text:
Ed Rampell: Voices: A Legacy to Remember does have a memorable story combined with snazzy costumes, great foot stomping choreography and finger snapping music, from traditional Negro Spirituals to Gospel to Jazz,
Progressive Media Advocates
By Ed Rampell
Carole Bartolotto: The problem with concluding that GMOs are safe is that the argument for their safety rests solely on animal studies. These studies are offered as evidence that the debate over GMOs is over. Nothing could be further from the truth.
Steve Hochstadt: The Republicans in Congress, virtually all of whom publically deny that climate change is occurring, have just proposed a budget which cuts or reverses all the programs which might reduce the pace of warming.
Joe Mathews: Thanks to Xi Jinping, Chinese millionaires have propped up the state's sagging real estate market, universities, and companies.


text:
Yoga FAQs (cont.)
Do I need special equipment for yoga practice?
Hardly. All you need is a nonslip yoga mat that is long enough to lay fully stretched on your back.
Medically Reviewed by a Doctor on 1/9/2015
Medical Author:
Rick Kulkarni, MD
Medical Editor:
(Page 8 of 14)
Must Read Articles Related to Yoga FAQs
Yoga Yoga is the systematic practice of physical exercise, breath control, relaxation, diet control, and positive thinking and meditation aimed at developing harmony...learn more >>
From WebMD
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Read What Your Physician is Reading on Medscape
Postexercise Muscle Soreness »
Hough gave the first detailed description of delayed-onset muscle soreness (DOMS) in 1902.

subset: commoncrawl


text:
Now is the time
Healing the Circle Lenten Devotionals
If we as a people of Christ are to atone for and reconcile with Native American people, we must first acknowledge the numerous injustices inflicted upon an entire people/culture/society. We are called to not only admit the wrong, but we are called to deeply search for the truth, to know (as best we can) their experiences when everything they knew and loved was torn from them permanently.
We are all 'in the circle'
Healing the Circle Lenten Devotionals
As I write this, the song "will the circle be unbroken" runs through my mind. This is true when it comes to or lives. We are part of so many circles. Family, church, friends etc….we are part of the circles. I look forward to being within the circle, to worship, to share, to celebrate and to comfort. We are all brothers in sisters in Christ, we are all related and we are all in the circle.
Another Lent ends
Some theologians say Lent ends on Holy Thursday. Others hold fast to Holy Saturday. Lent lasts 40 days. The first day of Lent is Ash Wednesday. Six weeks later excluding Sundays, Lent is over. Then, it's Easter. Christ rises triumphantly from the grave to reign forever. Looking back, did the richness of this liturgical season prepare us for Easter? Were the sacrifices promised in earnest kept in earnest? Or, did resolutions and/or sacrifices made in earnest slip away buried by the busyness of life and living? Maybe the one sacrifice kept became "living water." Has our prayer and self-denial led us to a closer walk with Christ?
We're All Related
Healing the Circle Lenten Devotionals
The man looked down then up at me, "I'm sorry my parents didn't treat them Indian folks better." I felt the Holy Spirit welling up in me, realizing the importance of what he was saying not for me but for his son and family. I smiled and said, "Wanishi ta (Thank you very much) for saying what you did and for remembering the story and that we're all related."
She Loves Before She Meets
Healing the Circle Lenten Devotionals
Carol is only 10 years old, but she's already taught me much about love and acceptance. I can hardly wait for our youths to meet Carol--along with others like her--in the Lake Traverse Reservation. And I hope our youths love them all even before they've met them.
Lift Him Up
If God can save the Hebrew children from snakebite by looking at Moses serpent of bronze on a pole; and God can save the world through his Son not condemn it through his perfect love; if amazing grace can save us through faith in Jesus Christ why is it so difficult to do what the Son of Man desires namely "Lift Him Up?" Lifting him up is a no brainer if one believes in the promise of John 12:32 "And I, when I am lifted up will (not might) draw all persons unto me."
Massacre site offers historic truth
Healing the Circle Lenten Devotionals
We envision Indians and non-Indians coming to the site to remember what happened at Sand Creek. We envision scholars and students, pastors and church folk coming to learn the truth of history and to continue raising the important questions lest we repeat the sins of our forebears. It is time for more than words.
When a Native American broke my heart
Healing the Circle Lenten Devotionals
The best way I know how to bestow worth is to convey in word and deed the worth God, through Jesus Christ, has placed upon them. "For God so loved the world."
The Resurrection Message
Healing the Circle Lenten Devotionals
The message of the death and especially the resurrection was a marvelous message for the Cherokee. In response to that message, Cherokee was the first language the Bible was translated into in the Western Hemisphere.
Designed by a Great Creator
Healing the Circle Lenten Devotionals
With what name shall we call our God, whose majesty triumphs over all the earth? Many names have been suggested. All fall short of God's glory. Some reveal at least a portion of God's character by describing one or more of God's attributes. One of those names is Creator.
Healing the Circle Lenten Devotionals
Clergy Assistance Program Blog
Black Methodists for Church Renewal
Cross Racial/Cross Cultural Appointments
Week of Prayer for Christian Unity


text:
43pl provides companies with an easy way to engage with the CRCSI.
Benefits include preferential networking; access to IP, people, and networks; first hand views of R&D projects; etc. But each company tends to extract its own value from the engagement. Importantly, if you are considering joining, remember that "the more you put in the more you get out."
The mechanism of engagement through "43pl", a bare trust structure of limited liability, is a familiar commercial instrument and offers uncomplicated exit. The trust allows access to CRCSI Intellectual Property.
The CRCSI provides administration activities through the 43pl Company Secretary Mel Plumb.
Confidential advice in engaging in the CRCSI is provided by the CRCSI Deputy CEO Graeme Kernich.
Each year 43pl members are asked what their respective expectations are from participating in the CRCSI, and how these have been met in the year elapsed.
The composite "value proposition for 43pl membership" listed below includes the mix of benefits drawn by current members
- Access to specific R&D initiatives and IP
- Neutral ground to meet government and research agencies
- Growing the business (technical, professional development)
- Meaningful networking into government & academia
- Market development; kudos
43pl Tenders - some CRCSI projects have components that are offered to 43pl members through a fair and transparent tender process 43pl
Business Roaming – support for companies that seek networks and B2B and other relationships internationally, and particularly with Canada (through TECTERRA) and Sweden (through Future Position X) and globally (through the Global Spatial Network)
Annual internal Conference – a highly valued, invitation-only event of premium networking and technology and business insights and contacts Access to CRCSI Intellectual Property
BRISBANE - 1 Oct 2:30-5:00pm. Novotel Hotel, 200 Creek Street, Brisbane
SYDNEY - 2 Oct 2:30-5:00pm, Vibe Hotel North Sydney, 88 Alfred Street, Milsons Point (opposite train station)
MELBOURNE - 3 Oct 2:30-5:00pm, Graduate House, Uni Melbourne, 220 Leicester St, Carlton
WELLINGTON - 6 Oct 2:30-5:00pm Novotel Hotel, 133-137 The Terrace Wellington
PERTH - 9 Oct 3:30-5:00pm, Function Centre, Technology Park Bentley
Register with Wendy Jackson


text:
Preview: Is 'huge style difference' good news for sisters?
Global Sisters Report: Basilian Fr. Thomas Rosica said the report "will hopefully be a very positive message for women religious in the United States."
Global Sisters Report: Basilian Fr. Thomas Rosica said the report "will hopefully be a very positive message for women religious in the United States."
The document, said Barcelona Cardinal Lluís Martínez Sistach, is "far from being complete," but it is a collection of what was said in the first week of the synod.
South African Cardinal Wilfrid Napier said the synod's relatio has put the prelates in "a position that is virtually irredeemable."
During this morning's discussions, "there was no sense of doom or gloom or despair," but a desire to share ideas that are working to promote church teaching.
The bishops at the synod on the family will not change any doctrines, according to reports from the Vatican Press Office on the second day of their discussions.
On the floor of the synod, "there was no language whatsoever of a need to change doctrine," reported Basilian Fr. Thomas Rosica who attended the closed sessions. Rather, the desire was "to repurpose what we know in a way that's accessible" to all.
"I didn't hear anything about changing doctrine, but I heard a great desire to deepen our understanding of doctrine," he told journalists.
One theme said to be included in the synod: how the prelates use labels that "are not necessarily words that invite people to draw closer to the church."
The Francis Chronicles: "There is no sickness whatsoever. If there was, we would be open about that and asking people to pray for him."
I have been predicting smooth sailing for the visit of President Barack Obama to the Vatican, but a story from Vatican Radio makes me more cautious.
The story, which ran Wednesday, is a typical pre-visit piece talking about visits to the Vatican by Obama and earlier presidents. But the last paragraph should cause heartburn in the White House.
At an event on the "Francis Factor" on Tuesday, the panelists repeatedly shifted attention away from the pope and onto Jesus.
Rev. Thomas Rosica, C.S.B., the English language assistant to Holy See Press Office, sent out an email clarifying what the pope said about "civil unions" in his interview in the Italian newspaper Corriere della Sera.


text:
'Butterfly' a bit slow to take wing
Production soars with solo in Act 2
"Madama Butterfly"
San Diego Opera
When: Tomorrow,7 p.m.; Friday, 8 p.m.; Sunday, 2 p.m.;May 20, 7 p.m.
Where: Civic Theatre, 301 C St., downtown
Tickets: $29 - $170
Phone: (619) 570-1100 or (619) 533-7000
San Diego Opera launched the final five performances of its 2009 season at the Civic Theatre on Saturday night with "Madama Butterfly," Giacomo Puccini's favorite among his artistic "children," even if its birth was both prolonged and painful.
Butterfly's 1904 La Scala premiere was a disaster (a combination of the opera's shortcomings and the musical politics of the day), and it was not until its 1906 French premiere that Puccini's revisions shaped "Madama Butterfly" into the masterpiece that has charmed, exalted and devastated audiences for 100-plus years.
Puccini was more, far more, than just a composer; he understood how every theatrical element – not only the words and the music, but the setting, costuming and lighting – combined to create a whole that not only framed the dramatic action but actually energized and propelled it. And "Madama Butterfly" stands among that handful of operas ("La bohème" is another, Mozart's "Così fan tutte" and "Figaro" two more) that embody the principles that undergird all great art works: unity of form and function, economy of means, and an organic melding of emotion and intellect.
But something hobbled "Butterfly's" musical flight and grounded its theatrical savvy on Saturday evening, and at first it was difficult to tell what it was. Rhythmic uncertainty from the pit kept the singers uneasily focused on conductor Edoardo MÜller, who either pushed the music ahead heedlessly or held it back arbitrarily.
The pallid, unnuanced playing of the San Diego Symphony made it hard to believe that this was the orchestra that had covered itself in glory a few weeks ago in "Peter Grimes." To lay the blame for the prevailing sense of disconnection at the conductor's feet, however, would be a mistake, for the real culprit – at least in the first half of the opera – is the wrong-headed "conception" that stage director Francesca Zambello imposed on the opera when this production was created in 1998.
By dismissing Puccini's careful choice of setting the first act outdoors – opting, for no discernible reason, to move Butterfly's wedding to Pinkerton into the consul's office – Zambello (whose staging was here remounted by Garnett Bruce) robbed the singers of the freedom to enlarge their performances to fill the space around them. When this sense of open-air expansiveness is missing, the opera is crippled.
But midway through Act 2, precisely at the moment when Butterfly must face the possibility that her beloved Pinkerton may never return, the production suddenly snapped into focus, caught fire and took flight, carrying everyone on both sides of the proscenium to the opera's emotional payoff, a shattering picture of sacrifice and remorse that makes eyes burn and throats tighten.
The standing ovation that greeted soprano Patricia Racette when the curtain rose on her solo bow affirmed the audience's awareness that they had just seen a great artist put her individual stamp on a role in which she has few competitors today. Plagued by pitch problems for much of the evening, she nonetheless unrolled a detailed conception Cio-Cio-San, whose emotional depth and textured personality were the work of an extraordinary singing actress.


text:
Tags: MICO 520 Series hydrualic-apply caliper disc brake
MINEXPO Must Sees
To help attendees maneuver through the wide assortment of mining equipment at the show, we've highlighted aggregate-related items on display.
ASGCO Booth # 923
ASGCO plans to display its Super-Skalper conveyor belt pre-cleaner. The all-stainless-steel system provides carry-back removal for high-speed belts, ... Read More...
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- Senate rejects amendment seeking to fund infrastructure by closing corporate tax loophole151 Views


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Quick update since i havent finished yet, busy getting married and such. Now that its over and we're back from the honeymoon I have some more time to finally finish this project up.
We found some nice wood at Lowes we actually like, its engineered hardwood(not laminate) bamboo flooring. They have a light and dark color and we went for the darker of the two as the other one is a bit too light and contrasts a bit much.
Just a few pieces put down to see what it looks like. Because of the already hefty weight of this keezer with all of the extra wood and potential storage we decided against tile, so we dont end up busting the caster wheels
Im waiting for my 24" drip tray to show up Thursday so i can finish cutting the wood, ****s expensive but ive already blown so much money on this project whats another $100 ...
Thankfully they are on sale at Beveragefactory, got the inset one with the stainless cover. Although i realized that this drip dray is .75" deep and the bamboo is only 5/8 thick. So ive come to the conclusion that because my top is plywood i will just use a chisel to cut away the top layer of plywood or so where the drip tray goes...i think this should give me adequate depth to get it flush with the top and not compromise the strength of the lid which is 3/4" thick and i only need another 1/8th or so of space. So it should end up looking something like this(only 2x longer as this is the 12")
Wow! This thing is amazing! I'm hoping to just figure out a simple collar on a freezer.
Nice work.
My lazy ass finally got around to ordering cabinet doors that came in today. Need to stain them and hopefully in another week or two it will be totally done and ill post pics


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Public Pickups big-tits-worship VIDEOS (6)
Check the game on the bro who submitted this clip of taking down a Hungarian cutie Felicia. After offering her money to show him around town, he discovered she was down to flash him her tits and ass, and horny to get her tight snatch stretched.
Public Pick Ups
95 %
81899 views - Mar 08, 2015
For some cash, Grace Joy showed off her tits and gave dude a blowjob, but that bisexual babe wanted to share his cock, so she talked Kira Queen into an amateur threesome! Watch these two busty sluts take turns sucking and fucking big cock!
Public Pick Ups
94 %
108098 views - Nov 30, 2014
Any poor student like the Italian hottie in this short amateur porn would take the cash guy offered to fuck. For a couple bucks, Valentina flashed him her perfect big tits in the park, and let him fuck her throat before he tapped that big juicy ass.
Public Pick Ups
96 %
88964 views - Oct 29, 2014
200 Euros to flash the juiciest big natural tits. 250 more to squeeze them, then he talked this open-minded waitress out of her clothes. Chintia was cash-hungry and thirsty enough to take his money and fuck him on the sofa where she works.
Public Pick Ups
95 %
94911 views - Oct 08, 2014
Dude locked himself in a change-room of the skating arena and banged the fuck out of this hottie he picked up. This amateur hottie called her tits "the world's best boobs" and she wasn't lying! Angel was willing to be so dirty for a couple ...
Public Pick Ups
95 %
105985 views - Jun 29, 2014
Dude found this amateur slut from St Petersburg wandering around the ski hill. She really wanted to hit the slopes, but she'd forgotten her boots, so that was a bust. Alexa jumped in his truck and came along for the ride of her life in the back seat
Public Pick Ups
91 %
81912 views - Apr 06, 2014


text:
Has anyone seen Karen?
asleep_in_the_gardenJuly 31, 2013
Can't help noticing she's hasn't been posting lately.
Karen if you see this please let us know that you're okay.
There's no way I'm the only one around here who misses you!
Thanks Rhizo,we can let this one "fall off" the board then!
Funny thing is that originally I was thinking I had posted this here(because this is one of her haunts),when in fact I had started the thread in the off topic side of this forum(which seldom see activity at all these days).
...Not that it wouldn't be perceived in the same way there(I remember the thread you speak of).
I just hope she's not gone...after all she's given over a dozen years to this place.
Having said that,I think I can safely say we can be done with this thread.
*whistles past the graveyard*


text:
LONDON (Reuters) - China's economy is likely to overtake the euro zone's this year, India is leapfrogging Japan and by 2030 the Asian pair will be bigger than the United States, euro area and Japan combined, the OECD said on Friday.
In a crystal-ball exercise to tease out long-term trends in the global economy, the Organisation for Economic Cooperation and Development said the combined gross domestic product of China and India was likely to exceed that of all the current Group of Seven rich economies by around 2025.
Their output in 2010 was less than half the G7's GDP.
The projections of the Paris-based OECD, a club of indutrial democracies, are based on 2005 purchasing power parities (PPP).
At market exchange rates, it will take emerging markets a bit longer to seize the crown - for example, Goldman Sachs reckons the BRICs quartet of Brazil, Russia, India and China will overtake the G7 by 2037.
Asa Johansson, one of the authors of 'Looking to 2060: long-term global growth prospects', said the report presented a hypothetical scenario rather than a firm projection.
Nevertheless, she said the extent of the expected shift in economic power away from developed countries was striking.
Measured in 2005 PPPs, China and India will account for 28 percent and 11 percent respectively of the output of 42 major economies by 2030, compared with 18 percent for the United States, 12 percent for the euro zone and 4 percent for Japan.
The OECD pencils in global growth of 3 percent a year over the next half-century, mainly driven - as in the past - by productivity improvements and a build-up in human capital.
Until 2020, China will have the highest growth rate among the countries studied, but it will then be surpassed by India and Indonesia as its working-age population rapidly declines.
However, China has a big start over India thanks to strong productivity growth and intensive investment in the past decade.
As a result, even though both economies will grow seven-fold in the next 50 years, China's per capita income will be 25 percent higher than current U.S. income by 2060, but India will languish at half the present American level.
Looking at it differently, China's GDP per capita is now just one-sixth that of the United States. But by 2060 it will have reached 60 percent of America's income level, putting China just behind Spain and France but ahead of Italy.
The OECD's exercise underlines the importance of demographics as a long-term driver of savings, investment and growth.
China's savings rate, which now exceeds 50 percent of GDP, is expected to plunge by no less than 40 percentage points by 2060, with about half of the drop due to ageing.
Italy, Greece and Portugal are likely to eventually run current account deficits in the order of 10-15 percent of GDP.
China, by contrast, is expected to see its current account surplus widen until the late 2020s as investment falls even faster than savings due to slowing potential growth.
Globally, current account imbalances will be back to pre-crisis levels by 2025-2030, potentially undermining growth in the absence of ambitious policy changes, the OECD said.
In keeping with the long-term focus of its report, the OECD assumes that the global financial crisis will have no permanent effect on trend growth rates.
(Reporting by Alan Wheatley; Editing by Catherine Evans)
Trending On Reuters
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Reuters India Mobile
Get the latest news on the go. Visit Reuters India on your mobile device Full Coverage


text:
I am trying to write a custom actions class library for my WiX install package. I've read that I should use CustomActionAttribute to mark the methods that I am planning to call from install package. There is no Votive plugin for VS2010, so when I type [CustomAction] above my method name, VS2010 doesn't find it. So what should I do to write a custom action method?
Take the 2-minute tour ×
Stack Overflow is a question and answer site for professional and enthusiast programmers. It's 100% free, no registration required.
There is Voitve support for Visual Studio 2010 in Wix 3.5 and up. With Votive you get a project template for creating managed custom actions which will set up all of the dependencies you need to add the CustomAction attribute.
You can add a reference to the Microsoft.Deployment.WindowsInstaller.dll to resolve the reference manually, but the project template sets up a few more needed steps to create the CA.
From this and other blog posts Rob has made, I thought WiX v3.5's Votive had VS2010 support, just not out of the box (last minute ship-decision?). Someone correct me if I'm wrong.


text:
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text:
Page 1
research papers
Acta Cryst. (2012). D68, 176–185
Acta Crystallographica Section D
Structure of the effector-binding domain of the
arabinose repressor AraR from Bacillus subtilis
Kater ˇina Procha ´zkova ´,aKater ˇina
Cˇerma ´kova ´,aPetr Pachl,a,b
Irena Sieglova ´,a,bMilan Fa ´bry,b
Zbyszek Otwinowskicand
Pavlı ´na Rˇeza ´c ˇova ´a,b*
aInstitute of Organic Chemistry and
Biochemistry, Academy of Sciences of the Czech
Republic, Flemingovo nam. 2, Prague 6,
Czech Republic,bInstitute of Molecular
Genetics, Academy of Sciences of the Czech
Republic, Videnska 1083, Prague 4, Czech
Republic, andcUT Southwestern Medical
Center, Dallas, Texas, USA
Correspondence e-mail:
# 2012 International Union of Crystallography
Printed in Singapore – all rights reserved
In Bacillus subtilis, the arabinose repressor AraR negatively
controls the expression of genes in the metabolic pathway of
of two domains of different phylogenetic origin and function:
an N-terminal DNA-binding domain belonging to the GntR
family and a C-terminal effector-binding domain that shows
similarity to members of the GalR/LacI family. The crystal
structure of the C-terminal effector-binding domain of AraR
in complex with the effector l-arabinose has been determined
at 2.2 A˚ resolution. The l-arabinose binding affinity was
characterized by isothermal titration calorimetry and differ-
ential scanning fluorimetry; the Kdvalue was 8.4 ? 0.4 mM.
The effect of l-arabinose on the protein oligomeric state was
investigated in solution and detailed analysis of the crystal
identified a dimer organization which is distinctive from that
of other members of the GalR/LacI family.
Received 1 September 2011
Accepted 15 December 2011
PDB Reference: AraR
effector-binding domain,
We would like to dedicate this
work to the memory of
Kater ˇina Procha ´zkova ´, who
tragically died in February
1. Introduction
Like many other bacteria, Bacillus subtilis can utilize various
carbohydrates as sources of carbon and energy. The enzymes
of the various metabolic pathways are in general synthesized
only in the presence of their substrate and in the absence of
the preferred carbon source. The transcription of the genes
coding for transporters and catabolic enzymes is thus tightly
regulated by numerous transcription factors (Henkin, 1996;
Stu ¨lke & Hillen, 2000).
As a soil bacterium, B. subtilis participates in the decom-
position of plant material and can utilize the l-arabinose
pentose carbohydrate that is present in plant polysaccharides.
The expression of 13 genes participating in the metabolism of
arabinose-, galactose- and xylose-containing polysaccharides
is negatively controlled by the transcription factor AraR.
AraR recognizes and binds to eight specific DNA operator
sites and thus blocks transcription from five promoters (Mota
et al., 1999, 2001; Sa ´-Nogueira & Mota, 1997). Binding of the
effector molecule arabinose modulates the binding of AraR to
DNA and results in derepression of the arabinose regulon.
AraR comprises 362 amino-acid residues and belongs to the
GntR family of bacterial regulatory proteins. These regulators
are characterized by a modular domain organization, with a
small N-terminal DNA-binding domain and a large C-terminal
effector-binding domain. The C-terminal domain typically
mediates homodimer formation; it receives the signal through
binding of the effector molecule and transmits it to the DNA-
binding domain.
contains the winged-helix–turn–helix domain typical of the
GntR family of bacterial regulatory proteins (Haydon &
Guest, 1991). This domain is responsible for specific DNA
Page 2
recognition and binding. The C-terminal domain of AraR is
comprised of 292 amino-acid residues (residues 71–363) and is
homologous to the GalR/LacI family of bacterial regulators
and sugar-binding proteins (Weickert & Adhya, 1992). This
domain is involved in effector (l-arabinose) binding and
protein dimerization (Franco et al., 2006).
Residues that are crucial for DNA recognition and effector
binding as well as for dimerization have been identified using
the mutagenesis approach and homology modelling (Franco et
al., 2006, 2007); however,no crystal structures are available for
AraR or its individual domains.
In order to obtain structural information on effector
recognition and dimerization, we produced recombinant
C-terminal domain of AraR and full-length AraR and char-
acterized the effector binding and its effect on protein
dimerization. We determined the crystal structure of the
C-terminal domain of AraR in complex with l-arabinose and
analyzed the structure in order to provide insights into the
structural mechanisms underlying the function of AraR.
2. Experimental
2.1. Protein expression and purification
The gene for full-length AraR (NCBI reference sequence
ZP_03593195.1) and the coding sequence for the effector-
binding domain of AraR (residues 71–363, referred to here as
C-AraR) were cloned into the pET151/D-TOPO vector using
Gateway cloning technology (Invitrogen). The vectors
included a sequence encoding an N-terminal His6affinity tag
followed by a V5 epitope and a sequence encoding the
tobacco etch virus (TEV) protease recognition site. Upon
TEV protease cleavage, the six-amino-acid sequence GIDPFT
remained at the N-terminus of the recombinant C-AraR as a
cloning artifact.
Protein preparation was carried out by a procedure used
previously for the production of other bacterial repressors for
crystallographic studies (Rˇeza ´c ˇova ´ et al., 2007, 2008). The
AraR and C-AraR proteins were overexpressed in Escher-
ichia coli BL21 (DE3) grown on LB broth (Sigma) supple-
mented with 0.8%(v/v) glycerol and 20 mg ml?1ampicillin.
The bacterial culture was grown at 310 K and protein
expressionwas inducedwith
thiogalactopyranoside at an OD600of about 1 for a total of 5 h
at 293 K. The cells were harvested by centrifugation, resus-
pended in five volumes of lysis buffer [50 mM Tris–HCl pH
7.5, 500 mM NaCl, 10 mM imidazole, 5%(v/v) glycerol] and
lysed by sonication after the addition of protease inhibitor
(1 mM PMSF). The lysate was clarified by centrifugation and
subjected to affinity chromatography using a His-Select Nickel
Affinity Gel (Sigma) column equilibrated with lysis buffer.
The His6-tagged protein was eluted with 250 mM imidazole in
lysis buffer.
The affinity tag was removed from C-AraR by incubation
with recombinant His6-tagged TEV protease overnight at
298 K while dialyzing against lysis buffer. The sample was
subjected to a second round of Ni–NTA metal-affinity
chromatography to remove the TEV protease and the cleaved
1 mMisopropyl
His6tag, extensively dialyzed against 20 mM Tris–HCl pH 7.5,
250 mM NaCl, 0.02%(v/v) ?-mercaptoethanol and concen-
trated using Amicon Ultra Concentrators (Millipore). Proteins
that were >95% pure as judged by SDS–PAGE were flash-
cooled in liquid nitrogen and stored at 203 K until further use.
2.2. Protein crystallization
Initial C-AraR crystals were obtained by the sitting-drop
vapour-diffusion method using the JCSG+ Suite (Qiagen)
crystallization screen with a Gryphon crystallization work-
station (Art Robbins); 0.2 ml protein solution was mixed with
0.2 ml well solution and the mixture was equilibrated against
50 ml reservoir solution. Crystals appeared after several days
in various conditions containing PEG as the precipitant.
Further optimization was performed manually and involved
changing to the hanging-drop mode in VDX48 crystallization
plates (Hampton Research). Optimal C-AraR crystals were
obtained by mixing 1 ml protein solution [16.8 mg ml?1in
10 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.02%(v/v) ?-mer-
captoethanol] supplemented with 50 mM l-arabinose with
1 ml reservoir solution [70 mM HEPES pH 7.5, 7%(w/v) PEG
8000, 6%(v/v) ethylene glycol, 20%(v/v) glycerol, 10 mM
spermidine] and equilibrating at 293 K against 150 ml reservoir
solution. Rod-shaped crystals appeared within a day and grew
to final dimensions of 0.3 ? 0.15 ? 0.15 mm within one week.
The crystals were flash-cooled by plunging them into liquid
nitrogen and were then stored in liquid nitrogen until use in
X-ray diffraction experiments.
research papers
Acta Cryst. (2012). D68, 176–185 Procha ´zkova ´ et al.
? Effector-binding domain of AraR
Table 1
Crystal data and diffraction data-collection and refinement statistics.
Values in parentheses are for the highest resolution shell.
Data-collection statistics
Wavelength (A˚)
Space group
Unit-cell parameters (A˚)
No. of molecules in asymmetric unit
Resolution (A˚)
No. of unique reflections
Completeness (%)
Rmerge† (%)
Average I/?(I)
Refinement statistics
Resolution range (A˚)
No. of reflections in working set
No. of reflections in test set
Rfree§ (%)
R.m.s.d. bond lengths (A˚)
R.m.s.d. angles (?)
No. of atoms in asymmetric unit
No. of water molecules in asymmetric unit
Mean B value, protein/solvent (A˚2)
Ramachandran plot statistics}
Residues in favoured regions (%)
Residues in allowed regions (%)
a = 62.1, b = 106.3, c = 111.8
30–2.2 (2.28–2.20)
33024 (2220)
6.6 (5.5)
86.8 (59.3)
5.7 (28.0)
35.9 (4.9)
30–2.2 (2.28–2.20)
18.42 (25.6)
22.6 (31.9)
† Rmerge=P
reflection hkl, with summation over all data.
where Fobsand Fcalcare observed and calculated structure factors, respectively.
is equivalent to R but is calculated for 5% of the reflections chosen at random and
omitted from the refinement process (Bru ¨nger, 1992).
(Chen et al., 2010).
ijIiðhklÞ ? hIðhklÞij=P
iIiðhklÞ, where Ii(hkl) is an individual
} As determined by MolProbity
intensity of the ith observation of reflection hkl and hI(hkl)i is the average intensity of
??jFobsj ? jFcalcj??=P
§ Rfree
Page 3
2.3. Data collection and structure determination
Diffraction data were collected at 100 K on beamline
MX14.2 of BESSY, Berlin, Germany. All diffraction data were
processed using the HKL-3000 suite of programs (Minor et al.,
2006). The crystals exhibited the symmetry of space group
P212121and contained two molecules in the asymmetric unit,
with a solvent content of ?56%. Crystal parameters and data-
collection statistics are given in Table 1. The crystals exhibited
anisotropic diffraction to a minimum Bragg spacing (dmin) of
about 2.2 A˚in one direction and to a dminof about 2.9 A˚in
the other direction. The low value of the completeness in the
highest resolution shell is a consequence of this anisotropy.
The structure of C-AraR was determined by molecular
replacement using MOLREP (Vagin & Teplyakov, 2010). The
polyalanine search model was derived from the structure of
the effector-binding domain (residues 55–329) of the E. coli
PurR repressor (PDB entry 1jft; Huffman et al., 2002), which
shares 26% sequence identity with C-AraR. Model refinement
was carried out using REFMAC v.5.2 (Murshudov et al., 2011)
from the CCP4 package (Winn et al., 2011) and was inter-
spersed with manual adjustments using Coot (Emsley &
Cowtan, 2004). The final steps included TLS refinement (Winn
et al., 2001) using 19 TLS groups.
The coordinates for the ligand were added after complete
refinement of the C-AraR and solvent model. The quality of
the final model was validated with MolProbity (Chen et al.,
2010). The final refinement statistics are given in Table 1. All
figures showing structural representations were prepared with
PyMOL (DeLano, 2002). The PISA (Krissinel & Henrick,
2005) and ConSurf (Landau et al., 2005) servers were used to
analyze the structures. Atomic coordinates and experimental
structure factors have been deposited in the Protein Data
Bank with code 3tb6.
2.4. Size-exclusion chromatography
Analytical size-exclusion chromatography was performed
on a Superdex 200 10/300 GL Tricorn column (Pharmacia)
equilibrated with 10 mM Tris pH 7.5, 250 mM NaCl,
0.02%(v/v) ?-mercaptoethanol. When the AraR and C-AraR
proteins in complex with l-arabinose were analyzed, the
equilibration buffer was supplemented with 50 mM l-arabi-
A sample of 50 ml AraR protein at 2 mg ml?1or of 50 ml
C-AraR protein at 3.2 mg ml?1(with or without the addition
of 50 mM l-arabinose) was loaded onto the column. The
apparent molecular weight was estimated by comparison with
protein standards (Sigma–Aldrich), which consisted of blue
dextran (2000 kDa), bovine serum albumin (66 kDa), carbonic
anhydrase (29 kDa), cytochrome c (12.4 kDa) and aprotinin
(6.5 kDa).
2.5. Differential scanning fluorimetry
The thermal shift assay was conducted in a PCR Roche
LightCycler 480 II. Solutions of 2 mM protein and 100 mM
l-arabinose were mixed and 1 ml 25? diluted (in water) 5?
SYPRO Orange (Bio-Rad) was added to 25 ml of this mixture
in the wells of a 96-well LightCycler 480 Multiwell Plate 96
and sealed using LightCycler 480 Sealing Foil (Roche). The
final concentrations of the AraR and C-AraR proteins were
1 mM. The final concentration of l-arabinose ranged from 0.1
to 50 mM. The measurements were performed in 20 mM Tris–
HCl pH 7.5, 50 mM NaCl, 0.02%(v/v) ?-mercaptoethanol. The
plate was heated from 293 to 363 K with a heating rate of
0.8 K min?1. The fluorescence intensity was recorded using
wavelengths for excitation and emission of 465 and 580 nm,
The data analysis is based on a published procedure (Lo
et al., 2004). The fluorescence values were fitted to the non-
linear equation (1) (Pantoliano et al., 2001) to obtain ?Hu,
?Cpu, Tm, Fminand Fmax,
FðTÞ ¼ Fmaxþ
Fmin? Fmax
1 þ exp
where F(T) is the measured fluorescence intensity at tem-
perature T, Fmin and Fmax are the fluorescence intensities
before and after the unfolding process, Tmis the midpoint of
the protein-unfolding transition, R is the gas constant, ?Hu
is the enthalpy of protein unfolding and ?Cpuis the heat-
capacity change of protein unfolding. The program GraphPad
Prism 5 was used to perform nonlinear fitting.
To carefully determine the parameters in the absence of the
ligand, ten parallel measurements were performed and fitted
separately. The mean values and their standard errors were
calculated as weighted arithmetic means, with the square of
the standard errors determined from each measurement used
as the weighting term. For each l-arabinose concentration
(0.1–50 mM), three parallel measurements were performed
and the weighted arithmetic means of Tmwere calculated.
2.6. Isothermal titration calorimetry (ITC)
Thermodynamic analysis by ITC was performed using VP-
ITC (MicroCal) at 298 K in 20 mM Tris–HCl pH 7.5, 50 mM
NaCl, 0.02%(v/v) ?-mercaptoethanol. 1.5 ml C-AraR protein
solution at a concentration of 35 mM was titrated against the
ligand solution at a concentration representing a 11.5-fold
molar excess over the protein concentration. At each injection
(of a total of 35), 10 ml ligand solution was injected into a
sample cell. Each experiment was accompanied by a corre-
sponding control experiment in which the ligand was injected
into the buffer alone. An accurate concentration of the protein
was determined by HPLC amino-acid analysis. The association
constants and the thermodynamic parameters were estimated
using Origin (v.7.0; MicroCal). The values reported are the
average and standard errors calculated from two titration
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Procha ´zkova ´ et al.
? Effector-binding domain of AraR
Acta Cryst. (2012). D68, 176–185
Page 4
3. Results and discussion
3.1. Overall structure description and quality
The structure of the C-terminal effector-binding domain of
AraR (C-AraR; residues 71–363) in complex with its effector
l-arabinose was determined by molecular replacement and
was refined using data to 2.2 A˚resolution. The data-collection
and model-refinement statistics are given in Table 1. The
orthorhombic crystal form contained two molecules in the
asymmetric unit, with a solvent content of 56%. The final
crystallographic model consisted of two molecules of C-AraR,
with residues 79–361 in polypeptide chain A and 80–358 in
chain B. The N- and C-terminal residues of both molecules
in the asymmetric unit could not be located in the electron-
density map and were thusnot included in the final model.The
two protein chains in the asymmetric unit are quite similar: the
r.m.s.d. for superposition of 292 C?atoms is 0.208, a value that
is within the range observed for different crystal structures of
identical proteins (Betts & Sternberg, 1999).
The electron density used for modelling l-arabinose into
the effector-binding site was of excellent quality in both
protein chains in the asymmetric unit (Fig. 1). Two glycerol
molecules and 246 water molecules were modelled into non-
protein electron density in positions allowing hydrogen-bond
There are two Ramachandran outliers ineachprotein chain:
His135 and Asp301. Residue Asp301 is an outlier with special
conformation in the effector-binding site which is conserved in
other transcriptional repressors: e.g. Asp275 in E. coli PurR or
Asp274 in E. coli LacI (Mowbray & Bjo ¨rkman, 1999). Residue
His135 is located in a flexible region at the dimer interface.
The effector-binding domain of AraR consists of two sub-
domains which have very similar ?/? topologies (Fig. 1). They
are composed of a core of six parallel ?-sheets flanked on each
side by two ?-helices. The N-terminal subdomain consists of
residues 71–182 and 318–346, while the C-terminal subdomain
consists of residues 183–317 and 347–363. Three cross-over
strands connect the two subdomains. The effector-binding site
is located at the interface of the two subdomains (Fig. 1).
A structural comparison with models deposited in the
Protein Data Bank using the program DALI (Holm & Sander,
1994) identified numerous structural homologues among
bacterial proteins belonging to the GalR/LacI family of tran-
scription regulators.
Despite their low sequence identity to C-AraR, the closest
from B. megaterium (sequence identity of 25%) and purine
repressor (PurR) from E. coli (sequence identity of 26%). This
structural similarity has been predicted previously based on
sequence homology and has also been used in homology-
modelling studies by others (Franco et al., 2006). The struc-
tures of CcpA and PurR superimpose with C-AraR with
r.m.s.d.s of 1.9 A˚for 269 equivalent C?atoms and 2.1 A˚for
273 equivalent C?atoms, respectively (Fig. 2a). The structural
similarity to the E. coli lactose operon repressor LacI, which is
the prototypical member of the GalR/LacI family, is also very
high (r.m.s.d. of 2.3 A˚for superposition of 267 equivalent C?
The locations of the binding sites for the effector in CcpA
(glucose 6-phosphate) and the co-repressor in PurR (hypo-
xanthine) coincide with the l-arabinose effector-binding site
in AraR (Fig. 2b).
3.2. Effector binding to AraR
3.2.1. Structural details of
effector-binding site is located in a deep cleft between the N-
and C-terminal subdomains of C-AraR. The side chains of
the following residues form the l-arabinose binding pocket:
Tyr92, Ile93, Phe94, Glu142 and Asn170 from the N-terminal
domain, Asp212, Gln214, Arg218, Tyr272, Asn273 and Asp301
from the C-terminal domain, and Asp185 and His318 from the
cross-over strands. The binding of the sugar at the interface of
the two subdomains restrains the overall protein conformation
substantially and stabilizes the protein. A change in the
molecular shape as well as increased thermal stability upon
l-arabinose binding was indeed observed in the size-exclusion
chromatography and differential scanning fluorimetry experi-
ments described below. Attempts to crystallize free C-AraR
protein failed, probably owing to protein dynamics and con-
formational instability in the absence of the effector molecule.
L-arabinose binding. The
research papers
Acta Cryst. (2012). D68, 176–185 Procha ´zkova ´ et al.
? Effector-binding domain of AraR
Figure 1
Ribbon representation of the overall structure of C-AraR. The protein is
coloured according to the secondary-structure elements, with ?-helices
in red, ?-strands in yellow and loops in green. The l-arabinose in the
effector-binding site is shown as sticks, with C and O atoms coloured grey
and red, respectively. On the right, the detail of the effector-binding site is
shown, with the 2Fo? Fcelectron-density map for l-arabinose contoured
at 1.0?. The detailed view is rotated by ?90?with respect to the overall
Page 5
The l-arabinose adopts the ?-configuration and is deeply
buried within the effector-binding site; it loses 92.5% (246 A˚2)
of its solvent-accessible surface upon binding to C-AraR. All
of the hydroxyl groups of l-arabinose are involved in polar
interactions with protein side chains: the O1 atom interacts
with the side chain of Gln214, O2 forms hydrogen bonds to
Arg218 and Asp301, O3 interacts with Asn273 and Asp301,
and O4 interacts with Asn273. In addition to direct hydrogen
bonds, the O1 atom is involved in a water-mediated inter-
action with the side chains of Arg218 and Asp185. All residues
in contact with l-arabinose are shown in Fig. 3.
Our structure confirmed the results of the mutational
analysis performed by Franco et al. (2006), identifying residues
Phe94, Asp212, Gln214, Arg218, Asp301 and His318 as being
in contact with the effector. Mutations
of all of these residues except His318
resulted in a non-inducible (suppressor)
phenotype (Franco et al., 2006).
3.2.2. L-Arabinose affinity and effect
on protein stabilization. The binding of
l-arabinose to AraR was investigated
using isothermal titration calorimetry
(ITC) and differential scanning fluori-
metry (DSF). While ITC provides
information on ligand-binding affinity
and thermodynamic parameters, DSF
follows the thermal stabilization of the
protein on ligand binding (Pantoliano et
al., 2001; Lo et al., 2004).
The ITC experiments were carried
out at pH 7.5 in 20 mM Tris–HCl,
50 mM NaCl, 0.02%(v/v) ?-mercapto-
ethanol. The ligand-binding event was
exothermic and the integrated ITC data
were best fitted by a single-site model
(Fig. 4). The fit provided an estimate of
the dissociation constant for binding of
l-arabinose to C-AraR in the buffer
described above as 8.4 ? 0.4 mM.
This value is comparable with those
determined using the same technique
for the binding of the strong inducer
isopropyl ?-d-1-thiogalactopyranoside
(IPTG) to the LacI lactose repressor
protein at pH 7.5 (Wilson et al., 2007).
The dissociation constant for IPTG
binding to LacI was 2.8 ? 0.2 mM, while
the affinity
2-phenylethyl ?-d-1-thiogalactopyrano-
side was characterized by a dissociation
constant of 50 ? 3 mM (Wilson et al.,
In the DSF experiments, the fluor-
escent dye SYPRO Orange was used to
monitor protein unfolding in response
to increasing temperature. The un-
folding process exposes the hydro-
phobic region of proteins and results in
a large increase in fluorescence, which is
used to monitor the protein-unfolding
proteins displayed melting profiles that
can be approximated by a two-state
model of thermal unfolding (Fig. 5b).
of the weakinducer
(Lo etal., 2004). Both
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Procha ´zkova ´ et al.
? Effector-binding domain of AraR
Acta Cryst. (2012). D68, 176–185
Figure 2
(a) Superposition of C-AraR (magenta) with CcpA (yellow) and PurR (green) structures. (b, c)
Detail of the effector-binding site with bound small-molecular effectors. Ligands are represented as
stick models with C-atom colours corresponding to the colour of the protein chain (O, N and P
atoms are coloured red, blue and orange, respectively). The coordinates used for the superposition
were those of CcpA from B. megaterium in complex with glucose 6-phosphate (PDB entry 2nzu;
Schumacher et al., 2007) and of PurR from E. coli in complex with hypoxanthine (PDB entry 1qpz;
Glasfeld et al., 1999).
Page 6
The melting profiles were used to estimate the midpoint
temperature of transition, which is also called the unfolding
transition temperature (Tm) as it correlates with the melting
temperatures determined using other methods (Ericsson et al.,
2006). The Tmvalues of C-AraR and AraR determined at
increasing concentrations of l-arabinose are summarized in
Fig. 5(a).
The thermal stability of AraR was 1.3 K lower than the
stability of the isolated effector-binding domain protein (323.6
versus 324.9 K). Addition of l-arabinose increases the thermal
stability of both proteins (Fig. 5). The increase in Tmvalues
upon ligand binding (?Tm) was very similar for both proteins.
We can thus conclude that the affinity of l-arabinose for the
effector-binding site in C-AraR is comparable with the affinity
for the binding site in the context of the full-length protein.
3.3. Oligomerization of AraR
3.3.1. Oligomeric state in solution. The effect of l-arabi-
nose on AraR oligomerization was investigated by analytical
size-exclusion chromatography (SEC). The molecular weight
of C-AraR as well as that of the full-length AraR protein was
estimated in the presence or absence of the effector molecule
Although exact molecular weight cannot be determined by
SEC, the apparent molecular weight calculated for globular
proteins can be used as a guide to evaluate the oligomeric
states of proteins when combined with the results of other
methods (e.g. protein crystallography). When a 50 ml sample
of purified C-AraR protein at 96 mM was injected onto a
Superdex 200 10/300 column, the protein eluted in two rather
broad peaks corresponding to apparent molecular weights of
276.5 and 119.4 kDa. Addition of l-arabinose at 50 mM to the
protein sample and column buffer produced an elution profile
with one sharp peak corresponding to an apparent molecular
weight of a globular protein of 70.6 kDa (Fig. 6). As the
theoretical molecular weight of the C-AraR monomer is
33.3 kDa, it can be concluded that the effector-binding domain
forms a homodimer in the presence of the effector. In the
absence of l-arabinose the protein forms a higher oligomeric
species with an apparent molecular weight corresponding to
octamers and tetramers or trimers (Fig. 6). The l-arabinose
acts as a conformational stabilizer which protects C-AraR
from aggregation.
The full-length AraR at 49 mM eluted as a single major peak
both in the absence and the presence of l-arabinose (Fig. 6)
which corresponded to the apparent molecular weight of a
homodimeric species (the theoretical molecular weight of the
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Acta Cryst. (2012). D68, 176–185 Procha ´zkova ´ et al.
? Effector-binding domain of AraR
Figure 3
Interactions of l-arabinose with C-AraR. The l-arabinose bound to
C-AraR is shown with yellow C atoms. The residues forming direct
hydrogen bonds (black dotted lines) to l-arabinose are highlighted by
green C atoms. Other residues within van der Waals distance (below 4 A˚)
of l-arabinose are shown with grey C atoms. Water-mediated hydrogen
bonds are represented by blue dotted lines. Residue His318, which is at a
van der Waals distance from l-arabinose, is not shown in the figure for the
sake of clarity.
Figure 4
Isothermal titration calorimetry (ITC) profile of l-arabinose binding to
C-AraR. The upper plot shows ITC results for 35 mM C-AraR titrated
against 420 mM l-arabinose. The lower plot shows the calorimetric trace.
The enthalpy of binding was obtained by integration of the upper curve
and subtraction of the curve from a control titration (not shown).
1 cal = 4.186 kJ.
Page 7
AraR monomer is 41.1 kDa). The minor peak that eluted at
17.9 ml represents a contaminating protein with an apparent
molecular weight of 13 kDa. The apparent molecular weight
of AraR in the presence of l-arabinose is slightly lower than
that of the free protein (85.9 kDa versus 93.8 kDa) and the
shapes of the elution peaks also differ. As the peak in the
presence of l-arabinose is sharper and more symmetric, we
can conclude that the presence of the effector stabilizes the
homodimeric form of the protein.
3.3.2. Oligomeric state in the crystal. For GalR/LacI
proteins in general, dimerization mediated by the effector-
binding domain is required for binding to the operator DNA
(Swint-Kruse & Matthews, 2009). Our SEC experiments
confirmed the dimerization of the full-length AraR as well as
of C-AraR in solution.
To identify the interfaces mediating AraR dimerization, we
performed a detailed analysis of the intermolecular contacts
observed in the crystal structures.
There are two major protein–protein interfaces in the
crystal which result in the formation of dimeric assemblies
(Fig. 7a). To deduce which of the two interfaces is likely to
reflect the dimeric interaction of C-AraR, these crystal inter-
faces and the architectures of the potential assemblies were
evaluated. Interface 1 has an interface area of 1431 A˚2, which
represents approximately 11% of the total solvent-accessible
surface area of a monomer. It contains 40 interacting residues
spanning both of the C-AraR subdomains (Fig. 7b). In the
N-terminal subdomain the interface comprises residues from
helix ?2 (153–162) and from the loop connecting the helix to
the preceding strand ?2 (147–153) and the residues forming
a loop connecting strands ?4 and ?5. In the C-terminal sub-
domain the following structural elements form the interface:
helix ?4 (residues 215–228) together with the subsequent loop
and 310-helix (residues 232–236) and strand ?7 (238–242).
Interface 2 has a smaller interface area of 885 A˚2, which
represents 6.5% of the total solvent-accessible surface area of
a monomer. The interaction is mediated almost exclusively
through the N-terminal subdomain by 25 interacting residues,
including the N-terminal residues, residues 88–92 from the
research papers
Procha ´zkova ´ et al.
? Effector-binding domain of AraR
Acta Cryst. (2012). D68, 176–185
Figure 6
Effect of l-arabinose on AraR oligomerization. The elution profiles from
size-exclusion chromatography of purified AraR and C-AraR in the
absence (solid line) and the presence (dotted line) of l-arabinose (l-Ara)
were monitored by absorbance at 280 nm. The apparent molecular weight
is assigned to each elution peak; the numbers in parentheses represent
the ratio between the apparent molecular weight and the theoretical
molecular weight of a monomer.
Figure 5
Thermal stabilization of AraR and C-AraR proteins by ligand binding.
(a) Plot of Tmdetermined by differential scanning fluorimetry (DSF) in
the presence of an increasing concentration of l-arabinose. (b) Examples
of the unfolding transition of AraR and C-AraR proteins in the absence
and presence of 50 mM l-arabinose (l-Ara). After reaching the plateau,
the fluorescence intensity starts to decrease owing to denaturation of the
protein–dye complex; these data were excluded from fitting and are not
Page 8
loop connecting strand ?1 to helix ?1, residues from helix ?1
(93–108), residues from strand ?2 (111–116) and residues from
helix ?2 (212–135). Only two residues from the C-terminal
subdomain of C-AraR (His304 and Ile308; Fig. 7b) are part of
interface 2.
Several pieces of evidence point towards the physiological
relevance of crystal interface 2 in AraR dimer formation. The
dimeric assembly produced by interface 2 allows the appro-
priate orientation of the DNA-binding domains for DNA
recognition and binding (Fig. 7a). In contrast, the dimeric
assembly produced by interface 1 has a head-to-tail orienta-
tion in which the the two N-terminal DNA-binding domains
would point in opposite directions, an architecture which is not
compatible with DNA binding (Fig. 7a). Interface 2 involves
residues in the vicinity of the effector-binding site (residues
88–92) and residues at the N-terminus (residue 81) which in
full-length AraR are directly linked to the N-terminal DNA-
binding domain.
The proximity of the effector-binding site would thus allow
an allosteric transition to the dimer interface upon effector
binding. Indeed, it has been shown for other members of the
GalR/LacI family that the N-terminal subdomain of the
effector-binding domain moves upon ligand binding, while
the C-terminal subdomain remains fixed (Swint-Kruse &
Matthews, 2009). The residues that participate in interface 2
are more conserved throughout the AraR orthologues than
those in interface 1 (Fig. 7c). Final evidence comes from the
mutation analysis of Franco et al. (2006), which showed that
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Acta Cryst. (2012). D68, 176–185 Procha ´zkova ´ et al.
? Effector-binding domain of AraR
Figure 7
C-AraR dimer interfaces observed in the crystal structures. (a) The dimeric assemblies formed through interfaces 1 and 2. Molecule A is coloured grey
and the two molecules interacting through interfaces 1 and 2 are coloured green and yellow, respectively. l-Arabinose is shown as spheres. (b) Details of
residues involved in interfaces 1 (highlighted in green) and 2 (yellow). (c) The solvent-accessible surface of the C-AraR dimer interfaces coloured by
residue conservation in closely homologous sequences. The surface is coloured in red to blue, with blue representing a sequence identity of 10% or lower
and red representing a sequence identity ofover 90%. Regions from neighbouring molecules which are in contact with the interface are shown as ribbons
and coloured green and yellow for interfaces 1 and 2, respectively.
Page 9
the mutation of residues Ile89, Tyr92, Glu102, Leu114 and
Phe305 resulted in severe defects in AraR function in vivo and
their role in dimerization was confirmed by classical trans-
dominant experiments. All these mutations are located in
interface 2 or in its close proximity (Fig. 7b).
We thus conclude that interface 2 is responsible for AraR
dimer formation. Crystal interface 1 might be involved in
the formation of higher AraR oligomers, as the cooperative
binding of two AraR dimers to two DNA operator sequences
that is crucial for efficient repression has been shown experi-
mentally (Mota et al., 2001).
Comparison of the C-AraR dimer formed through interface
2 with dimers of other proteins from the GalR/LacI family
revealed a similarity in the overall head-to-head dimer orien-
tation, but there is nevertheless a substantial difference in the
relative orientation of the monomers (Fig. 8).
The dimers of all GalR/LacI repressors for which structures
are available are formed around twofold axes (Fig. 8a). Such
an orientation is crucial for binding to the inverted-repeat
sequences of the operator DNA sites. The mutual orientation
of the effector-binding domains in the dimer is parallel and
the N-terminal DNA-binding domains point in one direction
(Fig. 8); in particular, the spacing allows
high-affinity interaction with the the
The crystal structures and molecular-
dynamics studies
binding of the effector molecule triggers
a conformational
transmitted into
domains and results in the misalignment
of the DNA-binding sites (reviewed in
Swint-Kruse & Matthews, 2009).
The dimeric assembly of the effector-
binding domains of AraR is structurally
different from that of other members of
the GalR/LacI family. The effector-
binding domains in the dimer are not
parallel; the longitudinal axes of the
monomers have an angle of about 40?
between them (Fig. 8). The position of
the DNA-binding domains in this dimer
cannot be predicted. AraR has a rare
modular structure with the C-terminal
effector-binding domain being homo-
logous to other members of the LacI/
GalR family but with the N-terminal
DNA-binding domain belonging to the
GntR family. The unique dimeric orga-
nization of the effector-binding domain
might be an indication of a different
mode of DNA recognition. Alter-
natively, the observed dimer archi-
tecture might also represent an induced
form of AraR in which the N-terminal
DNA-binding domains are in a position
with no affinity for DNA.
Further studies are required to investigate the structural
basis of the formation of the full-length AraR oligomer and
the modulation of the protein structure by binding of an
effector molecule.
DNA operator.
showed thatthe
This work was supported by the Ministry of Education of
the Czech Republic (research project No. ME08016) and
research projectsAV0Z40550506
awarded by the Academy of Sciences of the Czech Republic.
Diffraction data were collected on beamline MX14.2 at
BESSY, Berlin, Germany. The authors wish to thank Milan
Koz ˇı ´s ˇek of the Institute of Organic Chemistry and Biochem-
istry AS CR Prague for his help in performing the ITC
experiments and Devon Maloy for critical proofreading of the
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? Effector-binding domain of AraR
Acta Cryst. (2012). D68, 176–185
Figure 8
Comparison of the C-AraR dimer with a dimer of PurR. (a) The dimer of PurR from E. coli in
complex with hypoxanthine (PDB entry 1qpz; Glasfeld et al., 1999). The effector-binding domain is
represented as a surface, the DNA-binding domain is represented as ribbons and the co-repressor
hypoxanthine is represented as sticks. Longitudinal axes of the effector-binding domains are shown
as green lines. (b) The dimer of C-AraR. The effector-binding domain is represented as a surface
and the inducer l-arabinose is represented as sticks. Longitudinal axes of the effector-binding
domains are shown as maroon lines.
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? Effector-binding domain of AraR


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The Church On Love: Video 1 of 3
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In video 1 of 3 of "The Church On Love, Sexuality, And Marriage" I discuss what love is, and isn't, in the eyes of the Church using Christ's own teachings on it's meaning. Understanding the true nature of love is vital in order to approach topics like sexuality and marriage with something more than sentiment. This three part "Pray, Reflect, Act" series comes from questions for my blog that received on Facebook. To see the whole post including reference links and more visit http://www.catholicfamilyman.com
Love Sexuality Marriage Catholic Theology of the Body
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Thank you so much for this video. I am really struggling to grasp the full meaning and wholeness with "Love" in my life right now. When I stumbled across this, a lot of my thoughts and confusions became clearer. Thank you again.
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Google, Bing Hit All-Time Highs In June Search Market Share [comScore]
Google and Bing have reached all-time high levels, while Yahoo reached an all-time low in the latest comScore search market share numbers. They're due to be released publicly later today or tomorrow, but financial analysts have seen and are sharing their copies of the data.
According to comScore's explicit search market share report for June 2012 — which covers desktop searches only, not mobile — the numbers are as follows:
- Google: 66.8 percent in June, up from 66.7 percent in May; an all-time high for Google
- Bing: 15.6 percent in June, up from 15.4 percent in May; an all-time high for Bing
- Yahoo: 13 percent in June, down from 13.4 percent in May; an all-time low for Yahoo
ComScore's data has now shown Yahoo with 10 consecutive declining months, in which the company has fallen from 16.3 percent to the current 13 percent market share. Bing, meanwhile, has had 26 straight months of either flat or rising market share.
Postscript: ComScore has now formally announced the numbers above. Here's the chart version of the above for those who prefer it in graphical form:
Join the most accomplished search marketers in the world at SMX Advanced. Conference sessions and keynotes are designed for sophisticated search engine marketers. You'll choose from in depth sessions on organic search engine optimization (SEO), paid search advertising (PPC), and more. Lowest rates expire March 28. Register Now!
(Some images used under license from Shutterstock.com.)
The latest analyses, insights and strategies that inspire CMOs and marketers everywhere.


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To unearth the newest details about Tony Singh, check out PeopleFinders.com. We present an impressive array of records which will yield the responses you are seeking. You will also discover important facts like Tony Singh's age, address, aliases, and more.
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Follow My Lead! The Great Survival Challenge
Trivia, Quotes, Notes and Allusions
Quotes (16)
Iruka: Well, that's typical. Not happy unless he's giving people a heart attack.
Iruka: (Thinking) Naruto... Can't that little fool ever follow instructions?
Shikamaru: Iruka-sensei, I found one of their backpacks on an unmarked trail. Iruka: Naruto, you wouldn't...would you?!
Naruto: Hey! What are you doing over there? How'd you get out of this? Shikamaru: Hey, it wasn't my decision, but I'm glad they made it. I knew this mission would be a drag.
Tsunade: If you don't wanna do it, you don't wanna do it. I'll just re-assign your team. Naruto: Hey! Can't you take a joke? I was just Kidding! I was only pulling your leg, Grandma Tsunade, that's all. I mean, come on, hokage training? I'd never miss that. I was going to do it all along.
Naruto: I've got to get back to my training. Tsunade: That's right, because you're going to be Hokage someday. Right? I wouldn't think disappointing those eager young kids is something a future Hokage would do. Being a leader, that's what this mission is about. Achieving a goal while protecting the lives of the people on your team. In fact, of all the missions I could assign you, this one's the ideal training for becoming Hokage. Naruto: The ideal training, really?
Naruto: No way! No way! You've got to be kidding! I want to be sent on a real mission, not be stuck with babysitting. That's not what I signed up for! No way, forget it!
Lee: Good job. Everyone okay? Students: Yes sir, Lee-sensei! Lee: Huh? What did you just say? Students: Why, yes sir, Lee-Sensei! Lee: Lee-sensei! The sound of it, music to my ears! This is the power of youth! I can feel it! Forward men, forward into the setting sun! Students: Yes sir, Lee-sensei! Lee: Let its golden rays shimmer in the sweat of your armpits!
Notes (4)
YTV Airdate: November 8, 2008
Original Episode Title: Everyone, Come With Me! Great Sweat and Tears Survival Plan Romaji Episode Title: Minna Ore ni Tsuite Koi! Ase to Namida no Takurami Dai Sabaibaru Kanji Episode Title: みんなオレについて来い! 汗と涙のタクラミ大サバイバル
Original Airdate: November 2, 2005
Sakura didn't have a squad in this mission, although she is a genin.