But what happens with the sequences not selected by nature? And what possibilities lie in constructing entirely new sequences to make novel, or de novo, proteins bearing little resemblance to anything in nature?

That’s the terrain Princeton University’s Hecht Lab works in. And recently, their curiosity for designing their own sequences paid off.

They discovered the first known de novo protein that catalyzes, or drives, the synthesis of quantum dots. Quantum dots are fluorescent nanocrystals used in electronic applications from LED screens to solar panels.

Their work opens the door to making nanomaterials in a more sustainable way by demonstrating that protein sequences not derived from nature can be used to synthesize functional materials — with pronounced benefits to the environment.

Quantum dots are normally made in industrial settings with high temperatures and toxic, expensive solvents — a process that is neither economical nor environmentally friendly. But Hecht Lab researchers pulled off the process at the bench using water as a solvent, making a stable end-product at room temperature.

“We’re interested in making life molecules, proteins, that did not arise in life,” said Professor of Chemistry Michael Hecht, who led the research with Greg Scholes, the William S. Tod Professor of Chemistry and chair of the department. “In some ways we’re asking, are there alternatives to life as we know it? All life on earth arose from common ancestry. But if we make lifelike molecules that did not arise from common ancestry, can they do cool stuff?

“So here, we’re making novel proteins that never arose in life doing things that don’t exist in life.”

The team’s process can also tune nanoparticle size, which determines the color quantum dots glow, or fluoresce, in. That holds possibilities for tagging molecules within a biological system, like staining cancer cells in vivo.

“Quantum dots have very interesting optical properties due to their sizes,” said Yueyu Yao, co-author on the paper and a fifth-year graduate student in the Hecht Lab. “They’re very good at absorbing light and converting it to chemical energy — that makes them useful for being made into solar panels or any sort of photo sensor.

“But on the other hand, they’re also very good at emitting light at a certain desired wavelength, which makes them suitable for making LED screens.”

And because they’re small — comprised of only about 100 atoms and maybe 2 nanometers across — they’re able to penetrate some biological barriers, making their utility in medicines and biological imaging especially promising.

The research, “A de novo protein catalyzes the synthesis of semiconductor quantum dots,” was published this week in the Proceedings of the National Academy of Sciences (PNAS).

Why use de novo proteins?

“I think using de novo proteins opens up a way for designability,” said Leah Spangler, lead author on the research and a former postdoc in the Scholes Lab. “A key word for me is ‘engineering.’ I want to be able to engineer proteins to do something specific, and this is a type of protein you can do that with.

“The quantum dots we’re making aren’t great quality yet, but that can be improved by tuning the synthesis,” she added. “We can achieve better quality by engineering the protein to influence quantum dot formation in different ways.”

Based on work done by Sarangan Chari, Hecht Lab senior chemist and a corresponding author, the team used a de novo protein it designed named ConK to catalyze the reaction. Researchers first isolated ConK in 2016 from a large combinatorial library of proteins. It’s still made of natural amino acids, but it qualifies as “de novo” because its sequence doesn’t have any similarity to a natural protein.

Researchers found that ConK enabled the survival of E. coli in otherwise toxic concentrations of copper, suggesting it might be useful for metal binding and sequestration. The quantum dots used in this research are made out of cadmium sulfide. Cadmium is a metal, so researchers wondered if ConK could be used to synthesize quantum dots.

Their hunch paid off. ConK breaks down cysteine, one of the 20 amino acids, into several products, including hydrogen sulfide. That acts as the active sulfur source that will then go on to react with the metal cadmium. The result is CdS quantum dots.

“To make a cadmium sulfide quantum dot, you need the cadmium source and the sulfur source to react in solution,” said Spangler. “What the protein does is make the sulfur source slowly over time. So, we add the cadmium initially but the protein generates the sulfur, which then reacts to make distinct sizes of quantum dots.”

This research was supported by the National Science Foundation MRSEC program (DMR-2011750), the Princeton University Writing Center, and the Canadian Institute for Advanced Research. The research was also supported by NSF grant MCB-1947720 to MH.

“The Paleocene-Eocene Thermal Maximum is the closest thing we have in the geologic record to anything like what we’re experiencing now and may experience in the future with climate change,” said Lee Kump, professor of geosciences at Penn State. “There has been a lot of interest in better resolving that history, and our work addresses important questions about what triggered the event and the rate of carbon emissions.”

The scientists analyzed core samples from a well-preserved record of the PETM near the Maryland coast using astrochronology, a technique for dating sediments against orbital patterns that occur over tens to hundreds of thousands of years, known as Milankovitch cycles.

They found the shape of Earth’s orbit, or eccentricity, and the wobble in its rotation, or precession, favored hotter conditions at the onset of the PETM and that these orbital configurations together may have played a role in triggering the event.

“An orbital trigger may have led to the carbon release that caused several degrees of global warming during the PETM as opposed to what’s a more popular interpretation at the moment that massive volcanism released the carbon and triggered the event,” said Kump, the John Leone Dean in the College of Earth and Mineral Sciences.

The findings, published in the journal Nature Communications, also indicated the onset of the PETM lasted about 6,000 years. Previous estimates have ranged from several years to tens of thousands of years. The timing is important to understand the rate at which carbon was released into the atmosphere, the scientists said.

“This study allows us to refine our carbon cycle models to better understand how the planet reacts to an injection of carbon over these timescales and to narrow down the possibilities for the source of the carbon that drove the PETM,” said Mingsong Li, assistant professor in the School of Earth and Space Sciences at Peking University and a former assistant research professor of geosciences at Penn State who is lead author on the study.

A 6,000-year onset, coupled with estimates that 10,000 gigatons of carbon were injected into the atmosphere as the greenhouse gases carbon dioxide or methane, indicates that about one and a half gigatons of carbon were released per year.

“Those rates are close to an order of magnitude slower than the rate of carbon emissions today, so that is cause for some concern,” Kump said. “We are now emitting carbon at a rate that’s 5 to 10 times higher than our estimates of emissions during this geological event that left an indelible imprint on the planet 56 million years ago.”

The scientists conducted a time series analysis of calcium content and magnetic susceptibility found in the cores, which are proxies for changes in orbital cycles, and used that information to estimate the pacing of the PETM.

Earth’s orbit varies in predictable, calculable ways due to gravitational interactions with the sun and other planets in the solar system. These changes impact how much sunlight reaches Earth and its geographic distribution and therefore influence the climate.

“The reason there’s an expression in the geologic record of these orbital changes is because they affect climate,” Kump said. “And that affects how productive marine and terrestrial organisms are, how much rainfall there is, how much erosion there is on the continents and therefore how much sediment is carried into the ocean environment.”

Erosion from the paleo Potomac and Susquehanna rivers, which at the onset of the PETM may have rivaled the discharge of the Amazon River, carried sediments to the ocean where they were deposited on the continental shelf. This formation, called the Marlboro Clay, is now inland and offers one of the best-preserved examples of the PETM.

“We can develop histories by coring down through the layers of sediment and extracting specific cycles that are creating this story, just like you could extract each note from a song,” Kump said. “Of course, some of records are distorted and there are gaps — but we can use the same types of statistical methods that are used in apps that can determine what song you are trying to sing. You can sing a song and if you forget half the words and skip a chorus, it will still be able to determine the song, and we can use that same approach to reconstruct these records.”

Timothy Bralower, professor of geosciences at Penn State, also contributed to this research.

Other contributors were James Zachos, distinguished professor at the University of California Santa Cruz; William Rush, a postdoctoral associate at Yale University and the Cooperative Institute for Research in Environmental Science at the University of Colorado Boulder; and Jean Self-Trail and Marci Robinson, research geologists at the Florence Bascom Geoscience Center, United States Geological Survey.

The National Key R&D Program of China and the Heising-Simons Foundation provided funding for this work.

As the paper explains, the calculations are based on the hydrodynamic particle flow seen in head-on collisions of various types of ions at both the LHC and the Relativistic Heavy Ion Collider (RHIC), a DOE Office of Science user facility for nuclear physics research at Brookhaven Lab. With only modest changes, these calculations also describe flow patterns seen in near-miss collisions, where photons that form a cloud around the speeding ions collide with the ions in the opposite beam.

“The upshot is that, using the same framework we use to describe lead-lead and proton-lead collisions, we can describe the data of these ultra-peripheral collisions where we have a photon colliding with a lead nucleus,” said Brookhaven Lab theorist Bjoern Schenke, a coauthor of the paper. “That tells you there’s a possibility that, in these photon-ion collisions, we create a small dense strongly interacting medium that is well described by hydrodynamics — just like in the larger systems.”

Fluid signatures

Observations of particles flowing in characteristic ways have been key evidence that the larger collision systems (lead-lead and proton-lead collisions at the LHC; and gold-gold and proton-gold collisions at RHIC) create a nearly perfect fluid. The flow patterns were thought to stem from the enormous pressure gradients created by the large number of strongly interacting particles produced where the colliding ions overlap.

“By smashing these high-energy nuclei together we’re creating such high energy density — compressing the kinetic energy of these guys into such a small space — that this stuff essentially behaves like a fluid,” Schenke said.

Spherical particles (including protons and nuclei) colliding head on are expected to generate a uniform pressure gradient. But partially overlapping collisions generate an oblong, almond-shaped pressure gradient that pushes more high-energy particles out along the short axis than perpendicular to it.

This “elliptic flow” pattern was one of the earliest hints that particle collisions at RHIC could create a quark-gluon plasma, or QGP — a hot soup of the fundamental building blocks that make up the protons and neutrons of nuclei/ions. Scientists were at first surprised by the QGP’s liquid-like behavior. But they later established elliptic flow as a defining feature of QGP, and evidence that the quarks and gluons were still interacting strongly, even when free from confinement within individual protons and neutrons. Later observations of similar flow patterns in collisions of protons with large nuclei, intriguingly suggest that these proton-nucleus collision systems can also create tiny specks of quark-gluon soup.

“Our new paper is about pushing this to even further extremes, looking at collisions between photons and nuclei,” Schenke said.

Changing the projectile

It has long been known that that ultra-peripheral collisions could create photon-nucleus interactions, using the nuclei themselves as the source of the photons. That’s because charged particles accelerated to high energies, like the lead nuclei/ions accelerated at the LHC (and gold ions at RHIC), emit electromagnetic waves — particles of light. So, each accelerated lead ion at the LHC is essentially surrounded by a cloud of photons.

“When two of these ions pass each other very closely without colliding, you can think of one as emitting a photon, which then hits the lead ion going the other way,” Schenke said. “Those events happen a lot; it’s easier for the ions to barely miss than to precisely hit one another!”

ATLAS scientists recently published data on intriguing flow-like signals from these photon-nucleus collisions.

“We had to set up special data collection techniques to pick out these unique collisions,” said Blair Seidlitz, a Columbia University physicist who helped set up the ATLAS trigger system for the analysis when he was a graduate student at the University of Colorado, Boulder. “After collecting enough data, we were surprised to find flow-like signals that were similar to those observed in lead-lead and proton-lead collisions, although they were a little smaller.”

Schenke and his collaborators set out to see whether their theoretical calculations could accurately describe the particle flow patterns.

They used the same hydrodynamic calculations that describe the behavior of particles produced in lead-lead and proton-lead collision systems. But they made a few adjustments to account for the “projectile” striking the lead nucleus changing from a proton to a photon.

According to the laws of physics (specifically, quantum electrodynamics), a photon can undergo quantum fluctuations to become another particle with the same quantum numbers. A rho meson, a particle made of a particular combination of a quark and antiquark held together by gluons, is one of the most likely results of those photon fluctuations.

If you think back to the proton — made of three quarks — this two-quark rho particle is just a step down the complexity ladder.

“Instead of having a gluon distribution around three quarks inside a proton, we have the two quarks (quark-antiquark) with a gluon distribution around those to collide with the nucleus,” Schenke said.

Accounting for energy

The calculations also had to account for the big difference in energy in these photon-nucleus collision systems, compared to proton-lead and especially lead-lead.

“The emitted photon that’s colliding with the lead won’t carry the entire momentum of the lead nucleus it came from, but only a tiny fraction of that. So, the collision energy will be much lower,” Schenke said.

That energy difference turned out to be even more important than the change of projectile.

In the most energetic lead-lead or gold-gold heavy ion collisions, the pattern of particles emerging in the plane transverse to the colliding beams generally persists no matter how far you look from the collision point along the beamline (in the longitudinal direction). But when Schenke and collaborators modeled the patterns of particles expected to emerge from lower-energy photon-lead collisions, it became apparent that including the 3D details of the longitudinal direction made a difference. The model showed that the geometry of the particle distributions changes rapidly with increasing longitudinal distance; the particles become “decorrelated.”

“The particles see different pressure gradients depending on their longitudinal position,” Schenke explained.

“So, for these low energy photon-lead collisions, it is important to run a full 3D hydrodynamic model (which is more computationally demanding) because the particle distribution changes more rapidly as you go out in the longitudinal direction,” he said.

When the theorists compared their predictions using this lower-energy, full 3D, hydrodynamic model with the particle flow patterns observed in photon-lead collisions by the ATLAS detector, the data and theory matched up nicely, at least for the most obvious elliptic flow pattern, Schenke said.

Implications and the future

“From this result, it looks like it’s conceivable that, even in photon-heavy ion collisions, we have a strongly interacting fluid that responds to the initial collision geometry, as described by hydrodynamics,” Schenke said. “If the energies and temperatures are high enough,” he added, “there will be a quark-gluon plasma.”

“It’s conceivable that, in photon-heavy ion collisions, we have a strongly interacting fluid,” said Brookhaven Lab theorist Bjoern Schenke.

Seidlitz, the ATLAS physicist, commented, “It was very interesting to see these results suggesting the formation of a small droplet of quark-gluon plasma, as well as how this theoretical analysis offers concrete explanations as to why the flow signatures are a bit smaller in photon-lead collisions.”

Additional data to be collected by ATLAS and other experiments at RHIC and the LHC over the next several years will enable more detailed analyses of particles flowing from photon-nucleus collisions. These analyses will help distinguish the hydrodynamic calculation from another possible explanation, in which the flow patterns are not a result of the system’s response to the initial geometry.

In the longer-term future, experiments at an Electron-Ion Collider (EIC), a facility planned to replace RHIC sometime in the next decade at Brookhaven Lab, could provide more definitive conclusions.