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From the incredible wedding of Brian and Chad.
From the incredible wedding of Brian and Chad.
I've been kind of in a fashion-funk lately – I've been gravitating towards one single color palette only, which makes getting dressed quick and easy, but sometimes you can look kind of…..drab. Luckily there is a quick solution to that, and one of my favorite ways to add some awesome detail is in a big, statement necklace. Not only do they frame your face and draw the eye line up, it adds a quick punch of color where you need it.
A few of my favorites are above – (can you just tell I have a thing for gold and blues?) Shop the looks here:
We spend so much of the week working ourselves to the bone – and maybe it's just me, but rarely do we slow down and take the time to do things for ourselves, things that truly make us happy.
This weekend, I'd like everyone to take more than a few moments to indulge in yourself. Slow down, and do things that put a smile on your face. You know you deserve it!
A few things that make me happy are:
01: Fruity Cocktails | 02: Dining with Friends | 03: Sleeping In. Or Taking a Nap. Lots of them. | 04: Being with furry ones | 05: Ice Cream All Day.
What makes you happy?
The bridal shower is an occasion to be celebrated – usually, female members of the bride and grooms family (along with close friends!) come to celebrate the bride and "shower" her with gifts to help her stock her new home. Not to be mistaken with the bachelorette party, this event calls for attire that is slightly more pretty and demure (but I didn't say boring!). This outfit above makes my heart flutter – the lace keeps it feminine and girly, yet shows just enough skin – pair these with simple accessories for the perfect bridal shower look.
Shop The Look: One | Two | Three | Four | Five
So thrilled to announce that I will be speaking at the Be Sage Conference in Dallas this November! I cannot wait to share some of my secrets on designing and putting together destination weddings all around the world! If you're in the wedding industry and are curious to see how we work, I would be honored if you will join me! I hope to see you there!


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Started this discussion. Last reply by Stina Sep 12, 2009. 1 Reply 0 Favorites
I'll follow you too!http://mommyhastowork.blogspot.com/ThanksContinue
anne crane has not received any gifts yet
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A mean mother never allows candy or sweets to take the place of a well-balanced meal.
A mean mother insists on knowing where her children are at all times, who their friends are and what they do. A mean mother breaks the Child Labor Law by making her children work - washing dishes, making beds, learning to cook and doing other cruel and unpleasant chores.
A mean mother makes life miserable for her offspring by insisting that…
Posted on September 1, 2009 at 2:28pm 0 Comments 0 Favorites

subset: commoncrawl


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Moortown Lodge was originally a Georgian hunting lodge and is conveniently located ten miles from the Dorset coast, not far from Christchurch, and just five minutes south of Ringwood. It offers free W...More about Moortown Lodge
High quality, budget accommodation ideal for both families and business travellers. Spacious, en suite bedrooms feature tea and coffee making facilities, and Freeview TV in most hotels. Internet acces...More about Premier Inn Bournemouth/Ferndown
Dating back to the 16th century, this delightful farmhouse was gifted to Lord Nelson's family as part of the Trafalgar estate. The house has comfortable bedrooms, most with four-poster beds, and all a...More about Newton Farmhouse
This delightful Victorian home is situated in a quiet residential area within easy walking distance of the town centre. Bedrooms are attractively furnished and decorated, with many thoughtful extras. ...More about Amberwood
This charming 17th-century inn, within easy reach of Bath, Salisbury, Glastonbury and the Dorset coast, offers comfortable accommodation in a peaceful setting. Carefully prepared dinners are enjoyed i...More about Compasses Inn
This stunning 19th-century, mock-Elizabethan mansion is set in 40 acres of beautifully landscaped gardens and forest. Bedrooms are spacious and great consideration is given to guest comfort. The elega...More about Rhinefield House Hotel
The inn is part of the Bramshaw Golf Club and has tailored its style to suit this market, but it is also an ideal base for visiting the New Forest. Bedrooms are comfortable and attractively furnished,...More about The Bell Inn
Full of character, the Old Mill has an interesting history going back well over five hundred years. Located in tranquil water meadows, the medieval city of Salisbury is just a ten-minute walk along th...More about Old Mill
This popular hotel, dating from 1896, has gardens that overlook the water meadows and the cathedral. The attractive bedrooms vary in size, some offer excellent quality and comfort, and some rooms are ...More about Grasmere House Hotel
This 13th-century coaching inn, situated beside the river, enjoys picturesque views of Salisbury Cathedral, especially from the Pavilion Restaurant which provides a good range of dishes. Many original...More about The Legacy Rose & Crown Hotel


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MENLO PARK, CA–(Marketwired – May 21, 2013) – Delphix, the leader in agile data management, today announced CEO Jedidiah Yueh was chosen as a winner in the CEO of the Year category of the 2013 San Francisco Business Times Tech & Innovation Awards.
"Delphix has seen tremendous growth recently, including major new partnerships with SAP and In-Q-Tel and over 200 percent sales growth in 2012," said Yueh. "This award recognizes the impact our company is making for customers worldwide."
The Tech & Innovation Awards program accepts entries from private or public companies headquartered in the Bay Area. Editors from the San Francisco Business Times selected Yueh from a pool of nearly 200 nominees. Yueh is joined in the CEO of the Year category with several other notable executives from companies including Splunk, Eventbrite, Ask.com and SquareTrade.
Yueh has demonstrated a successful track record for identifying new market opportunities and staying ahead of industry trends. Prior to founding Delphix in 2008, Yueh also founded Avamar, a software company that pioneered data de-duplication and shipped a billion-dollar product in the backup and recovery industry, with over 20,000 current customers. EMC acquired Avamar in November 2006 for $165 million. He brings over a decade of experience in the software and data storage industries and has more than six patents or patents pending.
Delphix transforms the economics of database management, cutting application project schedules by up to 50% while reducing data storage costs by 90%. Delphix-powered virtual databases for Oracle and Microsoft SQL Server are more powerful than physical versions; with the ability to create complete copies in minutes in 1/10th the storage footprint.
About Delphix
Delphix enables agile data, transforming the economics of database and application management. Delphix software addresses the single largest source of inefficiency and inflexibility in the modern datacenter — provisioning, managing, and refreshing databases for business-critical applications. Leading global organizations use Delphix to dramatically reduce the time, cost, and risk of application rollouts, simplifying database operations by 100x, while consolidating redundant hardware by 10x. Delphix is located in Menlo Park, California.
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Delphix Announces the Winner of the 2014 Annual Technology Scholarship for Women
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Sequim man lauded for brave response to attack on pregnant woman
Joe Smillie/Peninsula Daily News
Sequim Police Chief Bill Dickinson, left, presents the city's Citizen Commendation Award to Raven Crawford with Mayor Candace Pratt, right, at last week's City Council meeting.
By Joe Smillie
Peninsula Daily News
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"In bare feet, and with little regard for his own safety, Mr. Crawford ran nearly a block to assist the female," Police Chief Bill Dickinson said.
Crawford wrestled the woman's assailant to the ground and held him there until police arrived.
"I got to play hero for about 10 minutes because I knew I could count on our paid, professional heroes to relieve me," Crawford said. "Which they did just in time."
For his actions, Crawford was given the city's Citizen Commendation Award by Dickinson and Mayor Candace Pratt at Monday's City Council meeting.
"What he saw was wrong, and he decided to do something about it," Dickinson said.
Standing ovation
The crowd of more than 30 at Monday's meeting gave Crawford a standing ovation after he received the award.
Crawford was in his home at a trailer court near the intersection of West Washington Street and Brackett Road when he heard the screams.
Without waiting to put on his shoes, he ran down the paved roadway, where he saw "a very pregnant" woman being assaulted by a man.
Crawford wrestled the man away, suffering abrasions and a puncture wound from what police believe was a needle in the assailant's pocket.
"Completely out of energy," Crawford held the assailant for about 10 minutes until officers arrived.
The assailant, who was not identified at the meeting, was later convicted on assault and domestic violence charges, Dickinson said.
The chief credited Crawford for acting quickly but advised others to contact police in such emergencies.
"Obviously, we don't want people trying to play vigilante, but Mr. Crawford was very brave to step in as other citizens were there watching," Dickinson said.
Sequim-Dungeness Valley Editor Joe Smillie can be reached at 360-681-2390, ext. 5052, or at email@example.com.
Last modified: August 16. 2014 6:04PM


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DetailsChina Glaze Neon Purple Panic Nail Polish
Take purple and drag it over into pink territory a little ways, then pump it full of neon and you get China Glaze Neon Purple Panic Nail Polish. This dynamic color is a youthful purple that is perfect for summer time.
For a full selection of China Glaze nail polish colors and other nail salon supplies as great low prices, shop Big Daddy Beauty today!
Manufacturer: China GlazeSKU: CHINA-70290Weight: 0.25 lbs100% Guaranteed Authentic
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StartUpHire is the place to find Systematic Portfolio Manager jobs. Review the list of Systematic Portfolio Manager career opportunities to the left. These Systematic Portfolio Manager jobs represent some of the most rewarding careers available.
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More information about this job Overview DataDirect Networks (DDN.com) is a world leader in massively-scalable storage platforms and solutions engineered for the Big Data and Cloud era. Our storage, processing solutions and professional services enable content-rich and high-data3/16/2015 Columbia, MD Save This Job
More information about this job Overview DataDirect Networks (DDN.com) is the world leader in scalable storage infrastructure for big data and cloud applications. We are the leading provider of data storage and processing solutions and professional services that enable2/14/2015 Colorado Springs, CO Save This Job
Ruckus Wireless is a pioneer in the wireless infrastructure market, enabling carriers and enterprises to stay ahead of the exploding demand for high-bandwidth applications and services. The Ruckus Smart Wi-Fi technology redefines what?s possible in wireless network performance2/7/2015 Sunnyvale, CA Save This Job


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Grafting onto old wood - Take 2
damiencwalkerJuly 18, 2013
Hi all
I posted a question here the other day about grafting onto old wood in cherry trees. I'm looking for advice about grafting buds onto the lower section of the trunk in order to get new growth low on the tree..
I've added a picture here that shows the approximate shape of my trees. As you can see, there are no existing low branches I can head back to encourage new growth. What I'd like to know is if there is any way to graft buds into the lower section of the trunk, remembering the trees are around 10 years old.


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Natural Talent Design Competition
The 7th Annual
The Minnesota Chapter of the USGBC supports the Natural Talent Design Competition to help facilitate a green building collaboration between aspiring architects and designers with the Chapter and the professional community.
What: This year's project is the renovation and expansion of the Jackson Elementary School in St. Paul, MN.
This is an educational facility that will accommodate hands-on activities, workshops and classes for students, teachers visiting school groups and the community .
View the complete PROJECT SCOPE
Why: To empower students and young professionals to become future leaders within the green building movement.
Who: Young professionals within two years of graduation and current college students. Teams can work in tandem with mentors, including project check-ins and professional development. Designs will be due October 31, 2014 and winners will be announced at the end of November. Teams will be recognized at our Annual Meeting.
Dates to Remember:
September 12, 2014 – Last day for free registration | $25 per team after
October 31, 2014 – Design submission due date
November – Judging & Award Ceremony (date to be determined)
All teams benefit greatly from participating in the competition. They gain valuable project experience and can add the competition to their CV. The top three teams will be awarded prize money. In addition to this, all teams are recognized in a press release following the ceremony.
Rebekka Jossart, Design Competition Chair | [email protected]
Sheri Brezinka, Executive Director | [email protected] | 612.328.9098


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Google Starts Scanning Android Apps 172
from the bender's-job-between-seasons dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
It is good that they are going to finally scan for malware. But in the end Android apps need better quality control. There are so many poorly-written apps with memory leaks. I end up having to reboot my Android at least 1x/day for no explainable reason. Android is really neat but I feel like it is one big beta test that I paid money to use.
...a more fine-grained security model and a firewall to android?
I understand it's a problem for Google if users can suddenly notice how much
is transferred to Google but I think it's the only way to go in the end.
You figured something like this would have been in place from day one. Let's sell apps, but not worry about if they are loaded malware or viruses.
Not likely. FTA:
Once an application is uploaded, the service immediately starts analyzing it for known malware, spyware, and trojans. It also looks for behaviors that indicate an application might be misbehaving and compares it against previously analyzed apps to detect possible red flags.
That's a pretty good description of proper scanning for bad code. As TFS stated, this isn't the Apple paradigm where they want to control their users. The purpose is to maintain a profitable marketplace and platform by protecting users who keep hearing about Android malware.
Thrashing is just virtual crashing.


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Mail: Clarity on the post office debacle
Davis at the Legislature: Rain, brand fees, state aid to schools
More opinion
Registration open for UNL ranch practicum
Governor appoints new District 4 representative to Nebraska Wheat Board
More Ag News
Click Here for theHOTTEST SHEETS!
North Platte police are looking for a hit and run driver who hit a pedestrian on Saturday.
Sarah Doherty, 32, was walking across the street to her car was parked on Fifth St., when another car ran into her and then drove off, westbound. Doherty's car was parked in a driving lane, according to a police diagram.
The collision happened around 6:40 p.m. near the intersection of Fifth and Carr.
Doherty, who lives at 1220 East Sixth, suffered visible but not disabling injuries, according to the police report. She was not taken to the hospital.
Flatrock Publishing, Inc.
1300 E 4th St., Suite F
North Platte, NE 69101


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What can I do to save my azelea?
I have an azelea next to my house hidden between overgrown burning bush and a huge peony. I admit, it's been neglected. I've lived there 5 years and have thought of moving it, but never got around to it. This spring we had several days of 20 degree weather in April while I was out of town and the poor thing has about had it. It's leafed out now, but all of it's sparce leaves are burned brown in patches. Obviously, it didn't bloom at all. With such sparce growth, it actually appears there are three distinct bushes, all very small, instead of the one large one I thought I had.
I don't want it to die altogether. I have a shady bed in front of the house where an inkberry bush formerly was that I want to move it to, but I don't want to shock it any worse right now and I expect this isn't the best time to move it anyway. I've been told to move it in the fall. But what can I do for it right now that might give it a boost? It's looking awfully bad.
For many of us, this past winter our plants took a beating with the very unusual winters some of us had with warm weather followed by late freezes. They have what is more typical of drought damage but in this case was caused by the warm weather followed by late freezes. It causes Botryosphaeria dothidea which causes leaves to turn dull green and then brown and roll and droop. Cankers form on branches which may girdle the branch.
This is the most common disease of rhododendrons in the landscape. A typical symptom of this fungal disease is scattered dying branches on an otherwise healthy plant. Leaves on infected stems turn brown, then droop and roll inward. These leaves often lay flat against the stem and will remain attached. The pathogen can infect all ages of stem tissue through wounds, pruning cuts, and leaf scars. Heat, drought stress, and winter injury can increase disease incidence. Cankers on branches can gradually grow through the wood until the stem becomes girdled. Diseased wood is reddish brown in appearance. Discolored wood viewed in longitudinal cross section often forms a wedge that points toward the center of the stem, and the pith may be darker brown than the surrounding wood.
Sanitation and applying a fungicide such as metalaxyl (Subdue) after pruning my provide some control. Plants should be grown in partial shade, with mulch and kept well watered during dry periods. All dying branches should be promptly pruned out in dry weather and all discolored wood should be removed. Plants should also be protected from rough treatment during maintenance activities to prevent unnecessary wounds.
Whether to transplant in spring or fall is always a question. Fall is normally the best time because roots on a new plant need help establishing themselves. The shallow root system can't take in all the water it may need to survive and a drought can spell disaster. Water them frequently in the morning. Mature plants are much hardier and Mother Nature seems to take good care of them under normal conditions. Care for new plants for 2-3 years to help them get established. One problem with fall transplanting is that it makes a plant more susceptible to frost heave in climates where freezing and thawing cycles are common. In that case, rhododendrons transplant best in the spring. It must be noted that maintaining the proper moisture level in the summer is very difficult after spring transplanting. Make sure you watch the plant after it was moved like you would a new plant. Its roots are compromised and it will need a reliable source of moisture. If the weather has a dry spell, make sure you water any newly planted rhododendrons, large or small.
Unfortunately miracid is mostly advertising hype from Scotts. It is actually mostly water soluable nitrogen which is exactly what azaleas don't need. Use a balanced azalea fertilizer like Hollytone in the spring. Use it at half the rate on the package.


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The Glacier Bay Lyndhurst Series 2-Handle Bridge Kitchen Faucet with Side Sprayer in Mediterranean Bronze will add old-world charm and functionality to your kitchen décor. Durable with brass construction in an elegant Mediterranean bronze finish, separate lever handles for hot and cold water allow you to conveniently adjust the water temperature and volume, while the high-arc spout allows plenty of clearance. The included side sprayer makes washing vegetables, clearing the sink or filling containers quick and convenient, while the ceramic disc cartridges ensure no drip performance. Pair with other Mediterranean bronze pieces from the Lyndhurst Collection for a complete, polished look. WaterSense certified. Limited Lifetime Warranty.
|Assembled Depth (in.)
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New Rugby 7s stadium in Dubai
As Dubai's newest rugby stadium prepares to open we catch up with the chairman of the Arabian Gulf Rugby Football Union to talk about the state of the game in the region. Discuss this article
Rugby in the desert. It's incongruous to say the least. Breezing about on a pitch may be one thing, but the prospect of sticking one's head into a heaving pit of an eight-man scrum leaves me less than stoked.
But Dubai rugby has forged a name for itself. Speak to anyone who's into their game and they'll tell you that the emirate is fast becoming synonymous with a hard, fast style of sevens rugby. So much so that Emirates airline has planted the flag for a stadium on Al Ain Road that will put rugby at the city's heart.
'Dubai rugby is getting a world-class facility,' says Dave Skidmore, chairman of the Arabian Gulf Rugby Football Union (AGRFU), which will be making its home at the new ground. The stadium has been named The Sevens in tribute to the competition that has kept rugby-driven masses flocking to Dubai for almost 40 years, and which will be played in November of this year. 'There aren't many independent facilities that offer six pitches, 14 changing rooms and a large social facility, including swimming pools and netball courts,' notes Skidmore. 'It's going to be a great community hub.'
For those unfamiliar with the faster variant of rugby, sevens uses union rules, but cuts the number of players from 15 per side to seven. A match lasts 15 minutes (down from a standard 80-minute game). It all adds up to a hard, fast game, where scoring tends to be easier (with more space on the pitch) than the traditional game. The added excitement, alongside a simpler format than its grown-up brother, means it's often a more attractive game to watch and appeals to a wider audience.
The Dubai Sevens has traditionally been held at the Dubai Exiles rugby ground and forms one leg of the IRB Sevens World Series. After New Zealand's win last year, this November will see the tournament will become the first event to use the new ground.
The Sevens will feature a permanent grandstand with seating for 4,000, along with temporary structures around the main pitch to cater for up to 40,000 sports fans. The stadium will also be the venue for the Rugby World Cup Sevens, which Dubai is hosting in March next year.
The Sevens is the sort of project you'd expect to find in a Rugby-mad country such as New Zealand. It suggests the sport has taken over the town. Yet it's cricket and football, not rugby, being played by groups of kids in parking lots or dominating the talk of taxi drivers. Does union really have the sort of appeal that such a structure demands, and can that extend beyond one annual competition and the expats it traditionally attracts?
Rugby found its way into Dubai's Right To Play programme last year. The project is designed to boost the amount of sport going on in schools around the world and has HRH Princess Haya bint Al Hussein (wife of Sheikh Mohammed) as its UAE patron. Since rugby was included in the curriculum, the appeal has grown rapidly. Already over 600 rugby lessons have taken place and 15,000 students have been scrumming down in gym classes in schools (many of which are Arabic) around Dubai.
'We're finding that Emiratis are really taking to the game,' says Skidmore. 'Once they find it's not quite as physical as they first thought – when they've bounced back up a couple of times – they realise it's not so bad.'
Surprisingly, AGRFU is finding that girls are often taking to it more than boys. 'Having rugby accepted in the curriculum has been a massive boost. Combine that with the new facility, which will offer coaching courses, and the future of rugby in the UAE looks bright,' Skidmore says.
Established teams, such as the Dubai Exiles, have been going for 40 years now, but a clear sign of the sport having any long-term future among locals will come with the emergence of more teams and more Emiratis appearing on the lineups. There are already teams with a strong Emirati base, such as the Dubai Falcons, but it's reckoned that Right To Play and the new Sevens stadium will provide the sort of grassroots backing that the sport needs to take root locally.
So is rugby the next stage in Dubai's endless quest for self-definition? Perhaps. The city has long been on the map for rugby's international audience, but whether it can forge a local standing remains to be seen. The response from Right To Play is promising, and The Sevens venue will offer space across its five other pitches to the various rugby union teams that have made their home in Dubai. For now, we'll have to wait and see…
The Sevens will open by November. For updates on the competition go to www.dubairugby7s.comBy Chris Lord
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Advanced Shiur: An Old Hut Made New
Rabbi Yonasan Sacks on the halachos of Sukkah Yeshana
September 16, 2013
Kol Nidrei: A Model For Teshuva
Kol Nidrei is not merely a halachic declaration, but rather a guide to the teshuva process of Yom HaKippurim
September 11, 2013
Yom Kippur is a Palace
On the inevitable slippage from the height of Yom Kippur and how to view that challenge.
September 11, 2013
Elitism and Condescension
Introspection for Aseres Yemei Teshuva
September 11, 2013
9/11: One Person Can Make a Difference
"Social work is about helping people get to a better place, including yourself. I'll be a great social worker. I love people—all shapes and sizes."
September 10, 2013
Rosh Hashana: A Humble Accounting
Dr. Kadish's Rosh Hashana letter to the Touro College & University System family.
September 03, 2013
Divrei Torah for Rosh Hashana
Some words of inspiration as we prepare for Rosh Hashana and the Yomim Noraim.
August 30, 2013
"I'm a DO, that's what we do, we touch people."
A "win" from a recent D.O. graduate.
August 26, 2013
Welcome to the Lander family
For all those just starting out at Lander.
August 22, 2013
Bring Your A-Game
Advice for the Touro College of Pharmacy Class of 2017
August 20, 2013
A Wet Welcome
TouroCOM Throws BBQ Block Party for Students and Staff
August 13, 2013
Winning the Lottery
My 17-Year Journey from Cuba to Curing Others
August 06, 2013
To the TouroCOM Class of 2017: Breathe
A few words of advice from the Class of 2013
August 06, 2013
The Best Medicine
Pre-med Student Jeffrey Herskovits Helps Comfort Sick Children at Camp Simcha
August 05, 2013
The Advocate
"I can relate to when you don't know who to turn to."
August 01, 2013
100 Percent of TouroCOM's 2013 Graduates Placed in Residency Programs
Several Graduates Matched to Most Prestigious Programs in U.S.
July 26, 2013
A Home Run
Touro Spirit Night with the Mets
July 24, 2013
Glückwünsche Touro College Berlin!
Touro College Berlin Celebrates its 10 Year Anniversary and 2013 Graduation Ceremony
July 11, 2013
Steven Elrod is always coming forward to give back
Touro College of Pharmacy Student Steven Elrod Receives Prestigious Award from U.S. Public Health Service
July 10, 2013
Taking Tea at 113th ASM Meeting
Touro Professor and Undergraduate Students Present Anti-Microbial Research at the 113th Annual American Society of Microbiology Conference in Denver, CO
July 10, 2013


text:
The sulky schoolboy who became Britain's great wartime leader: Earliest surviving original images of Winston Churchill are released
Seven extremely rare photographs of Churchill have been restored and rescued from a barn in Cirencester
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Seven extremely rare photographs of Churchill have been restored and rescued from a barn in Cirencester
Tuesday 07 January 2014
Jack Hammersley and Jorge Gill were criticised for using a pulley system to wakeboard in an underground carpark
Thursday 02 January 2014
BBC business journalist Liz Barclay is set to join the Financial Consumer Panel, which advises the Financial Conduct Authority.
Wednesday 18 December 2013
Biggs became famous after he escaped from prison and spent the next three decades on the loose in Brazil following 'The Crime of the Century'
Tuesday 10 December 2013
A freeze on council tax and cap on welfare will add to their misery
Tuesday 24 September 2013
A senior police officer today claimed that no lessons could be learned from the operation which ended with the shooting of Mark Duggan.
Sunday 22 September 2013
As a hack myself, I ought not to be surprised if I myself become the victim of hackery. But last week I was royally stitched-up and for a moment, I confess, I had a sense of humour failure.
Thursday 05 September 2013
Camden Council set to approve plans for the search engine's new base in King's Cross
Friday 30 August 2013
The project is inspired by a 1980 study of New York's urban plazas
Sunday 25 August 2013
Sabrina Moss, 24, died in hospital after being involved in the double shooting
Saturday 24 August 2013
Another woman, also 24, is in hospital with non-life threatening injuries
Tuesday 20 August 2013
Responses follow The Independent's disclosure that 40 schools had published policy statements echoing language of notorious legislation
Sunday 18 August 2013
Londoner recovers from sixth round knockdown to secure IBF crown
Wednesday 31 July 2013
Natalie Hynde, the daughter of The Pretenders' singer Chrissie Hynde and The Kinks' Ray Davies has been arrested after apparently gluing her hands to her boyfriend during a protest against fracking.
Friday 19 July 2013
A former activist in the radical Islamic group Hizb ut-Tahrir has been chosen to fight a marginal parliamentary seat for the Liberal Democrats.
Wednesday 03 July 2013
A well-established handbook for writers and artists is to include a chapter on writing erotic fiction for the first time.
Brilliant Barbados is an exciting promotion giving visitors to Barbados more than ever before. It's always a good time to visit Barbados.
Discover Tobago with our competition for one lucky reader to have the chance to win a seven night self-catering holiday for two. Enter here.
The technology, which is now being trialled by West Yorkshire Police, could lead to the biggest change in forensic fingerprinting in its 100-year history.
Whatever the result of the forthcoming General Election, tackling Britain's housing crisis should be at the top of a new government's to-do list.
Borrowing money is not a decision which should be taken lightly. Read this guide to help get the right information.
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Win anything from gadgets to five-star holidays on our competitions and offers page.
Watch the best in the business world give their insights into the world of business.


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A Wedding in Great Neck
The Silverstein family is coming together in Great Neck, Long Island, for the nuptials of the youngest daughter. Always considered the favorite, and the object of much envy and resentment. Angelica has planned a fairy tale wedding to her fiancé, a former fighter pilot. But there are storm clouds on the horizon. Gretchen, Angelica's sister, is dealing with a failed marriage and her moody teenage daughter, Justine. One brother is a callous businessman while the other is struggling with his search for love and a career. Her mother is in a battle of wills with the wedding planner, while her father, a recovering alcoholic, struggles to confront his ex-wife's lavish new life in the Long Island manor of her dreams. And her grandmother Lenore has decided it's high time to take charge and set her grandchildren on their proper paths. Then an impulsive act by Justine puts the entire wedding at risk and brings the simmering family tensions to the boiling point. Before vows are exchanged, this day will change more than one life forever.
Publisher: New York : New American Library, c2012.
Characteristics: 315 p. ;,21 cm.
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Feb 28, 2013
Not a bad little book to read. I recommend it for a little light reading. I enjoyed it.
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text:
Page 1
Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase
Bla2 from Bacillus anthracis
Megan J. Hawk1, Robert M. Breece2, Christine E. Hajdin1, Katherine M. Bender1, Zhenxin
Hu1, Alison L. Costello2, Brian Bennett3, David L. Tierney2,*, and Michael W. Crowder1,*
1Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, Ohio
2Department of Chemistry and Chemical Biology, B80 Clark Hall, MSC03 2060, University of New
Mexico, Albuquerque, New Mexico 87131-0001
3National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin, 8701
Watertown Plank Road, Milwaukee, Wisconsin 53226-0509
In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase
Bla2 from Bacillus anthracis, the enzyme was over-expressed, purified, and characterized. Metal
analyses demonstrated that recombinant Bla2 tightly binds 1 eq of Zn(II). Steady-state kinetic
studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was
unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-
Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analog. Rapid kinetics
and UV-Vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2
is distrubuted, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the
first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms,
underscoring the need to demonstrate transferability when extrapolating results on Co(II)-
substituted proteins to the native Zn(II)-containing forms.
Anthrax is a disease that commonly affects cattle and other herbivores, caused by the spore-
forming bacterium Bacillus anthracis.1 Anthrax infections in humans can be caused by
contact with infected animals, or by ingestion or inhalation of B. anthracis spores. The
potential for airborne transmission, together with the known lethality of Anthrax infection,2,
3 has led to the development of anthrax as a potential biological warfare agent,4 and B.
anthracis has been labeled as a category A bioterrorism agent by the Centers of Disease
Control and Prevention (CDC).3 Currently, the CDC recommends a 60 day antimicrobial
regimen of ciprofloxacin, doxycycline, or amoxicillin for anthrax infection.4
Cephalosporins, trimethoprim, and sulfamethoxazole are not prescribed, due to suspected
resistance to these drugs. Recently, the Sterne strain of B. anthracis has been reported to
produce both a class A (Bla1) and a class B (Bla2) β-lactamase.5
* To whom correspondence should be addressed: M.W. Crowder: firstname.lastname@example.com; phone: (513) 529-7274; fax: (513)
529-5715, D. L. Tierney: firstname.lastname@example.com; phone: (505) 277-2505; fax: (505) 277-2609.
Supporting Information Available. A description of materials, over-expression and protein purification is provided, along with ten
Figures and six Tables, including SDS-PAGE gels of Bla2 purification (Figure S1), structures of the substrates used in kinetic studies
(Figure S2), detailed EXAFS fitting results for 1Co-Bla2 (Figure S3 and Table S1), CoCo-Bla2 (Figure S4 and Table S2), 1Zn-Bla2
(Figure S5 and Table S3), ZnZn-Bla2 (Figure S6 and Table S4). Zn K-edge XAS of 1Zn- and ZnZn-CcrA are also presented and
discussed (Figures S7-S10 and Tables S5 and S6). This information is available free of charge at http://pubs.acs.org.
NIH Public Access
Author Manuscript
J Am Chem Soc. Author manuscript; available in PMC 2012 March 6.
Published in final edited form as:
J Am Chem Soc. 2009 August 5; 131(30): 10753–10762. doi:10.1021/ja900296u.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 2
Currently, over 50% of the antibiotics prescribed by physicians are β-lactam-containing
compounds, such as penicillins, cephalosporins, and carbapenems.6 However, an increasing
number of bacterial pathogens show resistance to β-lactam antibiotics, threatening their
efficacy into the future. The most common pathway for resistance is the production of β-
lactamases, which hydrolyze the four-membered ring in β-lactam antibiotics, rendering them
ineffective.7, 8 To date, over 400 β-lactamases have been described, and these enzymes
have been classified into four subgroups (A-D).8 While exhibiting different kinetic and
inhibition properties, all group A, C, and D β-lactamases utilize an active site serine to
perform nucleophilic attack on the substrate. The serine β-lactamases (SβLs) are the most
clinically-significant, and there are some clinical inhibitors that are active towards a majority
of the enzymes in these groups.8
In contrast, the group B β-lactamases, often referred to as metallo-β-lactamases or MβLs,
require 1-2 Zn(II) ions for catalytic activity, and these enzymes are unaffected by SβL
inhibitors.9, 10 Approximately 40 MβLs have been reported, leading to their further
classification into 3 subgroups.11 The B1 enzymes require 1-2 Zn(II) ions for full activity,
and prefer penicillins as substrates. They bind one Zn(II) in the Zn1 (or 3H) site, which is
made up of His116, His118, His196, and a solvent-derived ligand that bridges the two metal
ions, and one Zn(II) in the Zn2 (or DCH) site, made up of His263, Asp120, Cys221, the
bridging solvent, and a terminally-bound water. The B2 enzymes require only one Zn(II) for
full activity, prefer carbapenems as substrates and bind the catalytic Zn(II) in the Zn2 site,
while the B3 enzymes, which prefer cephalosporins as substrates, generally require two
Zn(II) ions for full activity, binding one Zn(II) in a canonical Zn1 site and one Zn(II) in a
modified Zn2 site, where Cys221 is replaced by His121.
Although the Sterne strain of B. anthracis has not yet caused human infection, transfer of the
Bla2 gene to a more pathogenic Bacillus is expected to be facile. Bla2 shares 89% sequence
identity and 92% sequence homology with the B1 MβL BcII from Bacillus cereus,5, 12 and
it would seem that all structural, mechanistic, and computational studies already reported for
BcII would be applicable to Bla2. However, steady-state kinetic constants reported for Bla2
and BcII are different,5, 13 and several active site amino acids (IIe39, Thr182 and Gly151)
in BcII are not conserved in Bla2. In addition, there are conflicting data on BcII regarding
the nature of metal binding and its relation to enzyme mechanism,14-17 and it is not clear
which set of data, if either, applies to Bla2.
We report here detailed characterization of recombinant Bla2 from B. anthracis. The metal
content of the enzyme was ascertained with ICP-AES, and the steady-state kinetic constants
of various analogs of the enzyme, with a range of substrates, were determined. To probe the
structure of Bla2, the mono- and di-Co(II) analogs were prepared and characterized by UV-
Vis, 1H NMR, EPR, and EXAFS spectroscopies. These studies are complemented by
EXAFS of the mono- and di-Zn(II) enzymes. Together the data demonstrate that metal
binding by Bla2 follows distinct pathways, dependent on the identity of the metal ion (Co(II)
or Zn(II)), and that the reactivity of the Zn- and Co-containing enzymes are quite different.
Experimental Procedures
Steady-State Kinetics
A detailed description of materials, over-expression, protein purification and metal analysis
methodology is included in the Supporting Information. Steady-state kinetic studies were
conducted at 25° C in 50 mM Hepes, pH 6.5, on a Hewlett-Packard model 5480A diode
array spectrophotometer. The molar absorptivities of the substrates used (structures shown
in Supporting Information, Figure S2) were Δε485nm = 17,400 M-1cm-1 for nitrocefin,
Δε280nm = -6,410 M-1cm-1 for cefaclor, Δε305nm = 7,600 M-1cm-1 for meropenem, and
Hawk et al.Page 2
J Am Chem Soc. Author manuscript; available in PMC 2012 March 6.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
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Page 3
Δε300nm = -9,000 M-1cm-1 for imipenem.18 Substrate concentrations ranged from 10 to 200
μM, and the enzyme concentration was held fixed at ca. 10 nM. Steady-state kinetic
constants were determined as described previously.18
UV-Vis Spectrophotometry
Apo-Bla2 (ca. 1 mM) in Chelex-treated 15 mM Hepes, pH 6.5, containing 100 mM NaCl
was titrated with CoCl2 (Strem Chemicals, 99.999 %). Samples of Bla2 with 1 or 2 eq of
added Co(II) were incubated on ice for 5 min. before being centrifuged (10 minutes at
14,500 × g) to remove precipitated protein. Difference spectra were obtained by subtracting
the spectrum of apo-Bla2 from those of the Co(II)-added samples.
Stopped-Flow UV-Vis Studies
Diode array UV-Vis spectra were collected (200 – 700 nm) at 4° C on an Applied
Photophysics SX18-MVR stopped-flow spectrophotometer. The buffer used in these
experiments was 50 mM Hepes, pH 6.5, and the substrates were nitrocefin, cefaclor, and
imipenem. Substrate concentrations ranged from 5 to 100 μM (5, 15, 25, 50, and 100 μM,
nitrocefin was also examined at 35 μM), and the concentration of 1Zn-Bla2 was held
constant at 20 μM. All experiments were conducted in triplicate, and the data presented here
represent the average of these multiple data sets. Stopped-flow absorbance data were
converted to concentrations as previously described,19 and the data were corrected for the
instrument dead time of 2 ms. The resulting progress curves were fitted to single
exponentials, and the resultant values of kobs were plotted against substrate concentration.
When using nitrocefin as substrate, these plots were hyperbolic and fitted to kobs = K1[S]k2 /
(K1[S] + k-2), as previously described.20 When using cefaclor and imipenem as substrates,
these plots were linear and fitted to kobs = k1[S] + k-1. The progress curves were simulated
using KINSIM, as previously described. 21, 22 Dynafit was used to verify the simulations,
23 and ProK was used to obtain errors for the rate constants, as previously described.21
EPR spectroscopy
EPR spectra were recorded using a Bruker EleXsys E600 EPR spectrometer equipped with
an Oxford Instruments ITC4 temperature controller and an ESR-900 helium flow cryostat.
Samples were 1 mM protein in 15 mM Hepes, pH 6.5, containing 100 mM NaCl. A Bruker
ER-4116DM cavity was used, with a resonant frequency of 9.63 GHz (in perpendicular
mode) and 10 G (1 mT) field modulation at 100 kHz was employed. Other recording
conditions are given in the legend to Figure 4. Computer simulations of EPR spectra were
carried out using the matrix diagonalization program XSophe24 (Bruker Biospin GmbH)
assuming a spin Hamiltonian H = βg.B.S + S.D.S, where S = 3/2 and D > 0 corresponds to
an MS = |±½〉 ground state Kramers' doublet, as previously described.21, 25, 26
NMR Spectroscopy
1H NMR spectra were collected at 298 K on a Bruker Avance 500 spectrometer operating at
a proton frequency of 500.13 MHz. Samples of Bla2 in either 10 % or 90 % H2O were 1
mM in Chelex-treated, 15 mM Hepes, pH. 6.5, containing 100 mM NaCl. All samples were
incubated for 30 min on ice after D2O addition and centrifuged, before being placed in the
NMR tube. Spectra were collected using a presaturation pulse sequence (zgpr) for water
suppression, a recycle time of 41 ms, and a sweep width of 400 or 800 ppm. Prior to Fourier
transformation, the FID was apodized with an exponential function that resulted in an
additional line broadening of 80 Hz.
Hawk et al.Page 3
J Am Chem Soc. Author manuscript; available in PMC 2012 March 6.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
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Page 4
X-Ray Absorption Spectroscopy
Samples of Bla2 (∼ 1 mM, including 20% (v/v) glycerol added as a glassing agent) were
loaded in Lucite cuvettes with 6 μm polypropylene windows and frozen rapidly in liquid
nitrogen. X-ray absorption spectra were measured at the National Synchrotron Light Source
(NSLS), beamline X3B, with a Si(111) double crystal monochromator; harmonic rejection
was accomplished using a Ni focusing mirror. Fluorescence excitation spectra for all
samples were measured with a 13-element solid-state Ge detector array. Samples were held
at ∼ 15 K in a Displex cryostat during XAS measurements. X-ray energies were calibrated
by reference to the absorption spectrum of the appropriate metal foil, measured concurrently
with the protein spectra. All of the data shown represent the average of ∼ 12 total scans,
from two independently prepared samples each. Data collection and reduction were
performed according to published procedures27 with E0 set to 9680 eV for Zn and 7735 eV
for Co. The Fourier-filtered EXAFS were fit to Equation 1 using the nonlinear least-squares
engine of IFEFFIT that is distributed with SixPack 28, 29
In Eq. 1, Nas is the number of scatterers within a given radius (Ras, ± σas), As(k) is the
backscattering amplitude of the absorber-scatterer (as) pair, Sc is a scale factor, φas (k) is the
phase shift experienced by the photoelectron, λ is the photoelectron mean free-path, and the
sum is taken over all shells of scattering atoms included in the fit. Theoretical amplitude and
phase functions, As(k)exp(-2Ras/λ) and φas (k), were calculated using FEFF v. 8.00.30 The
scale factor (Sc) and ΔE0 for Zn-N (Sc = 0.78, ΔE0 = -21 eV), Zn-S (0.85, -21 eV), Co-N
(0.74, -26 eV) and Co-S (0.85, -26 eV) scattering were determined previously and held fixed
throughout this analysis.27, 31 Fits to the current data were obtained for all reasonable
integer or half-integer coordination numbers, refining only Ras and σas2 for a given shell.
Multiple scattering contributions from histidine ligands were approximated according to
published procedures, fixing the number of imidazole ligands per metal ion at half-integral
values while varying Ras and σas2 for each of the four combined ms pathways (see Table 4).
27, 31 Metal-metal (Co-Co and Zn-Zn) scattering was modeled by fitting calculated
amplitude and phase functions to the experimental EXAFS of Co2(salpn)2 and Zn2(sapln)2.
Biochemical Characterization
Steady-State Kinetics of Bla2—Previous kinetic studies by Palzkill and co-workers
were conducted in 50 mM Hepes, pH 7.5, containing 50 μM Zn(II) and 20 μg bovine serum
albumin (BSA).5 In initial attempts to measure steady-state kinetics under these conditions,
we found the enzyme to be unstable. Inclusion of BSA did not stabilize the enzyme in our
hands. However, Bla2's predicted pI of 7.6 led us to speculate that lowering the pH from 7.5
would stabilize the protein. Reproducible steady-state kinetic data, summarized in Table 1,
were obtained at pH 6.5 in 50 mM Hepes. It is important to bear in mind that the steady-
state kinetic buffers contain ∼ 100 nM Zn(II),32 even if the buffers are Chelex-treated, while
the assayed enzyme concentration is 1-10 nM. Given this, it is difficult to state with
certainty the exact metal content of the Zn-Bla2 analogs in Tables 1 and 2. Within these
limits, as-isolated Bla2, which most closely resembles the mono-zinc enzyme, hydrolyzed
nitrocefin, imipenem, cefaclor, and meropenem with kcat values ranging from 24 to 92 s-1
and Km values ranging from 25 to 110 μM (Table 1).
Hawk et al.Page 4
J Am Chem Soc. Author manuscript; available in PMC 2012 March 6.
NIH-PA Author Manuscript
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Page 5
Apo-Bla2 was prepared by dialysis against EDTA, followed by exhaustive dialysis to
remove residual EDTA (see Materials and Methods). Apo-Bla2 prepared in this way
contained no detectable Zn, Co, Mn, or Fe, as indicated by ICP-AES. The apoenzyme
showed ca. 10 – 20 % of the activity of the most active enzyme under steady state
conditions, but no detectable activity in stopped-flow. Direct addition of 1 or 2 eq of Zn(II)
to the apoenzyme gave the 1Zn- and ZnZn-analogs of Bla2 that were used for metal-
dependent steady state kinetic studies (Table 2). 1Zn-Bla2 exhibited a kcat of 32 s-1 and a Km
of 28 μM at pH 6.5 using nitrocefin as the substrate. The kcat of this enzyme was ca. 30 %
less than that of as-isolated Bla2 (Table 1). Addition of a second eq of Zn(II) (ZnZn-Bla2)
resulted in an increase in both kcat and Km, while further additions of Zn(II) led to a steady
drop in kcat, consistent with the instability of the dilute enzyme in the presence of excess
Zn(II) noted above. The analogous mono- and di-Co(II) enzymes (1Co-Bla2 and CoCo-
Bla2, respectively) gave steady-state values for both kcat and Km that were similar to the
corresponding Zn(II) enzymes (Table 2).
Stopped-Flow UV-Vis Kinetic Studies—Stopped-flow kinetic studies were conducted
on 1Zn-Bla2 with three substrates (nitrocefin, cefaclor, and imipenem). Similar studies were
not performed on ZnZn-Bla2, due to its instability in dilute solution (see Supporting
Information). In the reaction of 1Zn-Bla2 (20 μM) with nitrocefin (5-100 μM), only two
distinct features are observed: one at 390 nm that decreases with time, corresponding to loss
of substrate, and one at 485 nm that increases with time, corresponding to product formation
(Figure 1). No transitory absorbance was observed at 665 nm, corresponding to a stable
intermediate previously observed in reactions of nitrocefin with CcrA33 and L1,19 but not
with BcII.34 Progress curves at 390 and 485 nm using 35 and 50 μM nitrocefin were fitted
to single exponentials to obtain kobs (Figure 2A). A plot of kobs vs. substrate concentration
was hyperbolic, suggesting a rapid-equilibrium, two-step binding mechanism (Scheme 1).20
The progress curves were simulated (solid lines in Figure 1) using KINSIM, the mechanism
in Scheme 1, and the rate constants in Table 3. The King-Altman method35 was used to
determine theoretical expressions for kcat and Km, based on the mechanism in Scheme 1, and
the resulting steady-state kinetic constants (kcat = 7.4 s-1 and Km = 62 μM) were similar to
those determined in steady-state kinetic studies (Table 1).
Stopped-flow kinetic studies were also performed on 1Zn-Bla2 with imipenem (Figure 1B)
and cefaclor (Figure 1C). In these reactions, only the substrates could be observed. Unlike
for nitrocefin, plots of kobs vs. imipenem or cefaclor concentration is linear, suggesting one-
step binding mechanisms for both substrates (Scheme 2).35 The resulting progress curves
were simulated using KINSIM, the mechanism in Scheme 2, and the rate constants in Table
3. The King-Altman-determined theoretical expressions for kcat and Km give theoretical
values for imipenem (kcat = 35 s-1 and Km = 194 μM) and cefaclor (kcat = 15 s-1 and Km =
150 μM) that are similar to those determined by steady-state kinetic studies (Table 1).
Metal Binding
Optical Spectroscopy—We first examined Co(II) binding by optical titration of apo-
Bla2. The UV-visible difference spectra (Co-bound – apo-Bla2, Figure 2) show the Cys-S to
Co(II) charge transfer transition at 340 nm (ε340nm = 608 M-1 cm-1) that is indicative of
metal binding at the Zn2 site, even at 0.5 eq of Co(II). Also apparent at 0.5 eq of Co(II) are
the ligand field transitions (500 – 650 nm, ε550nm = 218 M-1 cm-1) normally associated with
metal binding at the Zn1 site (Figure 2).14, 36 Each of these features increases linearly in
absorbance as a function of added Co(II) (Figure 2, inset), suggesting that Co(II) distributes
between the Zn1 and Zn2 sites with minimal difference in affinity. We were unable to obtain
the optical spectrum of Bla2 containing 3 eq of Co(II) because the protein quickly
precipitated, similar to prior observations from Co(II) titrations of CcrA.37
Hawk et al.Page 5
J Am Chem Soc. Author manuscript; available in PMC 2012 March 6.
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Page 6
EPR Spectroscopy—EPR spectra from 1Co-Bla2 and CoCo-Bla2 were recorded under
non-saturating, saturating, and rapid-passage conditions and are shown in Figure 3. The
spectra from 1Co-Bla2 (Figure 3A) and CoCo-Bla2 (Figure 3B) recorded under non-
saturating conditions exhibited no resolvable rhombicity or 59Co hyperfine structure, and
were characteristic of 5- or 6-coordinate Co(II) with at least one water ligand. Spectra
recorded at high power and low temperature (Figure 3D) did not reveal any MS = |± 3/2〉
signals that would indicate tetrahedral Co(II). Slight differences were observed between the
non-saturated spectra of 1Co-Bla2 and CoCo-Bla2. The difference spectrum (Figure 3C)
revealed the presence of a second Co(II) species in CoCo-Bla2. This component,
deconvoluted by direct subtraction, may itself be due to more than one species but its
isolation does at least indicate some degree of preference of Co(II) for one binding site over
the other. The signal from 1Co-Bla2 could not be satisfactorily simulated as a single species,
suggesting that both the spectra of 1Co-Bla2 and of CoCo-Bla2 may be mixtures that differ
only in the relative proportions of the species. The best estimate that can be obtained from
EPR for the degree of discrimination between sites for Co(II) binding comes from the
difference spectrum of Figure 3E, which was generated by subtraction of 1.7 × Trace 3A
from Trace 3B. This resulted in a residual that contained no features of either positive or
negative amplitude that corresponded to either of the experimental signals, and indicates that
85 % of the Co(II) in CoCo-Bla2 is likely indistinguishable from the Co(II) complement of
1Co-Bla2. The nature of the other 15 % in CoCo-Bla2 is consistent with only a small
difference in the affinities of the binding sites.
The rapid passage spectra of 1Co-Bla2 and CoCo-Bla2 (Figure 3F) were indistinguishable in
form and differed only in intensity, by a factor of 2. This indicates that the species that is
responsible for this spectrum is the same in both 1Co-Bla2 and CoCo-Bla2, and that twice as
much of it is present in CoCo-Bla2 than in 1Co-Bla2. Comparison of the normal, non-
saturated spectrum of 1Co-Bla2 and the (CoCo-Bla2 - 1Co-Bla2) difference spectrum
(Figure 3C) shows that the spectrum of 1Co-Bla2 is sharper in the g⊥ region, around 2000 G
(200 mT). The derivative of the rapid passage spectrum (of either 1Co-Bla2 or CoCo-Bla2)
is, in turn, sharper still than that of 1Co-Bla2; the former more likely represents a single
chemical species and provides some direct evidence that the signal from 1Co-Bla2 contains
more than one species.
Taken overall, then, the EPR data indicate that the signal from 1Co-Bla2 is likely due to two
components, one of which can be isolated by recording the spectrum under rapid passage
conditions for that signal, and another that is similar in line shape, but could not be
deconvoluted. Some 85 % of the signal from CoCo-Bla2 consists of Co(II) in the same
environments, with the same distribution among those environments, as in 1Co-Bla2. These
data indicate a binding mechanism that is largely either random or cooperative. A minor
fraction of the Co(II) spin density in CoCo-Bla2, 15 %, appears either to (i) indicate Co(II)
that resides in a third, distinct environment, or (ii) represent a slight difference in the
distribution of Co(II) between two binding sites, or else (iii) is indicative of a broadening
mechanism due to weak spin-coupling between Co(II) ions that is more pronounced in
CoCo-Bla2 than in 1Co-Bla2. While the latter possibility would suggest random binding to
sites with similar affinities, rather than cooperative binding, there was no additional
evidence from parallel mode EPR (B0‖B1, not shown) for weak magnetic coupling between
the two Co(II) ions in CoCo-Bla2.
NMR Spectroscopy—The 1H NMR spectrum of CoCo-Bla2 in 10 % D2O shows five
downfield shifted resonances between 35 and 85 ppm (Figure 4). The spectrum is nearly
identical in appearance to that previously reported for di-Co BcII,14 where each peak was
assigned to a single proton, despite significant differences in linewidth and intensity. The 1H
NMR spectrum of Bla2 containing one eq of Co(II) is identical to that of CoCo-Bla2,
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although of lower signal-to-noise (not shown). In 90 % D2O, four of the resonances of
CoCo-Bla2 (78, 65, 49, and 44 ppm) disappear. With analogy to the crystal structure of
BcII, we assign these four resonances to the NH protons of His116, His118 and His196 from
the Zn1 site and His263 of the Zn2 site. The resonance at 42 ppm is not solvent-
exchangeable and can be assigned to the meta proton of His118, which binds metal through
the δN in BcII. This is consistent with the multiple Co(II) binding sites identified by EPR,
and the observation of both the S6Co(II) CT band and the d-d bands, both with one eq of
Co(II), indicating that the first eq of Co(II) distributes between the two metal binding sites.
The NMR spectra of Bla2 are very similar to those previously reported for BcII,14 with the
notable exception of the absence of a resonance shifted beyond 100 ppm assignable to the β-
CH2 protons of a Co(II)-coordinated cysteinate (spectra were recorded to 400 ppm). One
possibility is that our procedure to prepare the 1H NMR samples resulted in oxidation of
Cys221 to the sulfoxide. Similar oxidation has been suggested for CcrA,31 and an oxidized
Cys221 has been observed in crystal structures of BcII38 and VIM-2.39
Co K-Edge XAS—Comparing the EXAFS of 1Co-Bla2 and CoCo-Bla2 (Figure 5) shows
little quantifiable difference, consistent with the optical, NMR and EPR studies described
above. That the data are indeed different favors independent, distributed binding to two sites
of comparable affinity, rather than cooperative binding. The major difference is in the height
of the main peak, reflective of either higher coordination number or lower overall disorder in
the first shell on addition of the second eq of Co(II). The shape, particularly the width, of the
main feature is largely unchanged, and curve-fitting results suggest the latter interpretation
(lower disorder) better reflects the apparent difference. Best fits for 1Co- and CoCo-Bla2 are
shown in Figure 5, and summarized in Table 4. Detailed fitting results, including fits to
unfiltered EXAFS, are presented in Supporting Information, Figure S2 and Table S1 for
1Co-Bla2, and Figure S3 and Table S2 for CoCo-Bla2.
The distributed binding indicated above leads to the expectation that both 1Co-and CoCo-
Bla2 would reflect an average of the Zn1 and Zn2 coordination spheres, with 4 N/O and 0.5
S comprising the first shell. In both cases, inclusion of a partial sulfur scatterer dramatically
improves the quality of the first shell fits. For 1Co-Bla2, the average first shell bond length
is reflective of 4- or 5-coordination (Fit S1-1). While the fit residual decreases nearly 3-fold
(63 %) when the fit includes a mixed first shell of nitrogen and oxygen (compare Fits S1-1
and S1-2), the difference in refined distances (0.15 Å) is at the resolution of the data (0.14
Å). An even greater improvement is seen on addition of a sulfur scatterer (70 %, compare
Fits S1-1 and S1-3). The three shell fit, including resolved N, O and S shells, results in an
overall 83 % improvement from the single scattering fit (Fit S1-1 vs. S1-4), and 43 % over
the N/O + S fit (Fit S1-1 vs. S1-4), although the refined N and O distances are outside the
resolution of the data. For this reason, multiple scattering fits were conducted using a first
shell of 4 N/O + 0.5 S and indicate an average of 2 His ligands per Co ion (Fit S1-5).
Inclusion of a Co-Co vector in this fit leads to only 8 % improvement in the fit residual,
suggesting a bridged bimetallic cluster is not present (Fit S1-6).
Similar results are obtained for CoCo-Bla2 (Fig. 5, Bottom, and Figure S3 and Table S2).
The average first shell bond length is unchanged from 1Co-Bla2 (compare Fits S2-1 and
S1-1). A mixed first shell of N and O leads to a 49 % improvement in fit residual with N and
O distances at the resolution of the data (Fits S2-1 and S2-2) and inclusion of a partial sulfur
scatterer leads to a 70 % improvement in the fit residual. Multiple scattering fits, using 4 N/
O + 0.5 S to model the first shell, again indicate an average of 2 His ligands per Co ion. The
appearance of the outer shell scattering would suggest some rearrangement on addition of
the second Co(II) ion, but this information is outside the scope of the current analysis.
Inclusion of a Co-Co vector in this fit leads to a 20 % improvement in fit residual, while
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increasing the number of variable parameters to 14 from 12 (a 17 % increase). The refined
Co-Co distance is shorter than the Zn-Zn separation in similar systems, including this one
(below), while it is expected, if present to be slightly longer due to the modestly larger size
of the Co(II) ion. We take this as evidence that the aquo bridge is replaced by one solvent on
each metal ion in fully loaded CoCo-Bla2, in accord with our previous studies of CcrA,31
and consistent with the lack of both resolved 59Co hyperfine structure and a parallel mode
signal in the EPR.
Zn K-Edge XAS—In contrast to the distributed binding observed with Co, the binding of
Zn by Bla2 appears to be sequential. The Fourier-transformed EXAFS for 1Zn-Bla2 shows
much higher outer shell intensity than its ZnZn counterpart, which shows much better
resolution in its outer shell scattering (Figure 6, Top). Qualitatively, this is consistent with
binding of the first equivalent of Zn(II) in the Zn1 site, bearing three His ligands. Population
of the Zn2 site would then be expected to (i) broaden the first shell with addition of the
sulfur ligand, (ii) lower the overall outer shell scattering, as the average number of His
ligands is reduced from 3 to 2 and (iii) sharpen the outer shell features as the dinuclear site
becomes better organized. This description is fully consistent with the Fourier transforms
presented in Figure 6.
The qualitative description presented above is also supported by the curve fitting results
(best fits are shown in Figure 6 and Table 4, detailed results are shown Figure S4 and Table
S3 for 1Zn-Bla2, and Figure S5 and Table S4 for ZnZn-Bla2), which show the Zn in 1Zn-
Bla2 is coordinated by 4 N/O donors, including 3 His ligands. Inclusion of a partial sulfur
scatterer does not significantly improve the fit (< 30 %), while resulting in an unreasonably
short Zn-S distance (2.23 Å, compare Fits S3-1 and S3-2). Similarly, a mixed first shell of N
and O donors leads to a minimal improvement in the fit residual, only slightly larger than
simply increasing the number of variable parameters (compare Fits S3-3 and S3-4 to Fit
S3-1), with a separation in Zn-N and Zn-O distances that is less than the resolution of the
data. Multiple scattering fits indicate an average of 3 His ligands per Zn, and inclusion of a
Zn-Zn vector in this fit results in only 18 % improvement in fit residual with a relatively
short refined Zn-Zn distance of 3.36 Å. Together, we take this as evidence that there is no
population of the Zn2 site in 1Zn-Bla2, and that there are not sub-populations of di-Zn and
In contrast, the coordination sphere of the average Zn(II) in ZnZn-Bla2 clearly includes 0.5
S donors, as well as 4 N/O. The average first shell bond length (in N/O only fits) increases
by 0.05 Å over 1Zn-Bla2 (compare Fits S4-1 and S3-1), suggesting an increase in average
coordination number and/or the presence of a larger scatterer. A mixed first shell of resolved
Zn-N and Zn-O leads to a 70 % decrease in the fit residual, though the distances are right at
the resolution of the data (Fits S4-1 and S4-2). Inclusion of 0.5 S with 4 unresolved N/O
donors leads to an 88 % improvement in the fit residual (compare Fits S4-3 and S4-1), and a
three-component N + O + S fit results in an overall 93 % reduction in residual. For
simplicity, multiple-scattering fits were restricted to a first shell of 4 N/O + 0.5 S donors.
The fits are consistent with the average number of imidazoles being reduced from 3 to 2 on
addition of the second eq of Zn(II). Inclusion of a Zn-Zn interaction leads to a 43 %
improvement in the fit residual, while increasing the number of variable parameters from 12
to 14. The refined Zn-Zn distance of 3.44 Å is the same as that seen in other resting state
dinuclear MβLs by both XAS40 and diffraction,41-43 but very different from the 3.6-3.7 Å
EXAFS-derived separation reported earlier for BcII.44
Stopped-Flow of 1Zn- and 1Co-Bla2—The distributed binding of Co(II) by Bla2, as
demonstrated above, cannot distinguish between potential populations of mono-Co(II) Bla2
(loaded randomly in either the Zn1 or the Zn2 site) and a mixture of apo- and fully-loaded
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di-Co(II) Bla2, and indeed all variants may coexist in the absence of substrate. As can be
seen in Figure 7, while 1Zn-Bla2 does not stabilize an intermediate with nitrocefin, 1Co-
Bla2 clearly does. The lack of accurate values of Km and kcat for 1Co-Bla2 preclude the
simulation of these data. However, in recent work with the dinuclear B3 MβL L1, we
showed that occupation of the Zn1 site was sufficient for catalysis, but both metal ions were
needed to stabilize the nitrocefin-derived intermediate.45 Thus, the data in Fig. 7 indicate
that there is a significant population of CoCo-Bla2 present at 1 eq Co(II) per protein, at least
under turnover conditions. The apparent concentration of the intermediate suggests that
substrate may shift the metal binding equilibrium in favor of the dinuclear enzyme. This is
consistent with previous work by Page and co-workers on BcII.15, 46
Biochemical Characterization
Steady-state kinetic studies on as-isolated Bla2 containing 1 equivalent of Zn(II), using
several different substrates, show that the as-isolated enzyme is catalytically-active (Table
1). Previously, Palzkill and co-workers reported that recombinant Bla2 (Zn(II) content
unknown) exhibited a Km of 75 ± 5 μM and a kcat 313 ± 21 s-1, when using nitrocefin as
substrate.5 In our hands, recombinant Bla2 containing 1 eq of Zn(II) exhibited a Km of 25 ±
4 μM and a kcat of 41 ± 3 s-1 with nitrocefin as substrate. These values compare favorably
with previous steady-state kinetic data on BcII, which exhibits a Km of 9 μM and a kcat of 43
s-1 with nitrocefin.47 The stark difference in steady-state kinetic behavior of the two
recombinant Bla2 preparations may be due to differing metal content and/or different assay
conditions, such as pH. Our assays were conducted at 25° C, while the preceding studies
were conducted at 30° C,5 although this should result in only a 1.25-fold difference in kcat.
Metal Binding
Steady-state kinetic studies revealed that Bla2 containing 1 equivalent of Co(II) is less than
½ as catalytically active as CoCo-Bla2 (Table 2), suggesting that (i) Co(II) binding to Bla2
is random and that both metal ions bind with similar KD values or (ii) that Bla2 exhibits
positive cooperative binding of Co(II). A distributed binding model is supported by optical
studies, which show both the d-d bands (Zn1) and the Scys6Co(II) LMCT (Zn2), even at a
loading of only 0.5 eq of Co(II), whose intensity maximizes above 2 eq of Co(II) per protein
(Figure 2). The EPR spectra of 1Co-and CoCo-Bla2 are nearly superimposable, and suggest
5- or 6-coordination for both, with at least one solvent ligand (Figure 3). The 1H NMR
spectra of 1Co- and CoCo-Bla2 are nearly identical, showing the same five
paramagnetically-shifted resonances. Four of these resonances are solvent exchangeable
(Fig. 4), indicating a total of four coordinated histidines with only 1 eq of Co(II) added. The
Co K-edge EXAFS are also consistent with a distributed binding model, with only an
apparent decrease in disorder accompanying addition of the second eq of Co(II).
In sum, the spectroscopic studies of Co(II)-binding by Bla2 overwhelmingly indicate
distributed binding of Co(II) by Bla2 in the absence of substrate. In addition, while the
electronic absorption, EPR and EXAFS data all indicate that the average Co(II) environment
in 1Co-Bla2 and CoCo-Bla2 bulk samples is substantially similar, the EPR and EXAFS
nevertheless detected differences between them. The appearance of an ill-defined
broadening in the EPR and of lower overall disorder in the EXAFS, upon binding a second
Co(II), argue strongly against a strictly cooperative binding mechanism and, therefore, favor
independent, distributed binding of Co(II) to sites with comparable affinities. Samples of
1Co-Bla2 would then be expected to contain a statistical distribution of [Co_(Bla2)],
[_Co(Bla2)], [CoCo(Bla2)] and apo-Bla2, and steady state kinetics are consistent with this.
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In contrast to Co(II), the binding of Zn(II) by Bla2 appears to be sequential. EXAFS of 1Zn-
Bla2 show the first eq of Zn(II) clearly binds to the Zn1 site. Addition of a 2nd eq of Zn(II)
to form ZnZn-Bla2 leads to the observation of a Zn-S interaction, consistent with Cys221
coordination (and metal bound at the Zn2 site), and a clear Zn-Zn interaction at ∼ 3.4 Å. The
EXAFS are consistent with the steady-state kinetics, which showed that ZnZn-Bla2 is not
twice as active as 1Zn-Bla2, suggesting that the second Zn(II) ion does not greatly affect
catalysis and that the first site of Zn(II) binding, Zn1 in this case, is more important
kinetically.14, 44, 48 To our knowledge this is the first documented example of a naturally-
occurring Zn(II) enzyme that follows distinct metal binding pathways, dependent on
whether the metal is Zn(II) or Co(II), and strongly underscores the need to demonstrate
transferability when studying Co(II)-substituted Zn(II) enzymes.
The stopped-flow kinetics studies are consistent with the difference in metal binding
mechanisms, and they suggest that substrate binding may alter the metal-binding
equilibrium for Co(II), but not for Zn(II). 1Zn-Bla2 does not stabilize the nitrocefin-derived
reaction intermediate. The lack of an intermediate suggests that the situation described for
resting 1Zn-Bla2 by EXAFS, a homogeneous population of mono-Zn enzyme, with Zn(II)
bound in the canonical Zn1 site, is preserved during turnover. In contrast, the appearance of
an intermediate is clear in 1Co-Bla2-catalyzed nitrocefin hydrolysis. Within the accuracy of
ε665nm for the intermediate, the limiting concentration of the Co-bound intermediate (ca. 12
μM) suggests that, under turnover conditions, 1Co-Bla2 may, in fact, contain nearly equal
populations of apo- and CoCo-Bla2, while all of the resting state studies presented clearly
indicate a statistical distribution. At the current level of the data we can only speculate on
this point.
Comparison with Other B1 MβLs
The metal-binding properties of two other B1 MβLs have been studied in detail. A set of
enzymological, optical, X-ray absorption and magnetic resonance studies of Co(II) binding,
similar to those presented here, by the B1 MβL CcrA indicated sequential binding, with the
first equivalent of Co(II) bound exclusively at the Zn1 site.31 Both EPR and XAS of 1Co-
CcrA and CoCo-CcrA were suggestive of 5/6-coordination, rather than the expected 4/5.
Neither XAS nor EPR showed evidence of a covalent bridge between the Co ions.
The EPR studies presented here also suggest 5/6-coordination for both Co(II) ions in Bla2,
with little or no evidence of a bridging interaction. However, the XAS-derived average first
shell bond lengths for 1Co- and 1Zn-Bla2 differ by only 0.02 Å, while those of 1Co-31 and
1Zn-CcrA (see Supporting Information) differ by 0.12 Å. A similar trend is apparent
comparing the di-Co and di-Zn enzymes. The average first shell bond lengths for CoCo- and
ZnZn-Bla2 differ by only 0.01 Å, while those of CoCo-31 and ZnZn-CcrA differ by 0.12 Å.
These data clearly indicate that Co(II)-substitution better preserves the coordination number
of the native Zn(II) in Bla2 than it does in CcrA, which clearly indicates an increase in
coordination number for both metal ions.
Recent studies have shown that BcII shows a very modest preference for binding Co(II) at
the Zn1 site, but evidence of a di-Co(II) population is apparent at very low (∼0.6 eq) Co(II)
stoichiometries.14 The present studies on Bla2 are not inconsistent with this model, though
they offer no direct evidence as to the speciation of resting 1Co-Bla2. Freeze quench
spectroscopic studies of di-Co(II) BcII indicate the presence of a covalent bridge in the
resting enzyme, based on the observation of a parallel mode EPR signal.49 The presence of
the bridge appears, at present, unique to di-Co BcII, as in both CcrA and Bla2, we see no
concrete evidence for Co-Co interactions in the Co K-edge XAS or significant magnetic
coupling in their EPR.31
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The distributed binding of Co(II) by BcII14 is consistent with an earlier EXAFS study of
Zn(II)-binding by the same enzyme.44 Using optical competition and fluorescence
spectroscopy, de Seny, et al suggested that the first equivalent of Zn(II) distributes between
the two sites in BcII. In contrast, both CcrA and Bla2 have been shown here to bind Zn(II)
sequentially. To the greater issue of Co(II)-substitution, it appears at present that the
substitution is completely faithful in BcII; it reproduces the order of addition in CcrA, but
not coordination number, without the formation of a bridged cluster; and it fails for Bla2
where distinct metal-dependent mechanisms are observed.
Co(II) substitution has long been used to study zinc enzymes and often furnishes
catalytically active forms of zinc enzymes.50 Tetrahedral Zn(II) active centers can be
substituted with Co(II) and, in some cases, Co(II) directly adopts the original Zn(II)
coordination.51-53 In other zinc-dependent metalloenzymes, four-coordinate Zn(II) is
replaced by five-coordinate,52, 54, 55 or even six-coordinate56 Co(II). The general
propensity of Co(II) to adopt higher coordination in a hitherto four-coordinate Zn(II) site has
perhaps been underappreciated,57 but the affinity of Co(II) for an extra solvent ligand
appears to have little effect on catalytic activity, metal ion affinity, the mechanism of
binding, or gross structural features.58 Indeed, the present case of Bla2 is the first that we
know of where Co(II) binding and Zn(II) binding clearly differ, and renewed vigilance
should be exercised in interpreting studies on Co(II)-substituted zinc enzymes.
Taken together, the present data demonstrate that 1Zn-Bla2 is catalytically-active and
EXAFS data on this analog demonstrate that Zn(II) binds in the consensus Zn1 site. The
EXAFS data also show that Zn(II) binding is sequential, as in CcrA31 and L1.40 In contrast,
all of the spectroscopic and steady-state kinetic data on Co(II)-containing Bla2 indicate that
Co(II)-binding is distributed in the absence of substrate. The stopped-flow data suggest that
substrate binding may affect the Co(II)-binding equilibrium. A summary scheme showing
how Zn(II) and Co(II) bind to Bla2 is shown in Figure 8. Further studies are required to
address why Zn(II) and Co(II) behave differently in Bla2, and X-ray crystal structures of
both analogs may yield information about the different metal binding properties. We believe
this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct
mechanisms, underscoring the importance of validating transferability when extrapolating
results on Co(II)-substituted proteins to the native Zn(II)-containing forms.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
This work was supported by the National Institutes of Health (AI056231 to BB; EB001980 to the Medical College
of Wisconsin; RR16480 from the BRIN/INBRE Program of the National Center for Research Resources to DLT),
the Volwiler Professorship at Miami University (to MWC) and the National Science Foundation (CHE0809985 to
DLT). Funds to purchase the 500 MHz NMR were provided by the Hayes Investment Fund (Ohio Board of
Regents). The National Synchrotron Light Source is supported by the US Department of Energy.
extinction coefficient
ethylenediaminetetraacetic acid
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electron paramagnetic resonance
extended X-ray absorption fine structure
Fast Protein Liquid Chromatography
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Inductively Coupled Plasma spectrometer with atomic emission spectroscopy
nuclear magnetic resonance
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Figure 1.
Progress curves for the reaction of 1Zn-Bla2 with nitrocefin (A), imipenem (B) and cefaclor
(C). (A) The concentration of product increased over time using 35 (□) and 50 (○) μM
nitrocefin, while substrate concentrations decreased over time using 35 (◊) and 50 (△) μM
nitrocefin. The solid progress curves were generated by KINSIM, using the mechanism in
Scheme 1 and the kinetic constants in Table 3. (B) Progress curves of the reaction of
imipenem and Bla2 containing 1 eq. Zn(II) at 4 °C. The concentrations of substrate were
(△) 50 μM, (□) 25 μM, and (○) 10 μM, and the concentration of Bla2 was 20 μM. The solid
lines were generated by using KINSIM, the mechanism in Scheme 2, and the kinetic
constants in Table 3. (C) Progress curves of the reaction of cefaclor and Bla2 containing 1
eq. Zn(II) at 4 °C. The concentrations of substrate were (△) 50 μM, (□) 25 μM, and (○) 10
μM, and the concentration of Bla2 was 20 μM. The solid lines were generated by using
KINSIM, the mechanism in Scheme 2, and the kinetic constants in Table 3.
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text:
Floor Plans and Pricing
1 Bedroom
|One Bedroom One Bath
|$1230 - $1435
|1 Bathroom
|704 Sq. Ft.
Vertical Blinds, Ceiling Fans, Cable Available, Dishwashers (selected units), Energy Saving Appliances; Balcony/Patio; Ample Wall Space; Accent Wall Available; Air Conditioning; Wall to Wall Closets;Bright & Airy.
2 Bedrooms
|Two Bedroom One Bath
|$1465 - $1690
|1 Bathroom
|895 Sq. Ft.
Vertical Blinds, Ceiling Fans, Cable Available, Dishwashers (selected units), Energy Saving Appliances; Balcony/Patio; Ample Wall Space; Accent Wall Available; Air Conditioning; Large Walk In Closets;Bright & Airy.
|Two Bed Two Bath
|$1475 - $1650
|2 Bathrooms
|900 Sq. Ft.
Vertical Blinds, Ceiling Fans, Cable Available, Dishwashers (selected units), Energy Saving Appliances; Balcony/Patio; Ample Wall Space; Accent Wall Available; Air Conditioning; Large Walk In Closets;Bright & Airy;
3 Bedrooms
|Three Bedroom Two Bath
|$1695 - $2250
|2 Bathrooms
|1104 Sq. Ft.
Best 3 bedroom floor plan ever. Huge bedrooms,two full baths, vertical blinds, accent wall available, large patio, entertainers kitchen with granite counter-tops, and walk in closets.
Property Features
- Property Overview
- Special Features
- Community Features
- Apartment Amenities
- Local Schools
- Pet Policy
The Courtyards in Norwalk, CA
When you choose The Courtyards or Tierra Palms Apartments in Norwalk CA as your home, you can enjoy spending afternoons by your swimming pool, barbeque sumptuous meals with friends at the outdoor grills, or let the kids work off some never ending energy at the playground. You'll enjoy a serene retreat that's also close to shopping, restaurants, and all the activities Norwalk has to offer including 14 city parks and walking paths. Both properties offer the conveniences of city life combined with the comforts of a suburban retreat.
Special Features
- Newly Renovated Norwalk Apartments
- Accent Walls
- 2 Sparkling Pools and Spa
- Largest 3 Bedroom in Norwalk!
- Courtesy Patrol
- Central Location: Close to 605 and 5 Fwys
- Reserved Parking
- BBQ Areas
- Two Laundry Facilities
- Gated Community
- Close to Shopping and Dining
- Remodeled Apartments
Community Features
- Accepts Credit Card Payments
- Accepts Electronic Payments
- Apartment
- Controlled Access
- Courtyard
- Disability Access
- Fitness Center
- Freeway Access
- Laundry Facility
- On Street Parking
- Playground
- Pool
- Public Transportation
- Smoke Free Community
- Spa
Additional Information
Apartments in Community: 153
Apartment Amenities
- Air Conditioning
- Cable or Satellite
- Ceiling Fan
- Garbage Disposal
- Hardwood Floor
- Large Bedrooms
- Microwave
- Patio or Balcony
- Private Bathroom
- Walk In Closets
- Window Covering
Local Schools
- Corvallis Middle School
- New River Elementary School
- Norwalk High School
We believe school data to be accurate, but do not guarantee that. Please be sure to verify where your children will attend school before making your housing decision.
Pet Policy
Pets are welcome. Pets accepted under 35 lbs. Please give us a call for more details.
- Cats Allowed
- Pets Allowed
- Small Dogs Allowed
Contact The Courtyards
The Courtyards
Office HoursS 11:00 AM-5:00 PM M 9:00 AM-6:00 PM T 9:00 AM-6:00 PM W 9:00 AM-6:00 PM Th 9:00 AM-6:00 PM F 9:00 AM-6:00 PM Sa 10:00 AM-5:00 PM
The Courtyards offers spacious one, two and three bedroom apartments with A/C, ceiling fans, large walk-in closets, energy-saving kitchen, dishwasher, and private patio/balcony. Our gated, pet friendly community provides controlled access, pool and spa, fitness center, BBQ/picnic areas and playground. Close to schools, hospital, dining and shopping options. Easy access to the 105 and 605 freeways.
Exit the 605 Freeway at Firestone Blvd. Go East on Firestone Blvd. and turn right on Studebaker Rd. We are on the right side.
As you search for your perfect apartment, please keep in mind that rental rates (including specials and discounts) and availability are very fluid in the apartment industry. Based on many factors, the rental rates (including specials and discounts) and availability can actually change not just daily, but hourly, if not even more quickly. While our advertisers do take steps to keep their information current, the rental rates or availability you may find here now at ForRent.com may not be the same as when you contact the communities.
Quick Links For Your Apartment Search
- Los Angeles metro
- Southeast LA area
- Norwalk
- 90650
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