Thomas Perkins

Dr. Surya Pratap Deopa Wins 2025 BioFrontiers Outstanding Contribution Award

Steven Burrows — Sat, 30 Aug 2025 01:07:56 +0000

Dr. Surya Pratap Deopa Wins 2025 BioFrontiers Outstanding Contribution Award Steven Burrows Fri, 08/29/2025 - 19:07

Steven Burrows / JILA Science Communications Manager

Dr. Surya Pratap Deopa

JILA is proud to announce that Dr. Surya Pratap Deopa, Postdoctoral Fellow in the lab of JILA Fellow and MCDB Professor Tom Perkins, has been honored with the 2025 BioFrontiers Outstanding Contribution Award. This prestigious recognition celebrates Dr. Deopa’s impactful research contributions within the BioFrontiers community.

Dr. Deopa was recognized for his innovative technical advances that revitalized a previously stalled research project, significantly enhancing both the data quality and throughput of cutting-edge atomic force microscopy (AFM) experiments. His work has not only pushed the boundaries of experimental precision but also established molecular dynamics simulations as a powerful computational companion to AFM measurement interpretation.

Beyond his technical achievements, Dr. Deopa has distinguished himself as a generous and collaborative colleague, contributing meaningfully through both formal partnerships and informal scientific exchanges. His dedication to fostering a supportive and productive research environment exemplifies the values of the BioFrontiers Institute.

As part of the award, Dr. Deopa receives a certificate of appreciation and a $1,000 prize in recognition of his outstanding contributions.

Dr. Surya Pratap Deopa, Postdoctoral Fellow in the lab of JILA Fellow and MCDB Professor Tom Perkins, has been honored with the 2025 BioFrontiers Outstanding Contribution Award.

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Probing Proton Pumping: New Findings on Protein Folding in bacteriorhodopsin (bR)

Steven Burrows — Mon, 05 Feb 2024 18:31:50 +0000

Probing Proton Pumping: New Findings on Protein Folding in bacteriorhodopsin (bR) Steven Burrows Mon, 02/05/2024 - 11:31

Kenna Hughes-Castleberry / JILA Science Communicator

Diagram of the experimental setup (not to scale): Photoactivation of a single molecule of bR. Image Credit: Steven Burrows / JILA

When it comes to drug development, membrane proteins play a crucial role, with about 50% of drugs targeting these molecules. Understanding the function of these membrane proteins, which connect to the membranes of cells, is important for designing the next line of powerful drugs. To do this, scientists study model proteins, such as bacteriorhodopsin (bR), which, when triggered by light, pump protons across the membrane of cells.

While bR has been studied for half a century, physicists have recently developed techniques to observe its folding mechanisms and energetics in the native environment of the cell’s lipid bilayer membrane. In a new study published by Proceedings of the National Academy of Sciences (PNAS), JILA and NIST Fellow Thomas Perkins and his team advanced these methods by combining atomic force microscopy (AFM), a conventional nanoscience measurement tool, with precisely timed light triggers to study the functionality of the protein function in real-time.

“The energetics of membrane proteins hast been challenging to study and therefore not well understood,” explained Perkins. “Using AFM and other methods, we can create ways to look into this further.” Armed with a better understanding of the energetics of these proteins, chemists can design drugs that are more potent towards specific symptoms and illnesses caused by protein misfunction

Measuring Millisecond Protein Dynamics

While bR is a microscopic protein, it can be seen by the naked eye, and even in satellite images, when archaeon microorganisms bloom, they leave vast amounts of it as residue in salt-water ponds. “The ponds become filled with what's called Halobacterium salinarum, the parent organism of bacteriorhodopsin,” Perkins elaborated. “These ponds are used to harvest salt, and because they’re warm and salty, the bacteria love to grow there.”

At the microscopic level, bR works with other membrane proteins to produce energy for the cell by creating a proton gradient on one side of the cell membrane, which ushers the proton through to the other side of the membrane. bR does this by folding and unfolding its helices into specific shapes to control how many protons pass through the membrane. During this process, the proton migration produces chemical energy in the form of adenosine-tri-phosphate (ATP).

For Perkins and his co-author David Jacobson (a former JILA postdoctoral researcher and now an assistant professor at Clemson University), bR presented an opportunity to design a new experimental method for looking at real-time functional energetics. To study proteins like bR, Jacobson, and Perkins utilize AFM, which acts like a tiny finger to pull on the protein gently, which helps the AFM to feel the protein’s surface, mapping out its structure and giving a better understanding of how the protein folds.

Because bR’s folding processes are triggered by light, Perkins and Jacobson added a lighting element to the AFM procedure. “We had this clever idea to glue super thin green LEDs—which trigger the bacteriorhodopsin—to a metal puck, which we can attach to the AFM,” Perkins elaborated. “These green LEDs are also cheap, like $1.00 apiece or $1.50 apiece. Compared to our AFM cantilever, which costs about $80 apiece, throwing away a $1.50 LED is hardly something we worry about.”

With this inexpensive add-on to their AFM, Perkins and Jacobson could induce the bR to fold and unfold with millisecond precision. After collecting their data, the researchers found that the protein correctly folded 60% of the time, allowing the protons to pass through the membrane.
To verify the energetics and real-time function of the protein folding, the scientists mutated the bR protein to remain always in the “open” or unfolded state. Using their new experimental setup, they could reproduce findings similar to what they observed before in the “open” phase of the bR photocycle.

“In biology, you might see something, but you need to ask, am I seeing what I think I'm seeing?” Perkins said. “So, by making a mutation and seeing the effect that we expected, we have increased confidence that we're really studying the process we think we are studying.”

The Mystery of the Misfolded Protein

While Perkins and Jacobson observed proper folding 60% of the time, the other 40% of cases surprised them, as the protein misfolded but could still pump a proton through the membrane. “The misfolding is actually stabilizing,” added Perkins. “And that was really surprising.” In many cases, protein misfolding does not result in stabilization.

Due to the energetic stabilization, Perkins and Jacobson theorized that the bR’s structural helices weren’t separating properly to provide a completely open tunnel for the proton, though it still wiggled through, a process difficult to detect with AFM imaging.

Trying to understand the underlying mechanisms for the misfolding better, Perkins and Jacobson lowered the force on the AFM pulling assay to zero to see if this would coax the protein to fold correctly. However, the results remained the same: 40% of cases resulted in misfolding.
These results, with the same amount of misfolding, puzzled the researchers. While Perkins and Jacobson couldn’t identify the cause of these misfolding cases, they hope to investigate further. Now, they are interested in seeing what the rest of the biophysics community makes of these results.

“There could be more subtle effects, or maybe some new science there,” Perkins added. “It could be that there's a pathway that perhaps people haven't been able to see before.”

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Celebrating 60 Years of JILA

Steven Burrows — Tue, 12 Jul 2022 19:46:03 +0000

Celebrating 60 Years of JILA Steven Burrows Tue, 07/12/2022 - 13:46

Kenna Hughes-Castleberry / JILA Science Communicator

Groundbreaking ceremony for the new JILA laboratory wing and the 10-story office tower, February 25, 1965 (l-r) Lewis Branscomb, Chair of JILA; Donald Hornig, Science Advisor to President Lyndon Johnson; Joseph Smiley, CU President, and Robert Huntoon, Director of the Institute for Basic Standards at NBS. Credit: University of Colorado Publications Service

This year, JILA celebrates its 60th anniversary. Officially established on April 13, 1962, as a joint institution between the University of Colorado Boulder and the National Institute of Standards and Technology (NIST), JILA has become a world leader in physics research. Its rich history includes three Nobel laureates, groundbreaking work in laser development, atomic clocks, underlying dedication to precision measurement, and even competitive sports leagues. The process of creating this science goliath was not always straightforward and took the dedication and hard work of many individuals.

The idea for JILA came from a 1958 meeting of the International Astronomical Union in Moscow. Dr. Lewis Branscombe, a founding member and the head of the atomic physics department of the National Bureau of Standards (NBS, which would later become NIST) proposed an institution for laboratory astrophysics to co-founder, and professor of astrophysics at ��Ҵ�ý�ƽ��, Dr. Richard Thomas. As Branscombe was directly funded by the government, and Thomas by the university, they realized that the best option for such an institution would be a joint establishment between the two entities. Together with the third founding member, Dr. Michael Seaton, a theorist at University College London, they toured nine universities in 1960 and 1961 to find a suitable home for the institution. Finally, the trio settled on ��Ҵ�ý�ƽ�� as the location for their new institution. This was in part due to the President of the university at the time, Quigg Newton, who was supportive of their cause.

In April of 1962, JILA was founded, standing for the Joint Institute of Laboratory Astrophysics. Laboratory astrophysics was of particular interest to the International Astronomical Union as it focused on topics ranging from studying the Sun’s visible light spectrum to developing retroreflecting mirrors.

Aerial view of the newly completed JILA tower situated on the University of Colorado at Boulder campus, 1967. Credit: University of Colorado Publications Service

Trying to find a building on the campus to house JILA, ��Ҵ�ý�ƽ��'s Chief Financial Officer Leo Hill worked with both the NBS and National Science Foundation to pay rent for two floors of the old State Armory building. The NBS also provided funds for laboratory equipment. JILA began construction for its own building shortly after, with the first part, the B-wing, completed in 1966, and the JILA tower finished in 1967. JILA added two more wings to its building, the S-wing (dedicated in 1988), and the X-wing in 2011. There are plans for further expansion with a Y-wing to be built, but nothing is currently in process.

Setting up in the Old Armory building, the JILA scientists (by the early 1960s there were seven scientists at JILA) established several rules that would help JILA function properly. These rules centered around leadership, funding, and fellowships. It was negotiated that with JILA's creation, the NBS would provide instruments and laboratories, while ��Ҵ�ý�ƽ�� would provide researchers and land for the institution. With its unique agreements and roles, JILA’s institute was relatively free to make its own way scientifically. In 1961, ��Ҵ�ý�ƽ��'s Board of Regents approved the title of professor adjoint for any NBS faculty who taught classes at the University. This further solidified the connection between the university and the NBS and made it easier for JILA to attract new scientists.

One of these scientists was Dr. John “Jan” Hall, who was an expert in laser systems and who had previously worked at the NBS location in Washington DC. Though JILA was created during the height of the space race, with the idea being to help the U.S. win this race, Hall helped move JILA in a new direction with laser development. JILA still had ties to astrophysics and astronomy, such as developing lunar lasers for the space race, but the times were changing, and JILA was shifting its research focus to other topics.

Close up of entrance to the old State Armory Building, JILA’s first home on the University of Colorado campus. Credit: JILA

By the late 1960s into the 1970s, JILA's fields were expanding to include laser physics, atomic physics, and others. Hall, at the helm of this shift, helped develop the first high-precision lasers at JILA. His work on these systems would later garner him a Nobel Prize in Physics in 2005.

The 1970s brought a deeper sense of community within JILA, as it was described as a “fun, fast, and free-spirited place.” It was during this time that, along with rafting or ski trips, JILAns also created their own sports leagues, including softball and volleyball. In 1974, JILA elected its first female chair, Katharine Gebbie. Gebbie would later move over to NIST and become their Chief of Quantum Physics Division in 1988, but before she did, she helped recruit and support other female JILA Fellows in JILA. The fields of study within the institution also diversified, as in 1977, the NBS changed the name of their JILA division to the “Quantum Physics Division,” predicting the role that quantum physics would play in JILA'S future.

In the 1980s, JILA was beginning to modernize with the help of the early internet. Thanks to JILA fellow Judah Levine and colleagues the Automated Computer Time Service was brought online, accessible through dial-up modems. This was a monumental first step in modernizing time transfer, as users had access to atomic clock time. By 1988, JILA’s population consisted of more than 200 people, including 23 Fellows. It was also the year that the National Bureau of Standards (NBS)became the National Institute of Standards and Technology (NIST), changing its infrastructure and goals.

More breakthroughs occurred in the 1990s, as JILA once more shifted its mission to reflect NIST's mandate for developing precision measurement, and educating graduate students in future technology. In 1994, JILA had become more than its previous name implied, and dropped the definition of its acronym as the Joint Institute of Laboratory Astrophysics in acknowledgement of the broader scope of science conducted there. In 1995, Nobel-prize winning research was performed by JILA Fellows Carl Weiman and Eric Cornell, as they discovered the Bose-Einstein-Condensate (BEC), a special state of matter helpful for studying quantum dynamics. Nineteen ninety-six brought the 500th Fellows’ meeting, as well as diversity initiatives to make the community more inclusive.

JILA scientists studying in the library on the 10th floor of the JILA tower. Credit: JILA

The 1990s was also an important decade for laser physics at JILA. By 1997, JILA identified seven fields of physics that researchers were studying: atomic physics, chemical physics, materials physics, optical physics, molecular physics, precision measurement, and astrophysics. Laser physics was an underlying study in many of these fields. In 1999, JILA Fellows Margaret Murnane and her husband Henry Kapteyn created what was then the fastest tabletop laser system. That same year, Fellows Jan Hall and Jun Ye developed the first optical frequency comb laser, a tool used by researchers to study optical physics. With these important developments, JILA was quickly establishing a reputation as a world leader in physics research. This reputation boosted JILA's success, as, by the late 1990s, the institution was producing 5–10% of the nation's new Ph.D. graduates in atomic, molecular, and optical (AMO) physics.

The success continued into the 2000s, as the decade brought three Nobel Prizes to JILA. In 2001, Eric Cornell and Carl Weiman were awarded the Nobel Prize in Physics for their work in 1995 on the BEC. The State of Colorado established March 6th as “Carl Weinman and Eric Cornell day” to honor the scientists. A few years later in 2005, Jan Hall also received the Nobel Prize in Physics for his work on laser systems and for developing the first optical frequency comb. JILA also added biophysics as a new field of study, which was helped by the addition of JILA Fellow Thomas Perkins, who worked in this area.

Three JILA Fellows were honored during the 2010s by being selected by then-President Obama to fill important leadership positions within scientific governing groups, including the White House Office of Science and Technology Policy. These Fellows included Carl Weinman, Margaret Murnane, and Carl Lineberger. JILA also celebrated its 50th birthday on April 13th, 2012.

JILA tower (circa 1966) under construction in front of the recently completed laboratory wing, now known as the B-wing of the Duane Physical Laboratories complex. Credit: JILA

Since then, JILA Fellows have received many prestigious scientific awards and grants. The decades of graduate students and postdoctoral researchers who have worked at the institution have gone on to lead successful careers and scientific efforts for other institutions around the world. JILA has also helped spawn many spin-off companies, including 12 companies based in Colorado. These companies range in their products and technology and include companies such as ColdQuanta, Hall Stable Lasers, High Precision Devices, KM Labs, Vescent, to name a few.

With 60 years of scientific research and groundbreaking discoveries, and many successful scientific careers launched, hundreds of lives impacted, it is no surprise that JILA continues to be a global leader in physics research and a pillar within the scientific community. As JILA celebrates its 60th anniversary this year, we look not only to past accomplishments but also to the future, excited to be carrying on such a rich and fulfilling legacy.

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JILA Fellows Thomas Perkins and Graeme Smith win the 2021 Outstanding Postdoc Mentor Award

Steven Burrows — Thu, 16 Sep 2021 20:25:09 +0000

JILA Fellows Thomas Perkins and Graeme Smith win the 2021 Outstanding Postdoc Mentor Award Steven Burrows Thu, 09/16/2021 - 14:25

Kenna Hughes-Castleberry / JILA Science Communicator

Photo of JILA Fellows Graeme Smith and Thomas Perkins

JILA Fellow Thomas Perkins has been awarded the 2021 Oustanding Postdoc Mentor Award. This award recognizes mentors who have gone above and beyond to support their postdocs. Perkins was nominated by postdoc David Jacobson, who praised Perkins' effort to help Jacobson apply and receive the prestigious NIH K99 “Pathway to Independence” Award.

JILA Fellow Graeme Smith also won the 2021 Outstanding Postdoc Mentor Award, being nominated by ��Ҵ�ý�ƽ�� postdoc Vikesh Siddhu and former ��Ҵ�ý�ƽ�� postdoc, Felix Leditzky. Leditzky said Smith “played an integral part in guiding me through the process and helping me achieve this career goal. I aim to pay forward the trust and support that I received from him.”

JILA Fellow Thomas Perkins has been awarded the 2021 Outstanding Postdoc Mentor Award. This award recognizes mentors who have gone above and beyond to support their postdocs. Perkins was nominated by postdoc David Jacobson, who praised Perkins' effort to help Jacobson apply and receive the prestigious NIH K99 “Pathway to Independence” Award.

JILA Fellow Graeme Smith also won the 2021 Outstanding Postdoc Mentor Award, being nominated by ��Ҵ�ý�ƽ�� postdoc Vikesh Siddhu and former ��Ҵ�ý�ƽ�� postdoc, Felix Leditzky. Leditzky said Smith “played an integral part in guiding me through the process and helping me achieve this career goal. I aim to pay forward the trust and support that I received from him.”

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The Forces involved in Folding Proteins

Steven Burrows — Mon, 22 Mar 2021 19:10:56 +0000

The Forces involved in Folding Proteins Steven Burrows Mon, 03/22/2021 - 13:10

Kenna Hughes-Castleberry / JILA Science Communicator

Model of the type three secretion system in Salmonella Bacteria. Image credit: Steven Burrows / JILA

Washing your hands after cracking an egg or touching raw chicken may seem common sense, as the possible resulting bacterial infections have been thoroughly studied. Yet, researchers at JILA have found something surprising and ground-breaking about the physics of bacterial infections. In a new paper, JILA physicist Thomas Perkins collaborated with CU Biochemistry Prof. Marcello Sousa to dissect the mechanisms of how certain bacteria become more virulent. The research brings together the Perkins lab expertise in single-molecule studies and the Sousa lab expertise in the type III secretion system, a key component of Salmonella bacteria.

The type III secretion system is shaped like a syringe, with a needle that’s only two nanometers in diameter (for reference an atom is around 0.1 to 0.5 nanometers in diameter). Through these needle-like structures, bacteria pump effector proteins directly into host cells, such as humans or livestock for example, to take control of various host cell functions, including suppressing the cell’s immune system or hijacking its DNA and RNA machinery. In order for effector proteins to pass into the host cell, the proteins must be partially unfolded as the 2-nm pore is only a few atoms wide. Proteins that don’t unfold clog the needle leading to the prevailing model that effector proteins have low thermodynamic stability and so are often unfolded while proteins that clog the needle have high stability.

Protein folding and protein stability

Protein folding has been studied for decades. In general, proteins fold into their most stable state. Historically, protein stability is measured using thermodynamics. According to JILA Fellow and Adjoint professor in Cellular, Molecular and Developmental Biology, Thomas Perkins, “The classic way biochemists measure protein stability is either to increase temperature or a denaturant, like urea, to a high level and then lower it back down. If a protein unfolds and refolds under these conditions, then you can measure the thermodynamic stability of the protein.” When applying this technique to effector proteins, the team found effector proteins have typical thermodynamic stabilities. Indeed, thermodynamic stabilities are indistinguishable from proteins that clog the needle. The team then looked to see if mechanical force might play a role.

Applying Force to Unfold Proteins

The Perkins lab has had lots of experience unfolding proteins using atomic force microscopy (AFM). They developed modified cantilevers that have improved force precision and stability. Nonetheless, it still took four years of effort to develop the ability to reliably apply force to the two model effector proteins (SptP and SopE2), larger and more complicated proteins than the model proteins the Perkins lab has studied in the past. The team needed to anchor the protein down to a surface—without it randomly sticking—and then pull on it from a specific point. After this development, they were finally able to look at the mechanics of effector protein unfolding.

Their data showed that the two model effector proteins were mechanically labile, meaning that they would easily unfold at low forces. Perkins said: “what is remarkable is that these proteins unfold at very low forces compared to a peer set of other proteins. That led us to the hypothesis that it is the mechanical unfolding of these proteins that governs whether a protein can actually make it through the type III secretion system.” Prior work by others showed that proteins known to clog the needle unfold at high force. Perkins went on to say “If our hypothesis is correct, most, if not all, effector proteins should unfold at low force.”

Evolution and Unfolding Forces

From their data, the team realized that the mechanics of how these proteins unfold might shed light on another outstanding question in the field. Proteins that have the same structural fold and carry out the same function are homologues. Homologues usually have very similar protein sequences, the linear list of amino acids that make up the protein. However, protein sequences of effector proteins have virtually no similarity with homologues that are not secreted. First author Marc-André LeBlanc suggested that it is the evolutionary pressures for these proteins to unfold at such low forces but maintain typical thermodynamic stabilities so they refold efficiently that causes their sequences to look so different. Perkins added. “Looking forward, one of the things we’re planning to investigate is if homologues that don’t go through the type III secretion system are more mechanically robust.”

If these non-secreted homologues are more mechanically robust, then there was likely evolutionary pressure for these effector proteins to diverge from their homologues in the case of folding mechanics. This possible evolutionary pressure may provide insight into the factors that control protein folding and stability, which is what the Perkins and Sousa Labs are currently studying by using AFM to investigate targeted mutations in their model effector proteins.

The research is still ongoing. LeBlanc is looking forward to using the newly developed technology to not only expand the research, but give the study possible medical applications. “Everything is based on basic research at this point.” LeBlanc said. “But you can play that forward and start to think about applications. There are some labs using the type III secretion system as a protein delivery mechanism. You can certainly imagine if you have a protein that you want to get into another cell, this might be a good way to do it.” While the study is still in early development, the researchers are excited to see the applications of their newly developed technologies and the impacts of their work on the field of biophysics as a whole.

This paper was published in Proceedings of the National Academy of Sciences on March 23, 2021.

In a new paper, JILA physicist Thomas Perkins collaborated with CU Biochemistry Prof. Marcello Sousa to dissect the mechanisms of how certain bacteria become more virulent. The research brings together the Perkins lab expertise in single-molecule studies and the Sousa lab expertise in the type III secretion system, a key component of Salmonella bacteria.

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Grabbing Proteins by the Tail: Using AFM tools, scientists can gently pull apart protein chains

Steven Burrows — Tue, 11 Aug 2020 16:50:49 +0000

Grabbing Proteins by the Tail: Using AFM tools, scientists can gently pull apart protein chains Steven Burrows Tue, 08/11/2020 - 10:50

Rebecca Jacobson / JILA Science Communicator

The Perkins Group uses atomic force microscopy tools to "unravel" amino acids in cell membrane proteins. Image credit: Steven Burrows / JILA

Cells are surrounded by a membrane containing carefully folded proteins. Those membrane proteins interact with the watery environment inside and outside the cell and the fatty environment of the membrane that keeps the inside and the outside of the cell separated.

That gives them an important role-they are how the inside of the cell talks to the outside of the cell, allowing viruses to attack or letting in medications to treat disease, said David Jacobson, a post-doc in the Perkins Group at JILA. That's why roughly half of proposed and current drugs target membrane proteins.

Studying these membrane proteins is crucial for biomedical research, but they are tricky to measure in a lab. Many biochemical techniques for measuring membrane proteins remove them from their native bilayer by washing them in a detergent-potentially altering how they interact with their surroundings or how they are folded.

The Perkins Group has worked out a method of measuring membrane proteins using atomic force microscopy (AFM) tools. In a new study published on August 5 in Physical Review Letters, the team uses AFM to unfold and refold bacteriorhodopsin-a membrane protein found in microorganisms called archaea-precisely measuring the free energy it takes to do so and demonstrating a new means of studying and manipulating the proteins.

Unfolding and refolding

While a membrane protein is linear chain of amino acids, it folds into a unique three-dimensional structure. This three-dimensional structure depends on interactions between amino acids that make up the protein, which is what the Perkins Group wants to measure. One way to measure the strength and nature of those interactions is to grab the end of the protein chain with the AFM tip and pull, unfolding the protein structure.

"If we exert enough force, it starts to unfold," Jacobson explained. "The unfolding force is related to the strength of the interaction that holds the proteins together. The stronger the stabilizing interactions, the more force it takes to pull it apart."

Biophysicists, including the Perkins Group, have been doing this for years, Jacobson said-usually with brute force, thrusting the AFM tip into the membrane protein. That works, but it doesn't stick very well, which makes it difficult to perform multiple measurements, Jacobson added. Previous studies measured the force to unfold the entire structure and divided it among the 248 amino acids in the protein.

To make a more precise measurement, the Perkins Group chemically treated the 10-nanometer AFM tip so it bonds with the amino acids at the end of the protein. Then, they gently touch the tip to the very end of the protein chain and pull-only enough to unravel five or eight amino acids.

As the amino acids unfold, the cantilever holding the AFM tip flexes. Then the scientists relax the tension, allowing the small segment of amino acids to fold back into their shape.

Measuring the cantilever's flex with each unfold and refold tells scientists how much free energy is required to manipulate this protein structure. And this method can be done in the membrane's native bilayer, replicating its native environment.

Rebuilding new structures

Precisely manipulating a few amino acids opens the door to understanding an individual amino acid's role in the cell's function, Jacobson pointed out.

"All the chemistry of the cell is driven by proteins and a lot of the structure of the cell is made of proteins. The proteins don't work if they are not folded up into their correct three-dimensional structure. The three-dimensional structure depends on the interactions between the amino acids," he said.

This method also allows scientists to make changes and mutations to those amino acids, and study the effect of those changes.

"In principle, this technique can be applied to any membrane proteins...It opens the door to measuring, making mutation studies to measure the contribution of these individual amino acids to the overall stability of the protein," Jacobson added.

This study was supported by the National Science Foundation, including the Physics Frontier Center grant.

A protein's function within a cell relies on how it folds, unfolds, and refolds. Using atomic force microscopy tools, the Perkins Group can precisely measure the free energy it takes to unfold and refold a few amino acids in the protein, which opens the door to making more precise measurements and alterations to a cell's membrane proteins.

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DNA imaging, ready in five minutes

Steven Burrows — Tue, 16 Jul 2019 18:37:00 +0000

DNA imaging, ready in five minutes Steven Burrows Tue, 07/16/2019 - 12:37

Rebecca Jacobson / JILA Science Communicator

A new technique from the Perkins group allows crisp, clear AFM images of DNA. The best part? It's ready in 5 minutes. Image credit: Steven Burrows / JILA

It’s tricky to get a good look at DNA when it won’t stick to your slide, or lay down in a straight strand.

CU biochemist Tom Cech came to Tom Perkins’ atomic force microscopy lab with this very problem. His group was trying to understand how certain proteins interact with DNA. They knew their proteins interacted with DNA but they couldn’t get a good image of that interaction in liquid, DNA’s native environment.

“You have this nanoscale string in a ball shape that's undergoing thermal fluctuations, and then take this three-dimensional configuration, and squish it down really fast onto a surface,” Perkins explained. That squished DNA is often an uninterpretable blob.

“I kind of think of it like a bowl of spaghetti noodles. If you drop them on the floor, they'd be kind of all globbed up and in weird shapes,” explained Patrick Heenan, a graduate student in Perkins’ group.

The goal was to develop a process that would separate those globs into individual noodles. Heenan started working on this problem about two years ago, and he developed a technique to get your slide perfectly prepared to image a DNA sample. The best part? It’s fast – ready in just five minutes.

Tiny globs of DNA

DNA is about 2 nanometers wide – 100,000 times thinner than a sheet of paper. To get a good look at it, you need to put it on a slide that is truly flat. Mica, a light, soft silicate mineral, is a great candidate for a slide. All it takes to get a flat, thin sheet of mica is a piece of Scotch tape and some deft hands, Heenan explained.

“The beauty is it's super, super flat, so it is the perfect substrate,” Heenan said. “You can see all the atoms, and in some cases, you can see voids in the lattice.”

However, mica and DNA are both negatively charged; they repel each other. Scientists had tried changing the surface of the mica to make the DNA stick – soaking it for hours or days in various coatings and rinsing it with distilled water.

But the DNA still ends up in globs. When the apex of the atomic force microscope (AFM) tip runs over the DNA, the picture comes out blurry. Scientists can’t see how proteins or other molecules are interacting with the DNA. Many had given up on imaging DNA in liquid on bare mica, figuring it didn’t work or using coatings that made the images blurrier but still yielded globs. So Heenan started digging through the literature to see what others had tried.

“I read lots of papers, and I prepped hundreds of samples,” Heenan said. “It was a little bit like whacking through the weeds.”

Ready in five

It turned out soaking the mica for too long was part of the problem. Plus, the DNA wasn’t able to equilibrate on the surface, resulting in the squished ball shapes rather than nice, separate strands. “If you let it sit for a long time, you're actually losing some of the surface charge and impurities in the water are being sucked down into the surface,” Perkins explained.

Here’s how the improved process works. The mica is pre-soaked in a concentrated nickel-salt solution, then rinsed and dried. Then the DNA is bound to the mica with a solution of magnesium chloride and potassium chloride. Those conditions closely resemble the salts found naturally in cells, allowing the proteins and DNA to behave the way it normally would. Finally, the mica is rinsed with a nickel-chloride solution, which helps the DNA structure stick to the mica more tightly.

“It's kind of like the whole surface is uniformly sticky, so it can stick wherever it wants, and therefore it's easier for it to move around,” Heenan explained. “Everything is equally sticky.”

And it’s all ready for imaging in five minutes. Not only is it ready, but it delivers a clear picture – so clear that the double helix of the DNA is visible.

This development will help scientists study how DNA, protein and other molecules interact, as well as how DNA repairs itself.

“Patrick took on the challenge to image in liquid at biochemically-relevant conditions rather than in air, which, prior to Patrick's work, we and others would have assumed was impossible,” Perkins said. “What Patrick has developed is something that once you learn the eight steps, almost anybody can do it. It's really simple. It uses common salts, it takes five minutes, and you get higher signal-to-noise ratio, so basically, all sorts of good things.”

The study was published in ACS Nano on April 2, 2019, and was supported by the National Science Foundation Physics Frontier Grant and NIST.

Researchers at JILA have developed a fast, simple method to prepare samples that enhances DNA imaging. The results are so clear that the double-helix shape of DNA can be seen clearly.

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Thomas Perkins wins Gears of Government Award

Steven Burrows — Tue, 14 May 2019 01:22:47 +0000

Thomas Perkins wins Gears of Government Award Steven Burrows Mon, 05/13/2019 - 19:22

Rebecca Jacobson / JILA Science Communicator

Sometimes, focusing on the little things can gain you recognition. Dr. Thomas Perkins won a Gears of Government Award for his work in atomic force microscopy. The President's Office gives the Gears of Government Awards to honor those in the federal workforce who have made a profound difference in American lives.

“Whether they are defending the homeland, inspecting our food, making scientific discoveries, or managing cyber risks, Federal employees underpin all the operations of our government and touch nearly every aspect of our lives,” Office of Management and Budget’s Deputy Director for Management Margaret Weichert said in a press release. “These awards recognize not just the front-line mission employees, but also those teams and individuals that are strengthening our country to be a more modern, effective government to better serve their fellow citizens.”

As a JILA Fellow and NIST physicist, Perkins' lab falls under the Department of Commerce. His lab has focused on single molecule measurements of biological systems, like proteins, DNA and RNA. To get those measurements, his team has advanced atomic force microscopy in the last decade. Their advances in AFM technology have led to better understanding of biology, such as completely mapping the energy landscape of a hairpin-shaped bend in HIV RNA, and understanding illnesses from cancer to brain disease. With this knowledge, scientists can develop better drugs and therapies. Perkins emphasized that their work is a team effort.

“I am delighted to receive this award that reflects a decade of innovation by members of my lab,” Perkins said.

Dr. Thomas Perkins won a Gears of Government Award for his work in atomic force microscopy.

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Pulling apart HIV: Advance AFMs Allow Study of the HIV Hairpin

Steven Burrows — Wed, 27 Feb 2019 19:53:07 +0000

Pulling apart HIV: Advance AFMs Allow Study of the HIV Hairpin Steven Burrows Wed, 02/27/2019 - 12:53

Catherine Klauss / JILA Science Communicator

Using a cantilever AFM (gray), JILA researchers are able to unfold and refold the HIV hairpin, a bend in the HIV RNA molecule which helps the virus take over the infected cell’s protein-making machinery. By leveraging a series of advancements to AFMs, researchers can now measure folding forces (red line) much more precisely and thereby determine free energy landscapes (blue line), which map the energy both of and between folded states. Image credit: Steven Burrows / JILA

There are many molecules within the virus known as HIV, but only one is shaped like a hairpin. This molecule, aptly named the HIV RNA hairpin, allows the virus to create its own proteins from host resources, which is key to the virus’s ability to take over an infected cell.

Scientists having been studying the details of this virus for decades in an attempt to improve diagnostics and treatments for AIDS and other HIV-induced diseases. And now, JILA researchers have demonstrated a much easier, faster and more precise way to understand the structure and function of the HIV RNA molecule, especially the HIV RNA hairpin. Furthermore, the techniques developed for this research promise to allow a wider range of users to study similar biological molecules, as they are built upon commercially available and user-friendly atomic force microscopes, or AFMs.

AFM Advances

AFMs are high-resolution microscopes that can resolve structures as small as atoms. But on top of imaging, AFMs can also pull apart, or unfold, biological molecules such as proteins and nucleic acids like DNA and RNA.

Over the last decade, JILA Fellow Dr. Thomas Perkins and his team have focused on improving AFM technology for biological applications. Their advancements in cantilever shape provided enhanced time resolution and force precision. Their advancements in chemistry enabled individual molecules to be stretched end-to-end. And their advancements in instrument automation allowed researchers to unfold and refold the same individual molecule over a thousand times (whereas previously molecules could refold only a handful of times).

According to Perkins, his team’s advancements to AFM technology allow more researchers to probe biological molecules.

“It’s a question about accessibility,” said Perkins. “The benefit to doing these experiments on a commercial AFM is you can train an undergraduate to do the experiment.”

Energy Landscapes

Equipped with an advanced AFM, Perkins quickly pushed past old limitations of biological probes.

“We’ve spent all of this time overcoming various technical issues—cantilevers, surfaces, alignment—and now it’s this exciting time where we are doing applications, and we are doing things that people never expected you’d be able to do with an AFM,” said Perkins.

And one of the things never expected from AFMs was the ability to probe a folding molecule with the precision required to map out the energy landscape, said Perkins.

The energy landscape of a molecule is like a 3D topographic map whose peaks and valleys represent the energy of the molecule’s configuration. Because there are many ways a molecule can fold, there are many paths a molecule could take through this landscape.

And driving the molecule through this landscape is its desire to reduce its energy. Generally, a more folded molecule has a decreased energy. But some folding directions can be stymied by the equivalent of a high-mountain pass, or dam, in the landscape. Likewise, other foldings can be encouraged by sloping gullies. Understanding these complex landscapes—and how molecules navigate them—can help us understand why certain folds occur and why critical proteins may misfold and cause disease.

But diseases are caused by more than just misfolded proteins. Some diseases, like HIV, are caused in part by proteins made by an RNA hijacker called a hairpin.

The HIV Hairpin

The HIV hairpin is a hairpin-shaped bend in the virus’s RNA molecule that enables HIV to use a host’s protein-making machinery to make copies of the virus rather than normal cell proteins. While many viruses have hairpins, HIV’s is particularly elusive.

“This is a really hard hairpin to study,” said Perkins. “It’s particularly small, unfolds at low forces and refolds fast… This is about as challenging as it can get.”

Perkins research, published last September, maps the first full-energy landscape of the HIV hairpin and demonstrates the first instance of an AFM-based probe measuring the full energy landscape of any nucleic acid.

Yet studying this hairpin is not, unfortunately, a direct answer to conquering HIV’s effects on the human body.

“The hairpin is just one step along its lifecycle,” said Perkins, citing entrance, infection, and dormancy as other lifecycle steps. “[But,] the better we understand the life cycle of HIV, the more opportunities there are to develop therapeutics.”

In the future, Perkins hopes to understand the HIV hairpin at an even faster timescale. Currently, Perkins’ group can observe the hairpin fold with 40 microsecond (40 millionths of a second) resolution. But with new advancements, Perkins expects to improve this resolution down to one microsecond. With this resolution, it would become possible to actually watch the RNA unfold.

In addition to Thomas Perkins, this research was completed by JILA postdoc Robert Walder, JILA undergraduates William J. Patten and Ty W. Miller, JILA Visiting Fellow Michael T. Woodside of the University of Alberta and his graduate student Dustin B. Ritchie, and former JILA Postdoc Rebecca K. Montage. The paper was published 20 September 2018 in Nano Letters.

JILA researchers have demonstrated a much easier, faster and more precise way to understand the structure and function of the HIV RNA molecule, especially the HIV RNA hairpin. Furthermore, the techniques developed for this research promise to allow a wider range of users to study similar biological molecules, as they are built upon commercially available and user-friendly atomic force microscopes, or AFMs.

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Tom Perkins elected AAAS Fellow

Steven Burrows — Tue, 27 Nov 2018 20:45:23 +0000

Tom Perkins elected AAAS Fellow Steven Burrows Tue, 11/27/2018 - 13:45

Catherine Klauss / JILA Science Communicator

JILA Fellow Tom Perkins has been elected a Fellow of the American Association for the Advancement of Science (AAAS). Perkins was elected for his pioneering advances in high-resolution studies of single biological molecules.

Perkins is among the 416 AAAS members elected Fellows by their peers. The honor recognizes distinguished efforts to advance science, either scientifically or socially. The newly elected fellows will be presented with an official certificate and a gold and blue (representing science and engineering, respectively) rosette pin at a ceremony on 16 February 2019 at the AAAS Annual Meeting in Washington, DC.

The tradition of electing AAAS Fellows began in 1874 to recognize members for their scientifically or socially distinguished efforts to advance science or its applications. The nomination process for the 2019 Fellow Cycle will open by the end of November 2018.

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