Corporate Banner
Satellite Banner
Scientific Communities
Become a Member | Sign in
Home>News>This Article

NMR Advance Brings Proteins into the Open

Published: Wednesday, June 26, 2013
Last Updated: Wednesday, June 26, 2013
Bookmark and Share
A key protein interaction had eluded scientists’ observation until a team of researchers cracked the case by combining data from four different techniques of NMR.

When working a cold case, smart investigators try something new. By taking a novel approach to nuclear magnetic resonance spectroscopy — a blending of four techniques — scientists have been able to resolve a key interaction between two proteins that could never be observed before. They report on their findings the week of June 24, 2013, in Proceedings of the National Academy of Sciences (PNAS).

The interaction, which the team first described, is nearly universal across all of life. A protein machine called a chaperone takes hold of a disordered smaller protein to help it find its proper folded conformation. In this case, the team set up test-tube experiments where they hoped to watch the capsule-shaped bacterial chaperone GroEL capture a disordered amyloid β (Aβ) protein, a molecule that in humans is central in Alzheimer’s disease.

The two proteins are well studied, but the motions they go through when they first meet — when the open GroEL capsule captures its target — have been invisible to scientists. Electron microscopy and X-ray crystallography are only good for taking snapshots of easily frozen moments in time. NMR is capable of sensing the interactions and kinetics of protein handshakes as they occur, but in some cases any single technique can provide only hints and whispers of what’s going on.

Brown University biologist Nicolas Fawzi, who was a postdoctoral researcher in the group of Marius Clore at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the National Institutes of Health (NIH), worked with co-authors and NIDDK researchers David Libich, Jinfa Yang and Marius Clore to piece together the story of the proteins by combining four different NMR techniques. They figured out what each one could tell them about the interaction and built the case presented in PNAS.

“None of the four techniques alone gave us sufficient information,” said Fawzi, assistant professor of medical science in Brown’s Department of Molecular Pharmacology, Physiology, and Biotechnology. “Only by using them all together would we be able to figure out the structure and motions of Aβ when it was bound to GroEL. By having four indirect measurements together, that was able to give us a complete picture.”

The researchers acted like a team of detectives working on a case in which no single witness saw everything. Instead they found three witnesses, each with something different to contribute, and then one more that could corroborate some of what the others revealed and rule out other possibilities. The NMR techniques they used were lifetime line broadening, Carr-Purcell-Meinboom-Gill (CPMG) relaxation dispersion spectroscopy, and exchange-induced chemical shifts.

“The fourth technique we employed was Dark-state Exchange Saturation Transfer (DEST) spectroscopy, which we had developed in my lab at the NIH in 2011,” said Clore, also the paper’s corresponding author. “We were able to more effectively conduct our research by using that tool to corroborate and extend the information afforded by the other three measurements.”

Bouncing with the chaperone

The mystery debated among molecular biologists was what the GroEL chaperone requires of its captives at the moment they engage. Does it force them into a particular conformation? Does it hold on tightly while it closes its capsule lid around the smaller protein, or does the captive stay in motion at all?

What the team observed is that the GroEL is a permissive captor. It bound Aβ at just two “hydrophobic” sites, leaving the smaller protein to otherwise dangle in a variety of conformations. It also didn’t keep it bound the entire time, letting it instead detach and re-bind. Essentially Aβ would bounce off and on within GroEL’s binding cavity.

“By using these four techniques together we were able to extract information about the structure of the protein while it binds as well as how fast it comes on and off and what it’s doing at each position,” Fawzi said. “Instead of forming more particular structure upon binding it appears to retain great conformational heterogeneity.”

The lifetime line broadening technique, for example, told them that the Aβ was interacting with something big (GroEL), while the CPMG and chemical shift observations combined to show the length of time Aβ spent on GroEL before unbinding, as well as the structural details of Aβ when it was bound to GroEL. DEST provided information that could confirm much of the story of the other techniques.

Fawzi said GroEL’s laid-back approach could be a matter of being able to bind many different proteins in disordered conformations, but also of saving energy. Forcing proteins into a specific conformation just to make and sustain the initial capture would require more energy than it’s worth.

Eventually, in moments after those the team resolved in this study, GroEL closes its lid and encapsulates its target proteins fully, Fawzi said. That’s when it invests in forcing them to fold the right way.

For molecular and structural biologists, the newly proven blend of NMR techniques could open a number of other cold cases of elusive interactions.

“We can now look at how these big machines can do their job while they are working,” Fawzi said. “This is not just limited to this GroEL machine.”

The research was supported by NIDDK Intramural Research Program and supplemented by the NIH Intramural AIDS Targeted Antiviral Program of the NIH Director.

Further Information
Access to this exclusive content is for Technology Networks Premium members only.

Join Technology Networks Premium for free access to:

  • Exclusive articles
  • Presentations from international conferences
  • Over 2,800+ scientific posters on ePosters
  • More Than 4,000+ scientific videos on LabTube
  • 35 community eNewsletters

Sign In

Forgotten your details? Click Here
If you are not a member you can join here

*Please note: By logging into you agree to accept the use of cookies. To find out more about the cookies we use and how to delete them, see our privacy policy.

Related Content

Tissue Engineers Recruit Cells to Make Their Own Strong Matrix
Extracellular matrix is the material that gives tissues their strength and stretch. It’s been hard to make well in the lab, but a Brown University team reports new success. The key was creating a culture environment that guided cells to make ECM themselves.
Tuesday, November 10, 2015
Proteins with ALS, Cancer Role Do Not Assume a Regular Shape
Our cells contain proteins, essential to functions like protein creation and DNA repair but also involved in forms of ALS and cancer, that never take a characteristic shape, a new study shows.
Monday, October 12, 2015
Study Backs Flu Vaccinations for Elderly
Brown University researchers found vaccines well matched to the year’s flu strain significantly reduce deaths and hospitalizations compared to when the match is poor, suggesting that vaccination indeed makes a difference.
Wednesday, August 26, 2015
Web App Helps Researchers Explore Cancer Genetics
Brown University computer scientists have developed a new interactive tool to help researchers and clinicians explore the genetic underpinnings of cancer.
Monday, July 27, 2015
Tapeworm Drug Shows Promise Against MRSA
A new study shows that a drug already approved to fight tapeworms in people, effectively treated MRSA superbugs in lab cultures and in infected nematode worms.
Monday, April 27, 2015
A New Wrinkle For Cell Culture
Researchers at Brown University have developed an advanced technique for cell culturing that uses sheets of wrinkled graphene to mimic the complex 3-D environment inside the body.
Friday, April 24, 2015
Gold By Special Delivery Intensifies Cancer-Killing Radiation
Researchers at Brown and URI have demonstrated what could be a more precise method for targeting cancer cells for radiation.
Wednesday, April 15, 2015
DNA ‘Cage’ Could Improve Nanopore Technology
Scientists at Brown University have designed a nanoscale cage that can trap a single DNA strand and allow before-and-after sequencing of the same DNA strand in research trials.
Wednesday, February 11, 2015
New Technology Makes Tissues, Someday Maybe Organs
A new device for building large tissues from living components of three-dimensional microtissues borrows on ideas from electronics manufacturing.
Wednesday, January 07, 2015
New Research Unlocks a Mystery of Albinism
A team led by Brown University biologists has discovered the way in which a specific genetic mutation appears to lead to the lack of melanin production underlying a form of albinism.
Thursday, December 18, 2014
If CD8 T Cells Take on One Virus, They’ll Fight Others Too
The findings suggest that innate immunity changes with the body’s experience and that the T cells are more versatile than thought.
Saturday, October 25, 2014
A ‘Clear’ Choice for Clearing 3-D Cell Cultures
A new study is the first to evaluate three chemical technologies for making animal tissues see-through side-by-side for use with engineered 3-D tissue cultures.
Thursday, September 04, 2014
Study Proposes New Ovarian Cancer Targets
Researchers from Brown University propose that TAFs may be important suspects in the progression of ovarian cancer.
Friday, March 14, 2014
Gold Nanoparticles Give an Edge in Recycling CO2
It’s a 21st-century alchemist’s dream: turning Earth’s superabundance of carbon dioxide into fuel or useful industrial chemicals.
Monday, November 11, 2013
Fly Study Finds Two New Drivers of RNA Editing
A new study in Nature Communications finds that RNA editing is not only regulated by sequences and structures near the editing sites but also by ones found much farther away.
Thursday, August 08, 2013
Scientific News
High Throughput Mass Spectrometry-Based Screening Assay Trends
Dr John Comley provides an insight into HT MS-based screening with a focus on future user requirements and preferences.
How a Genetic Locus Protects Adult Blood-Forming Stem Cells
Mammalian imprinted Gtl2 protects adult hematopoietic stem cells by restricting metabolic activity in the cells' mitochondria.
Genetic Basis of Fatal Flu Side Effect Discovered
A group of people with fatal H1N1 flu died after their viral infections triggered a deadly hyperinflammatory disorder in susceptible individuals with gene mutations linked to the overactive immune response, according to a recent study.
New Tech Vastly Improves CRISPR/Cas9 Accuracy
A new CRISPR/Cas9 technology developed by scientists at UMass Medical School is precise enough to surgically edit DNA at nearly any genomic location, while avoiding potentially harmful off-target changes typically seen in standard CRISPR gene editing techniques.
The MaxSignal Colistin ELISA Test Kit from Bioo Scientific
Kit can help prevent the antibiotic apocalypse by keeping last resort drugs out of the food supply.
"Good" Mozzie Virus Might Hold Key to Fighting Human Disease
Australian scientists have discovered a new virus carried by one of the country’s most common pest mosquitoes.
Non-Disease Proteins Kill Brain Cells
Scientists at the forefront of cutting-edge research into neurodegenerative diseases such as Alzheimer’s and Parkinson’s have shown that the mere presence of protein aggregates may be as important as their form and identity in inducing cell death in brain tissue.
Closing the Loop on an HIV Escape Mechanism
Research team finds that protein motions regulate virus infectivity.
New Class of RNA Tumor Suppressors Identified
Two short, “housekeeping” RNA molecules block cancer growth by binding to an important cancer-associated protein called KRAS. More than a quarter of all human cancers are missing these RNAs.
Potential Treatment for Life-Threatening Viral Infections Revealed
The findings point to new therapies for Dengue, West Nile and Ebola.
Scroll Up
Scroll Down
Skyscraper Banner

Skyscraper Banner
Go to LabTube
Go to eposters
Access to the latest scientific news
Exclusive articles
Upload and share your posters on ePosters
Latest presentations and webinars
View a library of 1,800+ scientific and medical posters
2,800+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
4,000+ scientific videos