text:
It is upgrade time for me. I currently have iphone 4s, but I am not sure if I want iphone 5s or Samsung s4/note.
What phone do you use, and do you recommend it?
It is upgrade time for me. I currently have iphone 4s, but I am not sure if I want iphone 5s or Samsung s4/note.
What phone do you use, and do you recommend it?
Live your life as if nobody is watching you.
i have an i phone 5. We have always had i phones. If it has an apple logo on it we probably have at least one at home! we made the mistake of buying a samsung tab 2 for our 3 year olds birthday and soon began to regret it as we soon realised why everything we have is apple! it all syncs together nicely and works as it should without being complicated ! its a great brand, why change ?
A HTC One but I'm starting to regret it. I really wish I'd have stuck with iPhone! I had a 4S previously. If I'm honest, I'll probably sell the HTC One and my old 4S and put it towards the iPhone 6 when it comes out (if it looks good that is).
As much as I like the HTC, it just not as refined as the iPhone. There's just little quirks that I miss which I didn't think I would! That'll teach me to jump into a 2 year contract with a phone I'd never even looked at, never mind used before.
Oh, and I REALLY miss the Audi cradle in my arm rest for the iPhone.
iPhone5, does everything I need it to. Wife has a Galaxy S4, does everything she needs it to.
99' Audi S3(APY) - Brilliant Black Click>>>Going Sideways vid!
You've got to have a laugh!
Samsung 4 s mini. Use to love my iPhone but prefer my Samsung now. More practice for what I want it for. Still love my apple products though
Moved on from an iPhone 4 to a Sony Xperia Z1. The phone is absolutely brilliant! 20.7MP camera, waterproof, large clear HD display and plenty other stuff too. I must admit if you don't like big phones then avoid it as it is fairly large and quite difficult to get used to if you're coming from Apple to Android. I hear they have a smaller version coming out soon?
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Im in the same situation with my free upgrade coming up. I have at the mo the HTC Incredible S. Not a bad phone but now lagging and needs replaced. I was thinking of going for the HTC One Mini.......its been getting good reviews. Mrs.B. has the S4 Mini and she loves it so I dunno what to do really. Any one got the HTC One Mini?
HTC One here. Had a Samsung Galaxy S3 a few months ago too. Before that I had iPhones.
First comparison..... Android is much "better" than IOS.
Second comparison..... The S3 is better feauture-wise than the iPhone (even though it is a big buggy etc).
Third comparison..... The HTC out does the Samsung as a package.... usability, stability, build, reliability.
I like the HTC One because it seems to have the best of all worlds........ i.e the quality build of an iPhone (arguably even better as it has a one piece metal body), and its Sense UI is a lot more stable and bug free than Samsung's Touchwiz.
Until Apple makes IOS a lot less restrictive and includes features that Android has I won't be going back to an iPhone. The gap between the two mobile OSs is widening by the update.
iphone 5, just like JoJo said.. does everything I need it to…. and more
The only thing I cannot do / get on my iphone is 4G, because im with O2
Two-year-old iPhone 4 with 16gb on the 3 network. I'm in upgrade territory now, but am not in a rush to commit myself to a £40-odd a month contract for another two years to get an iPhone 5, especially as it would mean shelling out for a new Brodit cradle and Lightning adapter for the car.
I could probably get a 4S for around £30 something a month, but the only ones being supplied now are 8gb.
Happy to hang on to the 4 and pay £18 a month on a one-month rolling basis. Then when I see a deal that suits me, I can take it.
Last edited by jdp1962; 29th November 2013 at 23:55.
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I'm still on my HTC Desire HD at the moment as I've found nothing to replace it with that I really want. Saying that I am very tempted by the Sony Xperia Z1 if I can get a reasonable deal.
Had a Samsung Galaxy S II until recently when I upgraded to the Sony Xperia Z1. Didn't think much of the S II but I am quite happy with the Z1. Camera is junk but the handset in general is quite nice, if a bit big.
The size does put me off a bit.
GSM arena is a good site to compare phones and their spec
I've been on upgrade territory since May, I dropped my tariff down to the minimum once the contract is up, only pay £7 a month on my plan, I never use all my minutes or data, until my iPhone5 starts to slow down like my 4 did, I doubt I'll be in a hurry to upgrade unless it breaks to be honest.
99' Audi S3(APY) - Brilliant Black Click>>>Going Sideways vid!
You've got to have a laugh!
Iphone S5 all the way
samsung S4... i love to carry an Imax cinema screen around in my pocket.....
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Gone - 2005 B7 A4 SE TDI Avant 2.0 140bhp
Last edited by Oranoco; 30th November 2013 at 18:35.
iphone5. Fantastic phone. x
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Iphone 5 for me, third iphone, tried moving to Android but the software is nowhere near the same quality
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Samsung Galaxy s3, I love it had the s1 before.
Never had an Iphone so can't compare, but the s3 does everything I need and can plug into pretty much anything because it uses a micro usb plug.
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Galaxy Note 2. Epic phone, can pretty much do anything. If like me you use your phone a lot for browsing and texting, the screen size is perfect. Couldn't use an iPhone now far too small. Note 2 also has a stylus which can be handy for some.
Im using an iphone 4s at the minute.
I always told myself I wouldn't get an Iphone due to the fact I hated the whole fanboy trend thing, cost and the fact they were fairly restricted.
The past phones I had before the Iphone 4s were; HTC One X, Sony Xperia, Nokia N8.
All of the above I had issues with, the Sony Xperia would pretty much grind to a halt and need a reboot every few days, the same with the others. I put it down to buggy carrier firmware.
This iphone 4s came up cheap from a family relative so I thought I would give it a go.
So far I have had no issues - its taken a little getting used to, but the build quality is great and the phone is responsive, dont have issues with battery life either.
I did try out the new Samsung Galaxy range as my contract was due for renewal a few months back - I found the S3/4 too big (I like to be able to hit all areas of the screen with one hand (if that makes sense) the Mini versions felt a little plastic'y.
At the moment iv moved onto O2 simplicity as this phone fits the bill fine.
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HTC One and Sony Xperia Z
Had enough of iPhones and am glad to have changed.
Nice to hear people are ditching apple crap
Had 4 iPhone's in a row and got bored. Got a HTC one X, which I liked but it only lasted a year as I get bored. Got a galaxy s4 now and still love it. Although I'll be bored soon enough no doubt. I was very tempted by the HTC One, as the camera appealed, but fancied a change. And I hate sense, it's awful.
Love the s4 for the battery life and card slot. I bought a 64gb card and I have all the space I need.
I currently have an iPhone 5 and when my next upgrade is due il no doubt be getting the latest iPhone at the time! They are in a league of their own!
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HTC One here brilliant phone that does everything I want very well withoit to many features that I would never use which is why I never went for an S4. For me iphones have never been a contender.
Dont get to drawn in to the mega geeky anal comparisons, you'll do your own head in lol
iPhone 4S. Not upgraded as bought it outright when it first came out. Maybe next year. Love it as syncs with iPad and Mac and can use it wih Apple TV. Also use it as my sat nav and cycle computer. Epic device.
S3 Sportback
I have a note 3, i use it alot for browsing and netflix if im travelling, battery life is pretty good (and can swap batteries) expandable memory, lots of nice little features with the stylus. I had a iphone 4 before but got pretty annoyed with it not being very compatible with anything that isn't apple so ended up getting rid, got a Galaxy S2 on the cheap which was ideal apart from poor battery life. The note ended up being a lot cheaper as well, i pay £34 a month with unlimited internet and tethering
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IMO all of the smart phones have the same level of features model for model I have used iPhones, samsungs, BB etc but it's really down to how you are going to use it ... If you are an Apple user ie Mac, TV etc then you would be crazy not to buy an iPhone but if you are just looking for a phone with web and maybe satnav the look at any of the newish smart phones will serve you well.
99' Audi S3(APY) - Brilliant Black Click>>>Going Sideways vid!
You've got to have a laugh!
Saying all smart phones have the same amount of 'must have' features is like saying that all cars are the same as they all get you from A to B. Travelling to your destination in style is also very important.
I will agree that the differences between the many Android phones isn't that wide, but between IOS and Android there is a gulf feature-wise.
The main (and for me, important) differences between IOS and Android... (read: Android has them and IOS doesn't):
- Widgets.
- Mass storage functionality... i.e. syncing/adding content, media, etc to phone without the need for itunes.
- Being able to plug your phone into ANY pc to copy files without the need for itunes. And even if that PC does have itunes, without it trying to delete your entire library because your phone has been sync'd with another PC!
- Being able to download ANYTHING from the internet using the phone's browser, not just what Apple sees fit.
- Ability to replace any stock app (email/sms/phone dialer/etc) with 3rd party apps with more functionality.
- Downloading stuff via torrents
And the list goes on, but for me the above are must have features.
And as for Apple hardware... being limited to a choice between the small screen of an iPhone or the huge screen of an iPad/iPad mini is not good for me. I need something in between.
ALSO... Apple's insistence on non expandable storage (lack of memory card slot) is ridiculous. Ok, it suits them because people are forced to spend big to buy the largest capacity iphone if they don't want to risk getting caught short.
Again, I could go on but I won't, as I'm boring myself now.
The next time you're on a bus/train/coach/etc have a look around at what people are fiddling with, and you should notice that gone are the days of iPhones in every hand. They are a Tethnic minority now.
Just got myself the Sony Xperia Z1. On the whole very impressed, feels beautifully made and is quick and smooth in operation. Downside is the onboard speaker is poor and I can't assign different tones to e-mail and text alerts unless I can find a way round that. I still think I prefer my 3 year old HTC Desire HD, if it didn't have a battery that needed charging 3 times a day and couldn't open a lot of video links I'm sent I wouldn't be changing. Found out last night how to assign an e-mail alert tone so all is good on that front
Last edited by Oranoco; 11th December 2013 at 12:28. Reason: Found out how to sort tones
Had an xperia s last year, terrible phone, started off good after a few months the battery got poor and it started rebooting after a while.
Anybody got an LG G2? Seems like the phone to beat at the moment.
Seriously considering getting one in the next week or so.
LG G2 Review | Stuff


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BeerAdvocate on your phone?! True story. Try the beta now.
Tickets for American Craft Beer Fest are on sale now! (May 30 & 31 in Boston, MA)
Papier (Brandy Barrel) - The Bruery
Displayed for educational use only; do not reuse. |
Ratings: 52 | Reviews: 20 | Show All Ratings:
Reviews by Halcyondays:
4.3/5 rDev -0.7%
look: 3.5 | smell: 4.5 | taste: 4.5 | feel: 4 | overall: 4
750 mL bottle, this has been in my possession for 11 months,
A: Pours a light mahogany with a small smattering of bubbles.
S: Sweet caramel, port wine, classic brandy barrel you can smell it.
T: Caramel and residual (brown) sugar. Some grape, big raisin with a noticable ethanol. Very tasty.
M: Full-bodied and viscous. Flat carbonation-wise, but the ethanol and viscosity carry it.
D: As with the other 2 BA Papiers, this is fantastic. Boozy still a bit, great flavours, I can only see this getting better.
Serving type: bottle
04-14-2010 04:28:33 | More by Halcyondays
More User Reviews:
4/5 rDev -7.6%
look: 3 | smell: 4 | taste: 4 | feel: 4 | overall: 4.25
750 mL bottle poured into a taster. Opened my Papier and then Diego pulled this out. Nice. Bottle 739 I think.
Appearance - No real head. Minute amount of a collar. Murky brown body.
Smell - Much like the base, but with a brandy aroma. Vanilla, tons of caramel, and brandy. Also slightly oxidized.
Taste - Stronger brandy than I expected. Lots of caramel. Less oxidized than regular Papier. More caramel as it warms. Fruity and nice complementary brandy.
Mouthfeel - Low carbonation. Slick feel. Oxidation is nice on this one too.
Overall - Not better than the regular Papier. Never a true brandy barrel fan.
Serving type: bottle
03-09-2013 03:07:43 | More by Alieniloquium
3.33/5 rDev -23.1%
look: 3 | smell: 3 | taste: 3.5 | feel: 3 | overall: 3.5
750 ml bottle generously shared by ipa247. Thanks Jeff. Pours a wuddy brown with pretty much no head and just a couple of bubbles around the edge of the glass. The aroma is lots of alcohol, brandy, plum, raisin, and caramel. Lots of brandy in the aroma with quite a bit of sugar. The taste is very sugary with lots of brandy, grape, plum, and caramel. The finish is boozy with some oak and brandy and is quite warming with a bit of astringency. Full bodied. Low carbonation. A bit too sweet and boozy for me.
Serving type: bottle
01-03-2013 04:14:34 | More by jethrodium
4.13/5 rDev -4.6%
look: 4 | smell: 3.5 | taste: 4.5 | feel: 4 | overall: 4
Thanks for sharing, Jeff! Reviewed from notes.
A: Pours a muddy reddish raisin brown with a thin bubbly head that quickly faded into a short lived collar.
S: Strong notes of dark fruits mixed with brandy. Very sweet smelling. I get maybe a hint of oxidization as well. Very rich, boozy and strong smelling.
T: Much tamer than the nose suggested. The flavor is neither as sweet nor as boozey as I had dreaded. I get tons of leather, vanilla and tobacco. Dark fruit notes are there as well. The beer is definitely brandy forward but it is very drinkable and smooth. Caramel, oak and chocolate in the aftertaste.
M: The alcohol is hidden extremely well for how high it is. Thick body, low carbonation, greasy, thick, smooth and heavy mouthfeel.
O: Drinks very well, I was surprised, especially for how potent the nose is. A few ounces is still all I could handle. Thanks again Jeff.
Serving type: bottle
12-30-2012 23:37:28 | More by scottfrie
4.08/5 rDev -5.8%
look: 3.5 | smell: 4.5 | taste: 4 | feel: 4 | overall: 4
Pours a darker muddled brownish/amber color, pretty much flat, some bubbles on top. Nose is very sweet and boozy, caramel, oak, burnt sugars...reminds me of a hybrid between a port & shot of brandy. Flavor is sweet & boozy, oak, dark fruits, caramel, burnt sugars. Medium body, flat, rich.
Drinking the Papier variants is like drinking SA Utopias...pretty much a hybrid between beer & spirit. Really enjoy these types of beer, so I would have to say I'd seek this one out again and look forward to the next time I can do so. Recommend!
Serving type: bottle
08-31-2012 20:57:19 | More by Sean9689
Papier (Brandy Barrel) from The Bruery
out of 100
based on 52 ratings.

subset: commoncrawl


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Shajada Khatun in London, N8
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The Progressive Music Series for Basal Use in Primary, Intermediate, and Grammar Grades, Volume 2 (Google eBook) |
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a-way Abbie Farwell Brown Adolf Weidig Birds blow blue bove bright Charles Keeler chil Christmas cold cresc dance dear dreams dren English Folk Song fair Fare Felix Borowski Felix Mendelssohn-Bartholdy fid-die flow'rs Frank Walcott French Folk Song Gaetano Donizetti glad good-night green hear Horatio Parker Composed J J J land light ling Louise Baum Ludwig van Beethoven Marshall Bartholomew Mary Mapes Dodge mer-ry moon morning Nellie Poorman nev-er night o'er Old English Otto Miessner Composed play pretty Progressive Music Quarter-Note Beat rain RHYTHM STUDY ring Ring-a-ting ring-ing round sail Sand-man Series sing sleep snow Spring stars sweet swing T. M. II tempo thee thou thro thro'the ting tree ve-ry W. A. Mozart wind wing Youth's Companion
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Floyd Music Textbooks: Indiana State University Library
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Gamma-ray burst breaks record for biggest cosmic explosion
Washington, Nov 22 : Cosmic explosion caused by the death of a star in a distant galaxy became the focus of astronomers around the world after it occurred in April.
The explosion, known as a gamma-ray burst and designated GRB 130427A, has topped the charts as one of the brightest ever seen.
A trio of NASA satellites, working in concert with ground-based robotic telescopes, captured never-before-seen details that challenge current theoretical understandings of how gamma-ray bursts work.
"We expect to see an event like this only once or twice a century, so we're fortunate it happened when we had the appropriate collection of sensitive space telescopes with complementary capabilities available to see it," Paul Hertz, director of NASA's Astrophysics Division in Washington, said.
Gamma-ray bursts are the most luminous explosions in the cosmos, thought to be triggered when the core of a massive star runs out of nuclear fuel, collapses under its own weight, and forms a black hole. The black hole then drives jets of particles that drill all the way through the collapsing star and erupt into space at nearly the speed of light.
Gamma-rays are the most energetic form of light. Hot matter surrounding a new black hole and internal shock waves produced by collisions within the jet are thought to emit gamma-rays with energies in the million-electron-volt (MeV) range, or roughly 500,000 times the energy of visible light.
The most energetic emission, with billion-electron-volt (GeV) gamma rays, is thought to arise when the jet slams into its surroundings, forming an external shock wave.
NASA's Swift Gamma-ray Burst Mission detected the burst almost simultaneously with the GBM and quickly relayed its position to ground-based observatories.
--ANI (Posted on 22-11-2013)
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Prime minister vows to do "whatever is required" to help people in flood-hit areas as government's "slow" response is forced into the spotlight
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Police hunt man over 'racist tirade' on the Jubilee Line
Police are hunting this man after a Tube passenger was subjected to a "tirade of racial abuse".
Old Bailey hears Princess Diana leaked Palace phone numbers to journalist Clive Goodman
Princess Diana sent the News of the World a book of Buckingham Palace internal phone numbers because she wanted an ally against Prince Charles, the Old Bailey heard.
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A memorial service today for the broadcaster Sir David Frost boasted a guest list every bit as glittering as the regular turnout at his famous summer parties.
Miles Aldridge interview: "The women in the pictures are often based on my mother"
Fashion and art photographer Miles Aldridge's images of women trapped behind Barbie girl glamour owe much to a shocking moment of childhood discovery, he tells Alison Roberts
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Published: 03 July 2013
When fashion photographer Miles Aldridge was 10 he discovered that his father Alan — a Sixties illustrator who designed album covers for Elton John and worked with the Beatles — had "another family around the corner", a long-term mistress and another child. Miles's perfectly happy home was no more than a fake, and as his father left London for Norfolk with the other woman, his mother struggled with the drudgery of single parenthood and little money.
Almost 40 years later Aldridge's distinctive, vivid and often erotic photographs of beautiful women owe much to that shocking moment of childhood discovery. Look closely and the trademark passivity of his models, with their blank unsmiling expressions and layers of heavy make-up, has a subversive quality, as though the women are trapped in a nightmare of stereotyped femininity and Barbie doll glamour.
Aldridge places his subjects in kitchens, supermarkets and playgrounds, surrounded by the detritus of a dinner party, or with children and babies whom they seem to ignore. The style of their clothes and hair is almost always nostalgic, which is no coincidence.
"The women in the pictures are often based on the mother I grew up with," he confirms. "She's not the wife of a successful illustrator, although she's glamorous enough to be that. Instead she's someone who continually cooks and washes and cleans and wears this unemotional, mask-like face... I think my mother felt trapped by me and my sister, but she was definitely a kind of muse to me."
Aldridge's work is all over London next month — at Somerset House in a major new show, at the Brancolini Grimaldi gallery in Albemarle Street, and on fashionista coffee tables in a glossy new book of more than 130 pictures. A new monograph, I Only Want You to Love Me (Rizzoli), has also been published.
He is thoroughly immersed in the world of high fashion — his model sister Saffron was once the face of Olay, and in 1997 he married American supermodel Kristen McMenamy — though they're currently in the process of divorce. But you can't help feeling as you look at his plastic, robotic models frozen in moments of cold detachment, that he's actually in the business of undermining that world, proving in fact that nice frocks really don't make you happy.
"I don't think the world is one great palazzo of people drinking champagne," he insists. "It's not like that. There's darkness there beneath the surface. My pictures always have to reflect my anxiety about the world."
Aldridge's career has involved some serious champagne-drinking, of course — but not at first. As a child he mythologised his absent dad, who later moved to LA and married yet another woman. "He finally left when I was 12 and communicated after that largely by postcard." Aldridge tried his hand at directing pop videos while living in a council flat in Bethnal Green. "My dad wasn't very good with money. He made lots and lost lots. We weren't from a moneyed background at all."
He eventually sent some photos of an aspiring model girlfriend to an agency, and fell into fashion when they called him as well as her. By then he'd hung out on shoots with Saffron but had no real idea how to do it. "I remember watching my sister, who I knew was this oik from Camden who served burgers at Dingwalls, being transformed in a morning into an amazingly glamorous lady on a yacht. And I was impressed right from the start by the fakery of it, which is what a lot of my work is actually about.
"But at the start of it all I didn't really have an idea in my head. Not a single idea. All I had was the worst stomach cramps you can imagine because I was so nervous." The oik from Camden, incidentally, is now married to the financier Ian Wace, who is worth a reported £300 million.
In the mid-Nineties Aldridge certainly looked the part — a pretty, skinny boy with badly bleached hair. It was a time when a British accent could still take you far in New York."I wasn't really accepted in London. I didn't work for The Face, for example, for ages, it was very cliquey. But in New York I was shooting covers almost immediately ... There was lots of money and there was a kind of madness to the fashion industry — but it was also great fun. I don't think we'll see that kind of excess again."
He met McMenamy on a shoot for W magazine, and theirs was a famously glossy wedding. Karl Lagerfeld gave McMenamy away and Naomi Campbell was a bridesmaid. Aldridge won't talk about the ongoing divorce proceedings but he and Kristen have two children, 10-year-old Arthur and seven-year-old Eddie, whom he sees every other weekend.
Today, Aldridge's work undoubtedly blurs the line between commercial photography and art, and his photographs send messages beyond the simple promotion of clothes. Some will also think that the nudes, and some of his overtly sexual imagery, also blur the line between eroticism and the objectification of women. He doesn't deny that he often looks at the women he shoots with desire, and that part of his project is "to make them look the best they possibly can from that point of view". "I do love women, and I am fascinated by them," he says.
There is one picture that brings me up cold, however — a photograph of a passive woman lying on a bed with a man wearing bikers' leathers and helmet standing next to her. It's chillingly close to a rape fantasy in my book — yet Aldridge looks baffled when I tell him this. In fact, he says, the picture is yet again based on memories of his mother, this time in her dying hours as she lay in a hospice at the age of 49, racked by cancer. The man in the helmet is "the angel of death" and the picture "a kind of metaphor of my mother's passing". Yet it's also the one photograph of his that Italian Vogue, which was expecting something very different, has ever refused to publish.
Aldridge admits that he treads a fine line between providing his clients with easy material and creating his own darker worlds beneath the glossy surface. It's why his photos are often so vibrantly coloured: "It's a trick. Pretty colours make the message more palatable," he says.
"All I know is that these images reflect how I feel about the world and what I've learned about it thanks to my life with my mother and with other women."
It's a world of jagged edges and murky undercurrents, all done up in bows and frills like a pretty doll. I'm not sure who he's playing the trick on, but it's a clever one.
I Only Want You to Love Me is at Somerset House, WC2 (020 7836 8686, somersethouse.org.uk) July 10-September 29.
Short Breaths is at Brancolini Grimaldi, W1 (020 7493 5721, brancolinigrimaldi.com) July21-September 28, and a new monograph, I Only Want You to Love Me (Rizzoli) is out now.
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Battleship Cathy
Battleship Cathy is on a mission to find a romantic partner in a casual social setting!
4.54 / 5.00 19,571 ViewsAfter 2 years in the making (ok so that's 2 years on and off 'cause I'm a lazy bastard) it's finally here: Sonic the LOLhog! Get ready for a LOLocaust of a time as Sonic and friends get up to some hilarious mishaps and what not!
*Hang about after the credits for an extra treat!
This is way too funny!!!! do you want everyone to laugh themselves half to death!!!!! oh, you do??? this comedy can kill!!! dun, dun, duuunnn!!!!! with laughter!!! muhahahahahahahaaaaa!!!!!


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Copyright © 1999 Elsevier Ltd All rights reserved.
Structure, Volume 7, Issue 2, 131-142, 15 February 1999
Research Article
Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs
RL Stanfield1, E Cabezas2, AC Satterthwait2, EA Stura3, AT Profy4 and IA Wilson5, *
1 The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
2 The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
3 The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA, Department d'Ingenierie et d'Etude des Proteines, BAT 152 CEA/SACLAY, 91191, Gif sur Yvette, Cedex France
4 Repligen Corporation, 117 Fourth Avenue, Needham, MA 02194, Procept, Inc., 840 Memorial Drive, Cambridge, MA 02139, USA
5 The Scripps Research Institute, Department of Molecular Biology, Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA e-mail: firstname.lastname@example.com
- Background: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop.Results: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II β turn.Conclusions: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.
Human immunodeficiency virus 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. The exterior of the membrane-enveloped virus is embedded with multiple copies of the proteins gp120 and gp41, which are synthesized as a single protein precursor (gp160) and then cleaved, but remain in a noncovalent association on the membrane surface [[1]]. These viral antigens have been implicated in viral cell entry; gp120 binds to CD4 and then to at least one other coreceptor on the cell surface, before gp41-mediated fusion of the viral and target cell membranes. Recently, several β-chemokine receptors have been implicated as the viral secondary receptors, with CXCR4 serving as the primary coreceptor for T-cell tropic (T-tropic) or T-cell line adapted (TCLA) syncytia-inducing (SI) isolates and CCR5 serving as the major coreceptor for macrophage-tropic (M-tropic) nonsyncytia-inducing (NSI) isolates (for reviews, see [[2],[3]]). Viruses that require CXCR4 for their coreceptors are termed 'X4' viruses, whereas viruses that require CCR5 are termed 'R5' viruses. Dual tropic viral strains have been identified that can use both coreceptors and, thus, are termed R5X4 viruses; some alternative coreceptors have also been identified that can be used by the virus to enter cells.
The third hypervariable (V3) domain of gp120 is a disulfide-linked loop of approximately 40 amino acids with a high degree of sequence diversity among different viral isolates [[4],[5],[6]]. The V3 loop is one of the major immunogenic sites on the virus and is sometimes called the principal neutralizing determinant (PND) [[7]]. The accessibility or exposure of the V3 loop on gp120 appears to vary depending on viral isolate type and increases significantly when the virus interacts with CD4 through a conformational change that is triggered in gp120 [[8],[9]]. The V3 loop then becomes more sensitive to neutralizing antibodies and proteases as a result of the gp120–CD4 interaction. Sequence changes in V3 can alter viral cell tropism [[10],[11]], antibody neutralization [[12],[13],[14]], neutralization of soluble CD4 [[15]], syncytium formation [[11],[16]] and chemokine receptor usage [[17]]. It has been noted that T-tropic V3 sequences are usually more basic in charge than M-tropic sequences through accumulation of positively charged residues in regions on either side of the GPGR tip of the loop [[18],[19]]. As no easily discernable pattern relates specific changes in V3 residues to phenotypic changes, it is likely that conformational variations in gp120 and the V3 loop that arise from particular V3 loop sequences could modulate gp120 function. Although the crystal structure of a ternary complex between gp120, CD4 and an antibody Fab fragment has at last been determined [[20],[21],[22]], the gp120 was modified by removing several loops including V3. Thus, the paucity of structural information on the V3 loop still needs to be addressed.
In early work by La Rosa et al. [[4],[5],[6]], an analysis of 245 different HIV-1 V3 loop sequences showed that some amino acid positions in the loop are highly variable in amino acid composition, whereas others, especially those near the tip of the loop, are highly conserved; for example, examination of the MN sequence IHIGPGRAFY reveals that these amino acids are found at their respective positions in 94, 47, 82, 98, 95, 98, 91, 83, 72 and 80% of the other 244 isolates. The GPGR sequence is highly conserved in all known sequences of subtype B, whereas other group M isolates are characterized by the highly related GPGQ sequence at this position. The high degree of conservation of some of these amino acid positions suggests that they have a key structural and functional role and are important in some aspect of virus infectivity, such as modulation of the conformation of the V3 loop, or for modulating the interaction with coreceptor. The LaRosa et al. study [[4],[5],[6]] also predicted the secondary structure for their V3 consensus sequence (CTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC) to consist of an extended β strand, a type II β turn around residues GPGR, a β strand and then an α helix. This secondary structural prediction was carried out for all 245 sequences, with 93% predicted to have the extended β strand around residues SIHI and 66% predicted to have the short helix at residues DIRQ. Pro, Gly and Arg are the most commonly found residues in positions i+1, i+2 and i+3 of a type II β turn, whereas Gln is the second most commonly found residue at position i+3 [[23]].
We have been investigating several HIV-1 neutralizing antibodies and their complexes with V3 peptides in order to determine the tertiary conformation of the V3 loop and to understand why some antibodies are viral-specific while others recognize many different viral strains. These neutralizing antibodies recognize the V3 epitope as a free peptide in the soluble gp120 protein and on the intact HIV-1 virus. Knowledge of the conformation of this loop may help to explain coreceptor usage and the changes that take place in the virus upon conversion from a primary M-tropic isolate to the T-tropic strains associated with disease progression. A panel of antibodies, which were raised against the same 40 amino acid V3 peptide RP70 [[24]] ( Figure 1 ), neutralize the virus by preventing viral–cell membrane fusion. It has long been thought that these and other V3-specific antibodies do not interfere with CD4 binding. Recent studies using intact viral particles rather than soluble monomeric gp120, however, indicate that V3 neutralizing antibodies may also prevent the binding of HIV-1 to CD4+ human cells [[25]].
Previously, we have reported the structures of two antibody Fab fragments, 50.1 and 59.1, in complex with V3 peptides [[26],[27],[28],[29]]. Fab 50.1 is reported to be highly specific for the MN viral strain (T-tropic, SI), whereas 59.1 is reported to strongly neutralize IIIB (T-tropic, SI) and weakly neutralize MN [[24]]. The X-ray structures for V3 peptides in complex with Fabs 50.1 and 59.1 agreed well with the La Rosa et al. [[4],[5],[6]] predictions up to the conserved GPGR tip of the loop. In the 50.1–peptide complex, eight amino acid residues were ordered in the antibody combining site (CKRIHIGPG). Residues CKRIHI were in an extended β conformation with the two Ile residues buried in tightly fitting hydrophobic pockets in the antibody combining site and the GPG forming the start of a turn. The first Gly residue had torsion angles in the epsilon (ϵ) region of Ramachandran space, a region usually only available to Gly residues, centered around φ = 90° and ψ = 180°. The PG had torsion angles appropriate for a type II β turn. In the 59.1–peptide complex, 10 amino acid residues were visible (HIGPGRAFYT). Five of these residues had structural counterparts in the peptide from the 50.1 complex (HIGPG), and displayed very similar mainchain torsion angles. Following the type II β turn around GPGR, we observed a double bend consisting of a type I turn for residues GRAF (in [[28]], this turn is referred to as type III; the type III turn is a variant of the type I turn and is usually classified as type I) and a type I turn around RAFY. The sidechain of the Arg residue is bound in a deep, negatively charged pocket on the antibody surface.
The individual peptide structures from the 50.1 and 59.1 complexes could be pieced or spliced together to make a more complete model for a 13-residue section of the V3 loop [[29]] that included the GPGR tip. Contrary to the previous predictions [[4],[5],[6]] of a single tight turn around GPGR, a much broader double turn was found that consisted of a type II turn around GPGR and a double bend (type I, GRAF; type I, RAFY) around GRAFY. The Ala residue in the double bend had α-helical torsion angles of φ = -60°, ψ = -45°, similar to one of the two preferred conformations for the conformationally restricted residue Aib [α-aminoisobutyryl residue -NHC(CH3)2C(O)], which normally adopts torsion angles of -57°,-47° or +57°,+47° [[30],[31]]. We surmised that replacement of the Ala residue with an Aib residue could constrain the peptide and provide a more rigid scaffold for vaccine or drug design around this template. Nuclear magnetic resonance (NMR) studies of such an Aib-containing peptide confirmed that additional structure was indeed conferred on the peptide in aqueous solution [[29]]; the X-ray structure determination of Fab 59.1 with the Aib-containing peptide showed that the Aib residue made no significant changes to the peptide conformation, as expected [[29]]. The Aib residue was not necessary for binding or for crystallization, as Fab 59.1 could be crystallized isomorphously with peptides that were identical except for the Ala→Aib mutation. Another attempt to restrict conformation of the V3 peptides involved using a synthetic hydrogen bond mimic incorporating a hydrazone bond. These hydrogen bond mimics are designed to replace a CO–HN hydrogen bond within a β-hairpin structure, thus cyclizing the peptide into a constrained hairpin structure.
Here, we describe the crystal structure of Fab 58.2, a highly potent and broadly neutralizing antibody that has been reported to neutralize both T-tropic and M-tropic viral strains, as well as several strains that have been passaged only once or twice [[24]]. We report the structures for Fab 58.2 in complex with one linear Aib-containing peptide and two cyclic, constrained peptides.
Results and discussion
Structure determination and refinement
The crystal structures for Fab 58.2 in complex with one linear (Aib142; Figure 1 ) and two hydrazone-linked cyclic peptides (His loop, Ser loop; Figure 1 ) have been determined to 2.0 Å (Aib142) and 2.8 Å (His loop and Ser loop). Data collection and refinement statistics are summarized in Table 1. All structures were determined by the molecular replacement (MR) method using previously determined Fab structures (see Materials and methods section). The crystals of the two Fab–cyclic peptide complexes were isomorphous, as a result of only a single amino acid difference in their peptides (His315→Ser). All structures were refined with the X-PLOR simulated annealing refinement protocol [[32]], using the Engh and Huber parameter set [[33]] and a bulk-solvent correction. The Fab molecules are numbered according to standard convention [[34]] with light and heavy chain identifiers L and H, respectively. The peptides are numbered according to the BH10 isolate sequence [[35]] with a P chain identifier (RP313IP314HP315IP316GP319PP320GP321RP322AP323FP324YP325). Note that the sequential numbering system is interrupted due to a two amino acid insert, after P316, that occurs in the IIIB isolate. The Fab 58.2–Aib142 coordinates contain Fab residues L1–L212, H1–H230 and peptide residues P313–P325 (11 residues). The Fab 58.2–His-loop coordinates contain Fab residues L1–L212, H1–H230 and the cyclic peptide with the complete hydrazone linker (P1–P12; see Figure 1 ). The Fab 58.2–Ser-loop coordinates contain Fab residues L1–L211, H1–H230 and the cyclic peptide minus the hydrazone linker (P2–P12).
Table 1 |
Peptide | Aib142 | His loop | Ser loop | ||
Space group | C2 | P21212 | P21212 | ||
Unit cell (Å) | 72.9, 71.9, 88.2 | 96.1, 114.7, 49.5 | 95.5, 115.5, 49.6 | ||
β = 98.3° | |||||
Number of crystals | 3 | 1 | 1 | ||
Resolution (Å) | 43.6–2.0 | 24.0–2.8 | 47.8–2.8 | ||
Last shell (Å) | 2.05–2.0 | 3.02–2.8 | 2.98–2.8 | ||
Observations | 183,103 (9505) | 44,763 (4678) | 47,266 (9337) | ||
Unique reflections | 30,460 (2014) | 12,923 (2287) | 13,951 (2259) | ||
Rmerge (%)/ Rsym (%) | 10.6 (54.7) | 7.6 (28.8) | 7.2 (21.8) | ||
Completeness (%) | 99.6 (98.5) | 91.9 (82.8) | 98.9 (97.7) | ||
<I/σI> | 29.5 (3.4) | 7.5 (1.0) | 13.2 (1.5) | ||
Generator | Siemens M18X; Rigaku RU-300 | Rigaku RU-200 | Siemens M18X | ||
Optics | Supper long mirrors; monochromator | Monochromator | Supper double mirrors | ||
Detector | Mar 30 cm; R-axis II | Xuong-Hamlin multiwire | Siemens X1000 | ||
Data reduction program | HKL [[81]] | Xuong-Hamlin [[82]] | XENGEN [[83]] | ||
Rcryst (%)∗ | 19.6 (31.8) | 18.5 (27.6) | 20.0 (37.7) | ||
Rfree (%)† | 25.6 (38.2) | 30.5 (36.6) | – | ||
Rmsd bond lengths (Å) | 0.010 | 0.012 | 0.008 | ||
Rmsd bond angles (°) | 1.82 | 1.87 | 1.59 | ||
Number of water molecules | 120 | 1 | 1 | ||
Values given in parentheses are for the highest resolution shell. ∗Rcryst = Σh ∣ ∣ Fobs(h) ∣ – ∣ Fc(h)∣ ∣/Σh ∣ Fobs(h) ∣, where Fobs and Fc are the observed and calculated structure-factor amplitudes for reflection h = (h,k,l). †Rfree is calculated with 10% (5% for the His loop–Fab complex) of the data never used in the refinement. Rfree was not calculated for the Ser loop complex. The Rcryst and Rfree are calculated using all data with F/σFF > 0. A bulk-solvent correction as implemented in X-PLOR [[72]] was applied to all data sets. |
The Fab–Aib142 peptide complex structure ( Figure 2a ) was determined to 2.0 Å resolution. Analysis of the Ramachandran plot by PROCHECK [[36]] shows that 89% of the residues are in most favored positions with three residues in disallowed regions. These 'disallowed' residues include two residues in a disordered loop in the constant heavy chain (residues H128–H136), which almost always displays high displacement parameters in other antibody structure determinations [[37]] and residue AlaL51 (φ = 69°, ψ = -53°), which is the i+2 residue in a distorted type I′ turn in most Fab L2 complementary determining region (CDR) loops [[38]].
Two Fab–cyclic peptide structures ( Figure 2b,c ) were determined, both to 2.8 Å resolution. These structures differ at only one residue in the peptide ( Figure 1 ). The Rfree was not monitored during the Ser loop refinement as this particular structure was determined prior to the introduction of the Rfree validator. Only one bound water was included in each model due to the moderate resolution. Ramachandran plot analysis of the His loop complex by PROCHECK [[36]] shows that 81% of the residues are in most favored positions, with three residues in disallowed regions (two residues from the H128–H136 loop and AlaL51). The Ser loop complex has 87% of the residues in most favored regions with five residues in disallowed regions (two in the H128–H136 loop, the C-terminal residue of the heavy chain, AlaL51 and LysH64, which is the i+1 residue of a type II′ turn).
Structure description and comparisons
Fab 58.2 is a mouse IgG1κ antibody. The elbow angles for Fab 58.2 are 174.3°, 144.8° and 144.3° in the Aib142, His loop and Ser loop complexes, respectively. The 58.2 CDR loops L1, L2, L3 and H2 belong to canonical classes 5, 1, 1 and 1, as classified previously [[39],[40]]. The 58.2 H1 CDR loop has a one amino acid insert following residue 35 and so should belong to canonical class 2. Other Fabs with H1 loops of this length are AN02 (PDB accession code 1baf; [[41]]) and N10 (1nsn; [[42]]); however, the H1 loops in both 58.2 and N10 differ from the expected canonical structure around several residues at their tips and also differ in this region from each other ( Figure 3 ). Not surprisingly, a tendency for an increase in flexibility is seen as the H1 loop increases in length. Fab 58.2 has a long CDR H3 with a nine amino acid insert after residue 100. Previously determined structures with H3 loops of this length include KOL, 3D6 and R45-45-11 (accession codes 2fb4 [[43]], 1dfb [[44]] and 1ikf [[45]], respectively). The conformation of the base of the H3 loop is dictated by residues AlaH93, ArgH94 and AspH101. This combination of residues results in a 'kinked' base structure, as expected [[39],[46]], with the sidechain of AspH101 forming a salt bridge with ArgH94 and the mainchain carbonyl oxygen of H100I (the residue preceding H101) forming a hydrogen bond to TrpH103 Nϵ1.
The linear Aib142 peptide is 24 amino acids with an Aib residue replacing AlaP323 ( Figure 1 ). Electron density is clearly visible for 11 of the 24 residues, RIHIGPGRAibFY ( Figure 4a,b ). The peptide has an extended conformation for residues RIHI, which connect to a type I (αR→αR) turn around residues GPGR. A type VIa (βP→αR) turn is found for residues GRAibF and a type I turn is found around residues RAibFY, making residues GRAibFY a double bend. Residue ArgP322 is bound in a deep pocket in a highly negatively charged antibody combining site ( Figure 5a ), forming charge–charge interactions with residues AspL94 and GluH95 ( Figure 5b ). The bound portions of the cyclic peptides have a very similar conformation to the bound portions of the linear peptide. Clear electron density is present for most of the cyclic peptides, with a small break in density around the hydrazone linker in the His loop complex, and weak density for the linker in the Ser loop complex ( Figure 4c,d ). The hydrazone linker has been left out of the Ser loop model because of its weak electron density.
The three peptides bind to the antibody in virtually identical orientations, with the peptide lying flat on the Fab surface and residue ArgP322 protruding into a deep, negatively charged pocket on the Fab surface ( Figure 5a ). The surface area [[47]] buried by the peptide on the Fab is 666, 609 and 597 Å2 for the Aib142, His loop and Ser loop peptides, respectively. The corresponding buried surface area on the peptides is 578, 540 and 487 Å2. The Fab uses five of its six CDR loops to bind peptide; CDR L2 is not used and CDR H1 makes only minor contacts. This pattern of CDR usage is typical for antihapten and antipeptide antibodies [[48]]. A total of 124, 103 and 77 van der Waals contacts are made between the Fab and peptide for the Aib142, His loop and Ser loop complexes, respectively ( Table 2). In each complex, charge–charge interactions are found between ArgP322 of the peptide and AspL94 and GluH95of the Fab ( Table 3). In the Aib142 peptide, an additional charge–charge interaction is made between ArgP313 and AspL28. A total of 11, 7 and 7 hydrogen bonds and salt bridge interactions are made between the peptides and Fab in the Aib142, His loop and Ser loop complexes, respectively ( Table 3). The size and features of this interaction surface are similar to those of other Fab–peptide complex structures [[49]]. The Fab conformations are very similar between the different complexes as shown by the small differences in VL–VH pairings (less than 1.4°) between the complexes and the root mean square deviations (rmsds) for the individual domains, which range from 0.31 Å to 1.20 Å. One loop in the constant heavy chain (H128–H136), which frequently shows high thermal displacement parameters in Fab crystal structures [[37]], accounts for the high rmsds (0.83–1.20 Å) for the CH1 regions. Removal of this loop from rmsd calculations results in rmsds of 0.61–0.72 Å.
Table 2 |
Peptide residue | In all complexes | Aib142 | His loop | Ser loop | ||
ArgP313 | AspL28 AlaH100D TyrH100E | |||||
IleP314/ArnP1 | TyrH100E TyrH100G | |||||
HisP315/SerP315 | TyrH100E | AspL28 AlaL30 PheL32 | AspL28 AlaL30 PheL32 TyrH100 | |||
TyrH100 AlaH100D TyrH100F TyrH100G | ||||||
IleP316 | PheL32 TyrH33 | TyrH100G | PheL27D TyrH100G | |||
GlyP319 | PheL32 HisL92 | SerL91 | SerL91 | |||
ProP320 | PheL27D HisL92 | |||||
GlyP321 | HisL92 GluL93 AspL94 | |||||
ArgP322 | SerL91 GluL93 AspL94 LeuL96 GluH95 | HisH35a TyrH50 | TyrH50 | |||
AlaP323/AibP323 | AspL94 | TyrH50 AsnH58 | AsnH58 | |||
PheP324 | TyrH50 HisH52 GlyH56ThrH57 | TyrH33 SerH54 | TyrH33 | |||
TyrP325/GlyP10 | PheL27D | TyrH33 HisH52 | TyrH33 HisH52 | |||
GlyP11 | TyrH53 | |||||
Van der Waals contacts were calculated with the program CONTACSYM [[84]] using standard van der Waals radii [[85]]. The Fab residues contacted in all complexes are shown first, followed by additional contacts that are specific to the individual complexes. |
Table 3 |
Peptide atom | Fab atom | Aib142 | His loop | Ser loop | ||||||
P313 | Arg | NH2 | L28 | Asp | OD2 | 2.8 | ||||
P313 | Arg | NH1 | L28 | Asp | O | 3.2 | ||||
P313 | Arg | O | H100e | Tyr | N | 2.9 | ||||
P315 | His/Ser | N | H100e | Tyr | O | 2.7 | ||||
P315 | His/Ser | ND1 | H100e | Tyr | O | 3.3 | ||||
P319 | Gly | N | L92 | His | O | 3.0 | 2.9 | 3.1 | ||
P321 | Gly | O | L94 | Asp | N | 3.3 | 3.3 | 3.3 | ||
P322 | Arg | NE | L94 | Asp | OD1 | 2.8 | 2.9 | |||
P322 | Arg | NH2 | H95 | Glu | OE1 | 3.0 | 2.8 | 2.8 | ||
P322 | Arg | NH1 | H95 | Glu | OE2 | 2.9 | 2.8 | 3.0 | ||
P323 | Aib/Ala | N | L94 | Asp | OD2 | 2.9 | 3.1 | 2.9 | ||
P324 | Phe | O | H33 | Tyr | OH | 2.8 | 2.7 | |||
P10 | Gly | O | H52 | His | NE2 | 3.0 | ||||
Hydrogen bonds were calculated with HBPLUS [[86]]. |
Although the resolution of the His loop and Ser loop structures is insufficient to allow accurate placement of ordered water molecules, one strong peak of density in the Fab binding site of all three structures was assigned as a water position. Additionally, 120 water molecules were included in the 2.0 Å resolution refinement of the Aib142 complex. Six of these water molecules are found buried at the Fab–peptide interface (W12, W41, W62, W67, W72, W84).
The conclusions from epitope mapping experiments of antibody 58.2 agree well with the structural results. The epitope specificity for 58.2 was determined previously by screening a peptide display library expressing 1.5 × 108 unique 20 amino acid peptide sequences against 58.2 [[50]], as well as by epitope mapping, where 266 unique 14-mer peptides were prepared, with each amino acid position replaced with all amino acids (except Cys) (peptide KRKRIHIGPGRAFY) [[50]]. Residues KRKRI (P310–P314) could be replaced by most of the other amino acids, indicating that they are not important for peptide binding to Fab 58.2. HisP315 could be replaced with any amino acid except Gly, Pro and Thr. IleP316 could only be replaced with Glu, Leu, Met and Val. GlyP319 could not be replaced by any other amino acid. ProP320 could only be replaced by Ile. The second GlyP321 was not so important, as it could be replaced with all amino acids except Ile, Pro, Ser, Thr, Val and Trp. ArgP322 could not be substituted, whereas AlaP323 could be replaced with any amino acid except Pro. The PheP324 could be replaced with Asp, Ile, Lys, Gln, Arg, Ser, Thr, Val and Trp. These epitope mapping results indicate that residues GlyP319, ProP320 and ArgP322 are especially important for antibody binding, in agreement with the crystal structure that shows that ArgP322 forms a charge–charge interaction with the Fab and residues GlyP319 and ProP320 are critical for determining the conformation of the tip of the V3 loop. Residue GlyP319, which of course is a highly conformationally flexible residue, may also be important for allowing the loop to adjust to more than one distinct conformation. GlyP321, the i+2 residue in the type I β turn of the peptide in the 58.2–peptide complexes, is not absolutely required, in agreement with the diversity of residues that are found at the i+2 position of type I turns [[23]]. This result contrasts with the alanine replacement experiment on peptide binding to Fab 59.1 [[24],[28]]. When GlyP321 is replaced by an Ala residue, the affinity of Fab 59.1 for the peptide is reduced to about 32% of that of the native peptide, in good agreement for the strong preference for a Gly residue at the i+2 position of a type II turn [[23]], as found in the 59.1–peptide complex.
Two different V3 conformations
The most notable feature of the V3 peptide conformation while bound to Fab 58.2 is that it differs from the two previously determined structures for the same or similar peptide in complex with two other Fabs. The previously determined V3 loop structures were for peptide complexes with Fab 50.1, where the peptide epitope was identified as CKRIHIGPG, and with Fab 59.1, where the peptide epitope was HIGPGRAFYT. The three antibodies were all generated against the same peptide immunogen, RP70 [[24]] ( Figure 1 ). The five residues (HIGPG) that are structurally ordered in peptides bound to both Fabs 50.1 and 59.1 could be superimposed with low rmsds ( Figure 6 ) [[28],[29]]. These two peptides both adopt torsion angles for a type II β turn around residues GPGR. Thus, it was surprising to observe that the peptide bound to Fab 58.2 had torsion angles for a type I β turn around these same residues. The mainchain torsion angles for these four residues (GPGR) differ significantly between the two peptide-conformational types. The residues before and after the GPGR sequence, however, share very similar mainchain torsion angles ( Table 4). The second β turn, around residues GRAibF (or GRAF), is type I in the 59.1 complex (type I [αR→αR] turns have average torsion angles of φ = -60°, ψ = -30° and φ = -90°, ψ = 0° for the i+1 and i+2 residues), and type VIa in the 58.2 complex (type VIa [βP→αR] turns have average torsion angles of φ = -60°,ψ = 120° and φ = -90°,ψ = 0° for the i+1 and i+2 residues) with the torsional angles of residues AFY being highly similar in all peptide complexes ( Table 4). So, the peptides bound to Fabs 59.1 and 58.2 both contain a β turn followed by a double bend, but the different torsion angles for the central GPGR residues change the precise character of these turns and bends. NMR studies of the Aib142 peptide in solution have provided evidence for populations of both type I and II turns around residues GPGR [[29]], which is in good agreement with the two peptide conformations that we observe in the crystal structures of the V3 loop with different Fabs.
Table 4 |
50.1 Peptide | 59.1 Peptide | 58.2 Aib142 peptide | ||||||
φ | ψ | φ | ψ | φ | ψ | |||
CysP311 | ||||||||
LysP312 | -119 | -169 | ||||||
ArgP313 | -144 | 135 | 163 | |||||
IleP314 | -67 | 124 | -122 | 144 | ||||
HisP315 | -89 | 134 | 144 | -107 | 110 | |||
IleP316 | -124 | 141 | -133 | -169 | -124 | 137 | ||
GlyP319 | 102 | 173 | 79 | -172 | -58 | 141 | ||
ProP320 | -63 | 170 | -73 | 165 | -63 | -24 | ||
GlyP321 | 86 | 60 | 9 | -95 | -1 | |||
ArgP322 | -64 | -41 | -65 | 141 | ||||
Ala/AibP323 | -60 | -46 | -50 | -42 | ||||
PheP324 | -94 | 19 | -107 | 40 | ||||
TyrP325 | -49 | -22 | -68 | 161 | ||||
ThrP326 | -126 | |||||||
Several other NMR studies of V3 peptide conformation have been carried out in solution for both linear and cyclic peptides of varying length and sequence. These studies have shown that the V3 loop by itself is largely disordered in water, but contains a population of tight turns for residues GPGR. The NMR studies show evidence for type II turns [[51],[52]], both I and II turns [[53],[54],[55],[56]], nonspecific β turns [[57]] and a double turn, similar to that seen in the 59.1-peptide X-ray structure [[58]]. NMR studies have also supported the proposal that glycosylation of the V3 peptide can affect its conformation [[59],[60]] and that sequence changes affect the conformation [[61]]. NMR studies of an antibody–V3 loop peptide complex have also been carried out [[62],[63]] but indicated only that the peptide forms a loop with residues GPGR at the tip, with no more detailed information being reported. Interestingly, a search of the Brookhaven Protein Data Bank [[64]] for proteins containing the sequence GPGR identified two different proteins, but neither were in a type I or II β turn. In scorpion toxin II (PDB codes 1ptx and 1aho[[65]]), these residues form a wide, undefined turn near the C terminus of the protein (i+1, φ = -65°, ψ = 150°; i+2, φ = 115°, ψ = 135°; βP→ϵ), whereas in chaperonin-10 (PDB code 1lep[[66]]) the GPGR residues are part of a β strand. Additionally, in a recently reported X-ray structure [[67]] of the major histocompatibility complex (MHC) class I H-2Dd–HIV-1 peptide (RGPGRAFVTI) complex, the GPGR is in an extended conformation, as expected for peptides complexed with MHC class I molecules.
Correlation of structure with function
We clearly observe two different conformations for the V3 loop while bound to different neutralizing antibodies, and two conformations for the tip of the loop in solution [[29]]. These V3 loop conformations are biologically relevant, as the antibodies bind to viral gp120 to effect their neutralization activity. These conformations may represent two different and distinct secondary structural motifs that may or may not relate to viral phenotype, or they could merely indicate that the V3 loop is flexible and able to adopt different conformations. The only reported crystals of gp120 to date have been obtained for protein where this loop was truncated at its base [[20],[21],[22]]; crystals of gp120 with the intact loop have not yet been reported.
Biological implications
Human immunodeficiency virus 1 (HIV-1) is a membrane-enveloped virus, the exterior of which is embedded with multiple copies of the proteins gp120 and gp41. The third hypervariable loop (V3) of gp120 is involved in many aspects of virus infectivity and has, therefore, been the subject of much interest. The high degree of conservation of the Gly-Pro-Gly-Arg/Gln motif at the tip of the gp120 V3 loop, surrounded by regions of high sequence diversity, suggests this structural conservation is related to biological function.
Many studies have shown that the alteration of one or more amino acid residues within the stem of the V3 loop leads to changes in viral tropism. However, it remains to be seen whether these individual residue changes alter the loop structure or effect a switch in β-turn conformation at the tip of the loop. Clearly, a direct consequence of different turn types around Gly-Pro-Gly-Arg is that the flanking regions of V3 would be in different environments based on which turn type was found at the loop tip. The determination of the crystal structures of Fab fragments in complex with V3 loop peptides, presented here, goes some way towards understanding the conformational flexibility of this loop, although the answers to some questions remain elusive. These unanswered questions provide an important direction for future studies designed to reveal the biological role of the V3 loop. Further knowledge of the V3 structure will open up the possibilities for small molecule drug design based on its three-dimensional structure, or vaccine development through stabilization of biologically relevant conformations.
Materials and methods
The linear Aib142 peptide was prepared by chemical synthesis using Nα-9-fluorenylmethoxyl-carbonyl (Fmoc) protected amino acids and Fmoc-Rink's amide resin on an Advance Chemtech ACT350 peptide synthesizer, following the manufacturer's protocol. The N terminus was acetylated and the sidechain of Cys was protected with an acetamido-methyl (Acm) group. A mixture of trifluoroacetic acid (TFA)/phenol/ethanedithiol/thioanisole/water (84:6:2:4:4 v/w/v/v/v) was used to cleave the peptide from the resin. Peptide was purified using a preparative Cosmosil C-18 reverse-phase column on a Gilson high-performance liquid chromatography (HPLC) system. The peptide was eluted with a water/acetonitrile gradient containing 0.1% trifluoroacetic acid; 0–20% acetonitrile over 5 minutes followed by 20–35% acetonitrile over 20 min at 8 ml/min. The pure peptide eluted as a single peak on analytical HPLC and its identity was confirmed by mass spectrometry. The cyclic peptides were synthesized by solid phase synthesis using modified amino acids to form the hydrazone link. They were purified to single peaks by HPLC and their identities confirmed by mass spectrometry.
All crystallizations were carried out using the sitting drop, vapor diffusion method at 22.5°C. Crystals of the Fab 58.2–His loop and Ser loop complexes were obtained with a well solution containing 16% PEG 4000, 0.2 M imidazole malate, pH 5.0. The sitting drop consisted of 2.5 μl well solution, and 2.5 μl peptide/Fab (15 mg/ml Fab), with a peptide:Fab molar ratio of 2.4:1. Crystals of the Fab 58.2–Aib142 complex were grown from a well solution of 16% PEG 10,000, 0.2 M imidazole malate, pH 6.3. The drops consisted of 2.5 μl peptide/Fab (15 mg/ml Fab), and 2.5 μl well solution with a peptide:Fab molar ratio of 1.7:1. Space groups and unit cell dimensions for all crystal forms were determined by precession photography. Data collection statistics and information are tabulated for all three crystal forms in Table 1.
The Fab 58.2/Ser loop complex structure was determined first, using coordinates of Fab 59.1 (1acy, [[28]]), minus its peptide, as the MR model. Fabs 59.1 and 58.2 have approximately 83% and 65% sequence identity respectively in their VL and VH domains; their CL and CH1 domains are identical (IgG1, κ). The structure was determined using the X-PLOR rotation function and PC refinement [[68]], and a modified Harada translation function [[69]] written in our laboratory by D Filman and J Arévalo, with the intact 59.1 as a model. The intact Fab was found to be a suitable starting model because its elbow angle (the angle between the pseudo twofold axes of the VL – VH and CL – CH1 domains) differed from that of the unknown Fab by only 11o (the elbow angle for 58.2 was 144° and for 59.1 was 135°). Alternating cycles of model building with the Alberta/Caltech version of TOM/FRODO [[70],[71]] and refinement with the slow-cooling protocol in X-PLOR [[72]] were carried out, with peptide electron density being clearly visible after several cycles ( Figure 4d ). The refined coordinates for Fab 58.2–Ser loop (minus the peptide) were used as a starting model for the isomorphous Fab 58.2–His loop complex ( Figure 4c ). The Fab coordinates were refined using rigid body refinement followed by alternating cycles of slow-cooling refinement and model building as for the Ser loop complex. The Fab 58.2–Aib142 structure was also determined using MR. To estimate the elbow angle for the Fab 58.2–Aib142 structure, the Crowther Fast Rotation Function, as implemented in Merlot [[73]], was used to carry out cross-rotation functions with over 100 available Fab coordinate sets, which had previously been superimposed on a reference position, with the elbow angle aligned along the z-axis. A plot of elbow angle versus peak height for the top solution for each model suggested an elbow angle of around 180° for the unknown Fab 58.2. The starting Fab 58.2 model coordinates (the refined 58.2–Ser loop coordinates, without peptide) were artificially given this approximate elbow angle. The rotation function, PC refinement and translation function from X-PLOR were used for the structure determination [[68]]. The actual elbow angle for the refined structure was 175°. The structure was refined and rebuilt in alternating cycles as for the two previous complexes. Density for the peptide was very clear after only one cycle of slow-cooling refinement and manual rebuilding of the starting Fab model ( Figure 4a,b ).
Accession numbers
The coordinates and structure factors for the three Fab 58.2–peptide complexes are deposited in the PDB with accession codes 1f58, 2f58 and 3f58, to become available at the time of publication.
We thank Xiaoping Dai, Ron Hamlin, Rashid Syed and Tim Osslund for help with data collection and reduction for the Aib142–peptide complex crystals, and Ying Su and Nguyen-Huu Xuong for help with data collection and reduction for the Ser loop peptide complex. This work was supported by NIH grants GM-46192 (IAW) and AI-37512 (ACS), and carried out while RLS was a Scholar of the American Foundation for Aids Research. This is publication #11968-MB from The Scripps Research Institute.
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Publication Information
Received: October 8, 1998
Revised: November 23, 1998
Accepted: December 1, 1998


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IHC Winches has presented its 50-ton fiber rope traction winch, known as IDsis, to the Dutch and Belgian offshore markets.
The name 'IDsis' is an abbreviation of IHC Deep-sea installation system which is a dedicated deployment system for handling fiber rope, developed to overcome the restrictions of using conventional steel and synthetic wires. Steel is durable but heavy, which limits its usage in deeper water. Currently used synthetics are light, but low-heat resistance and high-creep properties limit their application on large winches. The rope is designed for critical deepwater applications, such as deep-sea installation, abandonment and recovery (A&R) and offshore mining operations.
The development of IDsis has resulted in the combination of: a traction winch, which reduces wear of the composite cable for the given load situations, and a storage winch, which is capable of reeling large amounts of fibre rope. Both are combined with an intermediate tensioner for safe and reliable operation in the offshore environment. Following the completion of the Factory Acceptance Test, the new deep-sea installation system will be tested in operational circumstances in the North Sea later this year. This will take place in association with Seaway Heavy Lifting on one of its offshore vessels. The Dutch Maritime Innovation Program has funded the development of the IDsis deployment system. This R&D project is executed in conjunction with such companies as Bexco , Seaway Heavy Lifting and Reden , which joined forces with IHC Winches to combine their various areas of expertise, such as winch and fiber rope technology, rope construction, operational offshore knowledge, modelling, simulation and legislation.
MacGregor, part of Cargotec, has won an order for two 50-metric-ton SWL versions of its heavy-duty grab cranes. The K5036-4HD MacGregor cranes are destined for a 68m floating crane barge,
MacGregor has secured an order from Wuchang Shipbuilding Industry for a 100-metric-ton SWL active heave-compensated subsea crane. The MacGregor crane will be installed
MacGregor, part of Cargotec, saidi it won an order for two subsea cranes from South Korean shipyard Hyundai Heavy Industries Co Ltd. The active heave-compensated
Reuters - Brazil's state-run oil company, Petroleo Brasileiro SA, said on Friday that it had discovered new, good-quality oil deposits in the so-called sub-salt region.
Reuters - Britain's biggest oilfield Buzzard will undergo 25 days of planned maintenance starting in late July, its operator Nexen confirmed on Friday. During
By Henning Gloystein, Reuters Ukrainian plans for expanding Black Sea natural gas output have been thrown into doubt by Russia's seizure of Crimea, a region
McDermott International, Inc. informs that one of its subsidiaries has been awarded a large engineering, procurement, construction and installation ("EPCI") project by an operator in the Arabian Gulf.
EMAS, the operating brand of Ezra Holdings, a global offshore contractor and provider of integrated offshore solutions to the oil and gas industry, today announced
The Harwich Dock Company (HDC) and Offshore Marine Management (OMM) have secured a significant long term contract with DONG Energy, one of the leading energy groups in Northern Europe,
Deep-ocean exploration company Odyssey Marine Exploration, Inc., has been awarded the exclusive contract to conduct an archaeological excavation and recover the
Acteon company InterMoor completed decommissioning operations of the Innovator platform in the Gulf of Mexico's Gomez field, Mississippi Canyon Block 711. Conducted in water depths of 910 meters,
Salvage work to remove a capsized drilling rig lying in shallow waters off Angola's new liquefied natural gas (LNG) plant is due to start in the second quarter, Italian oil services firm Saipem said.


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The new legal head of Union Pacific aims to keep the company on the rails
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There are 3 private schools in zipcode 08550, New Jersey, serving 270 students.
Minority enrollment is 62% of the student body, which is more than the New Jersey state average of 42%.
The student:teacher ratio of 19:1 is higher than the state private school average of 11:1.
- Town School Students Grades
Princeton Junction New Horizon Montessori Of Princeton Junction
Montessori School60 students | Gr. PK-K
Zipcode Statistics
About This Zipcode (08550) | Zipcode Statistics | (NJ) State Statistics |
Population (Approximate) | 19,445 people | 8,686,416 people |
Age 25+ w/College Degree | 79% | 35% |
Median Household Income | $166,283 | $78,182 |
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Updated February 16, 2014
Here are 25 factoids about private K-12 schools written to make you want to explore the private school world further and in depth.
Why does one private school cost less than another in the same area? Why are certain areas of the country much more expensive than others? We examine tuition costs around the nation.
Aren't girls more likely to succeed in a coeducational setting? Are there advantages to educating girls in a single sex setting? We look at some answers.


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