three independent hybridizations and three dye flips for a total of six arrays (2 treatments x 2 dyes x 3 replications = 12 samples, for 6 arrays). Each replicate consisted of an independent biological sample isolated on separate days. Identification of series: There are 18 series in this experiment. The second number in each series record represents the slide number (there were six slides used in this experiment). The third number in each series record represents the set on a given slide. For instance, for series record 331_2_1, these data correspond to slide 2, set 1. Each slide has 3 series numbers assoicated with it, representing set 1,2, or 3 on the slide. Each slide was paired with another in a "dye flip" experiment; thus, slides 2 and 3 were paired, slides 25 and 28 were paired, and slides 29 and 30 were paired. Keywords = dietary shifts Keywords = ecological genomics Keywords: repeat sample

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Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift.

Publications

Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift.

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