A protein’s ‘dance steps’ affect its biological function, study shows

• Mitch Jacoby

http://acsmeetings.cenmag.org/a-proteins-dance-steps-affect-its-biological-function-study-shows/

A new microspectroscopy technique can track changes in the overall direction of complex protein vibrations. The method could enable researchers to determine how an enzyme responds when an inhibitor binds to it, for instance, or when the enzyme develops a mutation.

“Global vibrations” can be thought of as intricate dance steps performed by proteins. The new technique provides an unprecedented up-close look at how those dance steps shift when a protein’s conformation changes, a process that underpins important biological functions.

At the American Chemical Society national meeting in San Francisco on Tuesday, physicist Andrea G. Markelz of the University at Buffalo, SUNY, reported that by using her group’s technique, anisotropic terahertz microscopy, she and her coworkers observed something unexpected.

Working with grad student Katherine A. Niessen and others, Markelz found that a protein can undergo large changes in these dance steps even though the overall protein vibrational energy hardly changes at all (Biophys. J. 2017, DOI: 10.1016/j.bpj.2016.12.049). That’s noteworthy, Markelz explained during a session sponsored by the Division of Physical Chemistry, because some researchers have long speculated that changes in the directions of these global vibrations can boost the efficiency of biological functions such as enzymatic activity. But little experimental evidence has been available to support that idea.

Related story: Medical imaging turns to oft-neglected part of light spectrum

The Buffalo group now knows why. Researchers had assumed that biologically relevant changes in vibrations would be accompanied by obvious changes in a protein’s vibrational energy states. Because researchers—until now–have been able to only measure energy state distributions, and studies had shown that those didn’t really change, the scientists concluded that the dance steps didn’t change either.

The Buffalo researchers carried out several analyses and found out that those assumptions don’t hold true. First, they used the terahertz microscopy method to study the vibrations of chicken egg-white lysozyme, a natural antibiotic. They compared the free form of the enzyme to the enzyme bound to tri-N-acetyl-D-glucosamine, a compound that inhibits enzyme action. The dance steps of the two forms differed dramatically. Yet inelastic neutron-scattering measurements showed almost no difference in energy.

The team also compared regular lysozyme to a structurally altered form known as a double-deletion mutant. The mutations are located far from the enzyme’s catalytic site and would be expected to have no effect on enzyme action. Yet the mutant mediates catalytic reactions nearly 1.5 times as efficiently as the regular enzyme. The team’s analyses show that the vibrational energy distributions of the two forms are identical. The directions of the vibrations, however, differ markedly, indicating that the mutant’s distinct motions give it a catalytic advantage.

“This is absolutely superb work,” said Steven D. Schwartz, a specialist in theoretical and computational biochemistry at the University of Arizona. Schwartz explained that there is an important ongoing debate regarding the role global vibrations play in the catalytic functions of enzymes. Settling that debate requires direct measurement of these motions while enzymes are carrying out reactions. “That is precisely the promise of this of this work,” he said.

When proteins court each other, dance moves matter

An illustration of the different ways in which proteins vibrate. The figure on the left shows proteins in a clamping motion, while the figure on the right shows proteins in a twisting motion.

By CHARLOTTE HSU

https://www.buffalo.edu/ubnow/stories/2017/03/markelz-protein-dance.html

“The direction of motion — the direction in which different parts of the protein are moving — can really determine how well a protein performs its biological function.”

Andrea Markelz, professor

Department of Physics

At every moment inside the human body, a carefully choreographed dance is being performed.

Proteins shake their bodies and wave their limbs, all with the goal of optimizing their interaction with other molecules, including other proteins. These tiny motions, called vibrations, enable the molecules to change shape quickly to bind to one another, which in turn facilitates tasks like absorbing oxygen and repairing cells.

The body’s efficiency at performing these functions depends on how well proteins can interact.

But what makes one protein a better suitor than another?

A new study sheds light on this question, showing that in this biological courtship, dance moves matter.

“In the past, research on protein vibrations focused a lot on the energy of those vibrations,” says lead scientist Andrea Markelz, professor of physics, College of Arts and Sciences. “But what we found is that the direction of motion seems to matter more. The direction of motion — the direction in which different parts of the protein are moving — can really determine how well a protein performs its biological function.”

The findings help to lay a foundation for the development of drugs targeting molecular vibrations. Such pharmaceuticals would block proteins from carrying out tasks that contribute to disease.

“We conducted the research using a new technique we developed called anisotropic terahertz microscopy (ATM), which reveals how nature exploits protein motions to improve efficiency. We can then optimize these motions for medicine and biotechnology,” says first author Katherine Niessen, a UB PhD candidate in physics.

The research, published on March 14 in Biophysical Journal, was conducted by UB, the University of Perugia in Italy, and Hauptman-Woodward Medical Research Institute. It was funded by the National Science Foundation (NSF).

Foxtrot or tango?

The study focused on the chicken egg white lysozyme, a protein found in egg whites.

As a first step in their project, the scientists compared the regular vibrations of the lysozyme to the vibrations of the lysozyme when it was bound to a molecule whose presence blocked the protein from carrying out its usual biological duties.

What the scientists saw was that the free and inhibited lysozymes vibrated at similar energies, but with distinct directions of motion: The free lysozyme fluttered with a hinge-like flapping action — like the wings of a butterfly — while the inhibited lysozyme moved in a more scissor-like pattern.

“The result was a fundamental change from the conventional view. The vibrations changed their direction, even as the energy of the motions stayed the same,” Markelz says. (She adds that as an analogy, this is akin to two people performing different dances — the foxtrot and tango, for instance — but exerting the same amount of energy.)

The same dynamic emerged when the team compared the regular lysozyme to a mutant chicken egg white lysozyme that was more effective at performing its job. The mutant and normal lysozymes had the same vibrational energies, but different vibrational directions.

A turnkey instrument for measuring vibrations

Research on molecular vibrations could open new avenues for drug development and artificial energy harvesting (The vibrations may explain why photosynthesis is so efficient). But historically, the tiny pulses and palpitations within proteins have been very hard to study.

Markelz is hoping to change that by developing a turnkey instrument that scientists around the world can use to research the vibrations.

To study the chicken egg white lysozyme, her team employed the ATM technique that her research group developed in-house. Unlike other methods used to research protein vibrations, ATM enables scientists to observe not only vibrational energies, but also the direction of motions.

The NSF recently awarded Markelz a nearly $400,000 grant to commercialize an easy-to-use ATM instrument that would expand the capacity of the scientific community to explore molecular vibrations. The device would represent a great advance over other existing methods that provide only a coarse overview of the vibrations and require extremely dry and cold environments and expensive facilities, Markelz says.

UB’s Technology Transfer office filed a patent application with the U.S. Patent and Trademark Office and is seeking an industry partner to commercialize the instrument.

Markelz receives $1.35 million to study molecules’ vibrations, opening new possibilities for an emerging field

From left: Andrea Markelz, UB professor of physics, with graduate students Mengyang Xu and Katherine Niessen. Credit: Douglas Levere

UB physicist is one of the world’s top experts in her research area

By Charlotte Hsu

http://www.buffalo.edu/news/releases/2016/12/020.html

Release Date: December 15, 2016

Andrea Markelz. Credit: Douglas Levere

BUFFALO, N.Y. — In the same way that a church bell resonates after it’s struck, the molecules that comprise the world around us vibrate in specific patterns.  

These tiny tremors, invisible to the naked eye, support life: They enable proteins in the human body to change shape quickly, a necessity for performing critical biological functions. And in plants, the motions are thought to be involved in photosynthesis.

Research on molecular vibrations could open new avenues for drug development and artificial energy harvesting. But historically, these pulses and palpitations have been very hard to study.

University at Buffalo physicist Andrea Markelz is hoping to change that.

Markelz, PhD, a top expert in the field, has received three new grants totaling more than $1.35 million to probe the nature of protein vibrations and to develop instrumentation that will enable other researchers to do the same.

“What’s really exciting to me is that in nature, these vibrations — these protein dynamics — have been optimized to improve how organisms function,” Markelz says. “So what can we learn from nature, and how can we use the principles established by nature to develop new technologies?”

Markelz’ new funding — from the National Science Foundation (NSF) and the U.S. Department of Energy (DOE) — will support the following work:

Measuring the motion of photosynthesis proteins

Funding agency: DOE
Grant amount: $339,998
Project details: In this project, Markelz and her team will measure the vibrations of proteins involved in photosynthesis, the life-sustaining process that allows plants and some bacteria to convert sunlight into the chemical energy that fuels an organism’s day-to-day activities. The scientists will describe the vibrations of molecules that help shuttle energy from one part of an organism to another during photosynthesis, and analyze how these oscillations make photosynthesis more effective. The research could spur the development of organic solar cells that harvest sunlight efficiently.

Pinpointing the movements of important proteins

“What’s really exciting to me is that in nature, these vibrations — these protein dynamics — have been optimized to improve how organisms function.”

Andrea Markelz, professor of physics

University at Buffalo

Funding agency: NSF
Grant amount: $615,646
Project details: In this project, Markelz and partners will identify the motions of proteins that carry out vital biological functions. The team will look at photoactive yellow protein (PYP), which is thought to influence how photosynthesizing bacteria called cyanobacteria respond to light, and dihydrofolate reductase, which helps to regulate the levels of chemicals involved in cell growth and proliferation in various organisms. The project will examine how the motions of these molecules impact their function within organisms, providing information on how proteins can change their shape so efficiently — a crucial step toward developing pharmaceuticals that inhibit or take advantage of protein vibrations to treat disease.

Collaborators include researchers at the Hauptman-Woodward Medical Research Institute; Oklahoma State University; the University of California, Santa Barbara; and the University of Wisconsin-Milwaukee. This work was previously supported by UB seed funding for the National Science Foundation Biology with X-ray Free Electron Lasers (BioXFEL) Science and Technology Center, a national partnership between institutions that is headquartered at UB.

Creating a turnkey instrument for measuring molecular vibrations

Funding agency: NSF
Grant amount: $395,534
Project details: This funding will allow Markelz to develop an instrument that researchers around the world can use to measure the vibrations of proteins and other large molecules. She led a team that produced a terahertz microscope that is capable of making measurements that isolate specific vibrations, and her goal is to expand on this work by building an instrument with similar capabilities that is easier to replicate and maintain. While other methods exist to study molecular vibrations, these methods provide a coarse overview of the vibrations and require extremely dry and cold environments and expensive facilities.

The technique the Markelz group developed is called anisotropy terahertz microscopy (ATM), and it gives researchers the unprecedented ability to isolate vibrations moving in specific directions. The technique is table-top and is typically performed at room temperature. The method involves shining terahertz light on a molecule, then measuring the frequencies of light the molecule absorbs (this works because molecules vibrate at the same frequency as the light they absorb). The goal is to commercialize an easy-to-use ATM instrument, greatly expanding the capacity of the scientific community to conduct research on molecular vibrations.

This work was previously supported by the Bruce Holm Memorial Catalyst Fund at UB. UB’s Technology Transfer office has invested in patenting the technology and is now seeking an industry partner to commercialize the instrument. Currently, a patent application has been filed with the U.S. Patent and Trademark Office.

Media Contact Information

Charlotte Hsu
News Content Manager
Sciences, Economic Development
Tel: 716-645-4655
chsu22@buffalo.edu
Twitter: @UBScience
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University of Buffalo-The symphony of life, revealed

http://www.buffalo.edu/news/releases/2014/01/012.html

Using a new imaging technique they developed, scientists have managed to observe and document the vibrations of lysozyme, an antibacterial protein found in many animals. This graphic visualizes the vibrations in lysozyme as it is excited by terahertz light (depicted by the red wave arrow). Credit: Andrea Markelz and Katherine Niessen.

A new imaging technique captures the vibrations of proteins, tiny motions critical to human life

By Charlotte Hsu

Release Date: January 16, 2014

BUFFALO, N.Y. — Like the strings on a violin or the pipes of an organ, the proteins in the human body vibrate in different patterns, scientists have long suspected.

Now, a new study provides what researchers say is the first conclusive evidence that this is true.

Using a technique they developed based on terahertz near-field microscopy, scientists from the University at Buffalo and Hauptman-Woodward Medical Research Institute (HWI) have for the first time observed in detail the vibrations of lysozyme, an antibacterial protein found in many animals.

The team found that the vibrations, which were previously thought to dissipate quickly, actually persist in molecules like the “ringing of a bell,” said UB physics professor Andrea Markelz, PhD, wh0 led the study.

These tiny motions enable proteins to change shape quickly so they can readily bind to other proteins, a process that is necessary for the body to perform critical biological functions like absorbing oxygen, repairing cells and replicating DNA, Markelz said.

The research opens the door to a whole new way of studying the basic cellular processes that enable life.

“People have been trying to measure these vibrations in proteins for many, many years, since the 1960s,” Markelz said. “In the past, to look at these large-scale, correlated motions in proteins was a challenge that required extremely dry and cold environments and expensive facilities.”

“Our technique is easier and much faster,” she said. “You don’t need to cool the proteins to below freezing or use a synchrotron light source or a nuclear reactor — all things people have used previously to try and examine these vibrations.”

The findings appear today in Nature Communications.

To observe the protein vibrations, Markelz’ team relied on an interesting characteristic of proteins: The fact that they vibrate at the same frequency as the light they absorb.

This is analogous to the way wine glasses tremble and shatter when a singer hits exactly the right note. Markelz explained: Wine glasses vibrate because they are absorbing the energy of sound waves, and the shape of a glass determines what pitches of sound it can absorb. Similarly, proteins with different structures will absorb and vibrate in response to light of different frequencies.

So, to study vibrations in lysozyme, Markelz and her colleagues exposed a sample to light of different frequencies and polarizations, and measured the types of light the protein absorbed.

This technique, developed with Edward Snell, a senior research scientist at HWI and assistant professor of structural biology at UB, allowed the team to identify which sections of the protein vibrated under normal biological conditions. The researchers were also able to see that the vibrations endured over time, challenging existing assumptions.

“If you tap on a bell, it rings for some time, and with a sound that is specific to the bell. This is how the proteins behave,” Markelz said. “Many scientists have previously thought a protein is more like a wet sponge than a bell: If you tap on a wet sponge, you don’t get any sustained sound.”

Markelz said the team’s technique for studying vibrations could be used in the future to document how natural and artificial inhibitors stop proteins from performing vital functions by blocking desired vibrations.

“We can now try to understand the actual structural mechanisms behind these biological processes and how they are controlled,” Markelz said.

“The cellular system is just amazing,” she said. “You can think of a cell as a little machine that does lots of different things — it senses, it makes more of itself, it reads and replicates DNA, and for all of these things to occur, proteins have to vibrate and interact with one another.”

Left to right: Andrea Markelz and Katherine Niessen, two of the study's University at Buffalo coauthors. Credit: Douglas Levere

Media Contact Information

Charlotte Hsu
Media Relations Manager, Architecture, Economic Development, Sciences, Urban and Regional Planning
Tel: 716-645-4655
chsu22@buffalo.edu
Twitter: @UBScience
Pinterest: UB Science

Abstract-Improved Mode Assignment for Molecular Crystals Through Anisotropic Terahertz Spectroscopy

Rohit Singh , Deepu Koshy George , Jason B. Benedict , Timothy Michael Korter , and Andrea G. Markelz

We report the anisotropic terahertz response of oxalic acid and sucrose crystals in the 0.2-3.0 THz range using terahertz time domain spectroscopy on large single crystals. We compare the observed anisotropic response with the response calculated using solid-state density functional theory (DFT) and find good agreement in the orientation dependence and relative intensities of the crystal phonons. It was found that oxalic dihydrate can be reversibly converted to anhydrous by controlled relative humidity. In addition, oxalic acid was found to have a large birefringence with n = 0.3, suggesting the material may be useful for terahertz polarization manipulation. Sucrose has a smaller birefringence of n = 0.05, similar to that of x-cut quartz. The anisotropic measurements provide both mode separation and symmetry determination to more readily achieve mode assignment for the more complex sucrose spectrum.

SLAC Researchers Explore Terahertz Realm

SLAC's Alan Fisher leads a talk, "Presentation and Future Concepts for Intense Terahertz from SLAC Accelerators," during the Sept. 5-6 "Frontiers of THz Science"... (Photo by Matt Beardsley)

https://news.slac.stanford.edu/features/researchers-explore-terahertz-realm

September 18, 2012

by Glenn Roberts Jr.

From detecting concealed weapons and other security threats to manipulating and studying molecules and nanomaterials, potential applications for terahertz (THz) radiation are varied and growing, noted scientists who participated in this month's "Frontiers of THz Science" workshop at SLAC.

The international workshop, held Sept. 5 and 6, drew 130 attendees from as far away as China and Germany for discussions about the latest techniques to generate and use THz radiation, which sits in a largely untapped band of the electromagnetic spectrum between far-infrared and microwave radiation.

In final sessions at the conference, scientists considered the most promising areas for discovery and the types of terahertz research that could best benefit from use of SLAC facilities and expertise.

There was general consensus that experiments using powerful sources of X-ray radiation, including SLAC’s Stanford Synchrotron Radiation Lightsource and Linac Coherent Light Source, could provide insights into how terahertz radiation can drive and control biomechanical, chemical and electrical processes, for example.

Mark Sherwin, a physicist at the University of California, Santa Barbara, who served as a co-chairman for a 2004 conference titled "Opportunities in THz science," said terahertz research since that time has become "a very broad worldwide community." 

Sherwin said SLAC, with its powerful X-ray facilities, is well-positioned to assist this community in exploring some of the previously unreachable areas of terahertz science. 

Because terahertz research is still a fledgling field, "There's a very problem-rich environment" to delve into, Sherwin told SLAC organizers of the 2012 workshop.

Andrea Markelz, a physicist at New York's University at Buffalo, suggested that using terahertz radiation to introduce structural changes, such as controlling a biomolecular function to study its various states, is one promising area of research. 

In a summary presentation, Aaron Lindenberg of SLAC and Stanford concluded that experiments can use terahertz radiation to study a wide range of condensed-matter physics and materials-science problems, from phase transitions to high efficiency, non-contact measurements of electrical properties of nanomaterials. 

And SLAC’s Kelly Gaffney said experiments using terahertz radiation as a "pump" to excite changes in sample materials, followed by X-rays to probe those changes, are perhaps the "most important opportunity to pursue." 

Also, he said, "If terahertz is an important spectral range to measure the properties of materials, it should be an important range to manipulate those properties and how they change over time."

Conference organizers, including SLAC's Chief Scientist Z-X Shen, Norbert Holtkamp, director of the Accelerator Directorate, and Jo Stöhr, LCLS director, will prepare a report based on the discussions and conclusions at the workshop. The report will chart progress in terahertz research since the 2004 conference and plot a course for the future of terahertz science.

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Shen will give a summary of this workshop at the LCLS/SSRL Annual Users' Meeting on Oct. 5. The deadline for registration and poster abstracts for LCLS/SSRL 2012 has been extended until Sept. 27.