Scientists Snap Artificial Proteins With Atomic Precision / Gli scienziati fabbricano le proteine artificiali con precisione atomica
Scientists Snap Artificial Proteins With Atomic Precision / Gli scienziati fabbricano le proteine artificiali con precisione atomica
Segnalato dal Dott. Giuseppe Cotellessa / Reported by Dr. Giuseppe Cotellessa
Peptoid nanosheets are a single layer of crystals made from the spontaneous stacking of peptoid chains into parallel rows. Individual nanosheets floating in water were rapidly frozen and imaged by cryogenic electron microscopy (cryo-EM) to reveal their atomic structure. Computer modeling was used to fit the peptoid structure to the imaging data. Individual atomic positions were determined for the peptoids, enabling researchers to visualize their molecular shape and organization within the lattice. Distinct bromine atoms (magenta) on the side chains were directly visualized. / Le nanoschede peptoidi sono un singolo strato di cristalli realizzati dall'impilamento spontaneo di catene peptoidi in file parallele. I singoli foglietti galleggianti in acqua sono stati rapidamente congelati e ripresi con la microscopia elettronica criogenica (crio-EM) per rivelare la loro struttura atomica. La modellazione computerizzata è stata utilizzata per adattare la struttura peptoidea ai dati di imaging. Le posizioni atomiche individuali sono state determinate per i peptoidi, consentendo ai ricercatori di visualizzare la loro forma molecolare e l'organizzazione all'interno del reticolo. Atomi di bromo distinti (magenta) sulle catene laterali sono stati visualizzati Credit: Berkeley Lab.
Protein-like molecules called "polypeptoids" (or "peptoids," for short) have great promise as precision building blocks for creating a variety of designer nanomaterials, like flexible nanosheets - ultrathin, atomic-scale 2D materials. They could advance a number of applications - such as synthetic, disease-specific antibodies and self-repairing membranes or tissue - at a low cost.
To understand how to make these applications a reality, however, scientists need a way to zoom in on a peptoid's atomic structure. In the field of materials science, researchers typically use electron microscopes to reach atomic resolution, but soft materials like peptoids would disintegrate under the harsh glare of an electron beam.
Now, scientists at the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) have adapted a technique that enlists the power of electrons to visualize a soft material's atomic structure while keeping it intact.
Their study, published in the journal Proceedings of the National Academy of Sciences , demonstrates for the first time how cryo-EM (cryogenic electron microscopy), a Nobel Prize-winning technique originally designed to image proteins in solution, can be used to image atomic changes in a synthetic soft material. Their findings have implications for the synthesis of 2D materials for a wide variety of applications.
"All materials we touch function because of the way atoms are arranged in the material. But we don't have that knowledge for peptoids because unlike proteins, the atomic structure of many soft synthetic materials is messy and hard to predict," said Nitash Balsara, a senior faculty scientist in Berkeley Lab's Materials Sciences Division, and professor of chemical engineering at UC Berkeley, who co-led the study. "And if you don't know where the atoms are, you're flying blind. Our use of cryo-EM for the imaging of peptoids will set a clear path for the design and synthesis of soft materials at the atomic scale."
Taking a hard look at soft materials
For the last 13 years, Balsara has been leading an effort to image soft materials at the atomic scale through Berkeley Lab's Soft Matter Electron Microscopy Program. For the current study, he joined forces with Ronald Zuckermann, a senior scientist in Berkeley Lab's Molecular Foundry who first discovered peptoids almost 30 years ago in his search for new polymers -materials made of long, repeating chains of small molecular units called "monomers" - for targeted drug therapies.
"This study comes out of many years of research here at Berkeley Lab. To make a material and see the atoms - it's the dream of my career," said Zuckermann, who co-led the study with Balsara.
Unlike most synthetic polymers, peptoids can be made to have a precise sequence of monomer units, a common trait in biological polymers, such as proteins and DNA.
And like natural proteins, peptoids can grow or self-assemble into distinct shapes for specific functions - such as helices, fibers, nanotubes, or thin and flat nanosheets.
But unlike proteins, the molecular structure of peptoids is typically amorphous and unpredictable - like a pile of wet noodles. And untangling such an unpredictable structure has long been an obstacle for materials scientists.
Pinning down peptoids with cryo-EM
So the researchers turned to cryo-EM, which flash-freezes the peptoids at a temperature of around 80 kelvins (or minus 316 degrees Fahrenheit) in microseconds. The ultracold temperature of cryo-EM locks in the structure of the sheet and also prevents the electrons from destroying the sample.
To protect soft materials, cryo-EM uses fewer electrons than conventional electron microscopy, resulting in ghostly black-and-white images. To better document what's going on at the atomic level, hundreds of these images are taken. Sophisticated mathematical tools combine these images to make more detailed atomic-scale pictures.
For the study, the researchers fabricated nanosheets in solution from short peptoid polymers made of a chain of six hydrophobic monomers known as "aromatics," connected to four hydrophilic polyether monomers. The hydrophilic or "water-loving" monomers are attracted to the water in the solution, while the hydrophobic or "water-hating" monomers avoid the water, orienting the molecules to form crystalline nanosheets that are only one-molecule thick (around 3 nanometers, or 3 billionths of a meter).
Lead author Sunting Xuan, a postdoctoral researcher in the Materials Sciences Division, synthesized the peptoid nanosheets and used X-ray scattering techniques at Berkeley Lab's Advanced Light Source (ALS) to characterize their molecular structure. The ALS produces light in a variety of wavelengths to enable studies of samples' nanoscale structure and chemistry, among other properties.
Xi Jiang, a project scientist in the Materials Sciences Division, captured the high-quality images and developed the algorithms necessary to achieve atomic resolution in the peptoid imaging.
David Prendergast, senior staff scientist and interim director of the Molecular Foundry, modeled atomic substitutions in the peptoids, and Nan Li, a postdoctoral researcher at the Molecular Foundry, performed molecular dynamics simulations to establish an atomic-scale model of the nanosheet.
At the heart of the team's discovery was their ability to rapidly iterate between materials synthesis and atomic imaging. The precision of peptoid synthesis, coupled with the researchers' ability to directly image the placement of atoms using cryo-EM, allowed them to engineer the peptoid at the atomic level. To their surprise, when they created several new variations of the peptoid monomer sequence, the atomic structure of the nanosheet changed in a very orderly way.
For example, when one additional bromine atom was added to each aromatic ring, the shape of each peptoid molecule remained unchanged yet the space between rows increased by just enough to accommodate the additional bromine atoms.
Furthermore, when four additional variants of the peptoid nanosheet structure were imaged, the researchers noticed a remarkable uniformity across their atomic structure, and that the nanosheets shared the same shape of peptoid molecules. This allowed them to predictably engineer the nanosheet structure, Zuckermann said.
"To have so much control at the atomic scale in soft materials was completely unexpected," said Balsara, because it was assumed that only proteins could form defined shapes when you have a specific sequence of monomers - in their case, amino acids.
A team approach to new materials
For close to four decades, Berkeley Lab has pushed the boundaries of electron microscopy into fields of science once considered impossible to explore with an electron beam. Pioneering work by scientists at Berkeley Lab also played a key role in the 2017 Nobel Prize in chemistry, which honored the development of cryo-EM.
"Most people would say it's not possible to develop a technique that can position and see individual atoms in a soft material," said Balsara. "The only way to solve hard problems like this is to team up with experts across scientific disciplines. At Berkeley Lab, we work as a team."
Zuckermann added that the current study proves that the cryo-EM technique could be applied to a wide range of common polymers and other industrial soft materials, and could lead to a new class of soft nanomaterials that fold into protein-like structures with protein-like functions.
"This work sets the stage for materials scientists to tackle the challenge of making artificial proteins a reality," he said, adding that their study also positions the team to work on solving a diversity of exciting problems, and to "raise people's awareness that they, too, can begin to look at the atomic structure of their soft materials using these cryo-EM techniques."
To understand how to make these applications a reality, however, scientists need a way to zoom in on a peptoid's atomic structure. In the field of materials science, researchers typically use electron microscopes to reach atomic resolution, but soft materials like peptoids would disintegrate under the harsh glare of an electron beam.
Now, scientists at the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) have adapted a technique that enlists the power of electrons to visualize a soft material's atomic structure while keeping it intact.
Their study, published in the journal Proceedings of the National Academy of Sciences , demonstrates for the first time how cryo-EM (cryogenic electron microscopy), a Nobel Prize-winning technique originally designed to image proteins in solution, can be used to image atomic changes in a synthetic soft material. Their findings have implications for the synthesis of 2D materials for a wide variety of applications.
"All materials we touch function because of the way atoms are arranged in the material. But we don't have that knowledge for peptoids because unlike proteins, the atomic structure of many soft synthetic materials is messy and hard to predict," said Nitash Balsara, a senior faculty scientist in Berkeley Lab's Materials Sciences Division, and professor of chemical engineering at UC Berkeley, who co-led the study. "And if you don't know where the atoms are, you're flying blind. Our use of cryo-EM for the imaging of peptoids will set a clear path for the design and synthesis of soft materials at the atomic scale."
Taking a hard look at soft materials
For the last 13 years, Balsara has been leading an effort to image soft materials at the atomic scale through Berkeley Lab's Soft Matter Electron Microscopy Program. For the current study, he joined forces with Ronald Zuckermann, a senior scientist in Berkeley Lab's Molecular Foundry who first discovered peptoids almost 30 years ago in his search for new polymers -materials made of long, repeating chains of small molecular units called "monomers" - for targeted drug therapies.
"This study comes out of many years of research here at Berkeley Lab. To make a material and see the atoms - it's the dream of my career," said Zuckermann, who co-led the study with Balsara.
Unlike most synthetic polymers, peptoids can be made to have a precise sequence of monomer units, a common trait in biological polymers, such as proteins and DNA.
And like natural proteins, peptoids can grow or self-assemble into distinct shapes for specific functions - such as helices, fibers, nanotubes, or thin and flat nanosheets.
But unlike proteins, the molecular structure of peptoids is typically amorphous and unpredictable - like a pile of wet noodles. And untangling such an unpredictable structure has long been an obstacle for materials scientists.
Pinning down peptoids with cryo-EM
So the researchers turned to cryo-EM, which flash-freezes the peptoids at a temperature of around 80 kelvins (or minus 316 degrees Fahrenheit) in microseconds. The ultracold temperature of cryo-EM locks in the structure of the sheet and also prevents the electrons from destroying the sample.
To protect soft materials, cryo-EM uses fewer electrons than conventional electron microscopy, resulting in ghostly black-and-white images. To better document what's going on at the atomic level, hundreds of these images are taken. Sophisticated mathematical tools combine these images to make more detailed atomic-scale pictures.
For the study, the researchers fabricated nanosheets in solution from short peptoid polymers made of a chain of six hydrophobic monomers known as "aromatics," connected to four hydrophilic polyether monomers. The hydrophilic or "water-loving" monomers are attracted to the water in the solution, while the hydrophobic or "water-hating" monomers avoid the water, orienting the molecules to form crystalline nanosheets that are only one-molecule thick (around 3 nanometers, or 3 billionths of a meter).
Lead author Sunting Xuan, a postdoctoral researcher in the Materials Sciences Division, synthesized the peptoid nanosheets and used X-ray scattering techniques at Berkeley Lab's Advanced Light Source (ALS) to characterize their molecular structure. The ALS produces light in a variety of wavelengths to enable studies of samples' nanoscale structure and chemistry, among other properties.
Xi Jiang, a project scientist in the Materials Sciences Division, captured the high-quality images and developed the algorithms necessary to achieve atomic resolution in the peptoid imaging.
David Prendergast, senior staff scientist and interim director of the Molecular Foundry, modeled atomic substitutions in the peptoids, and Nan Li, a postdoctoral researcher at the Molecular Foundry, performed molecular dynamics simulations to establish an atomic-scale model of the nanosheet.
At the heart of the team's discovery was their ability to rapidly iterate between materials synthesis and atomic imaging. The precision of peptoid synthesis, coupled with the researchers' ability to directly image the placement of atoms using cryo-EM, allowed them to engineer the peptoid at the atomic level. To their surprise, when they created several new variations of the peptoid monomer sequence, the atomic structure of the nanosheet changed in a very orderly way.
For example, when one additional bromine atom was added to each aromatic ring, the shape of each peptoid molecule remained unchanged yet the space between rows increased by just enough to accommodate the additional bromine atoms.
Furthermore, when four additional variants of the peptoid nanosheet structure were imaged, the researchers noticed a remarkable uniformity across their atomic structure, and that the nanosheets shared the same shape of peptoid molecules. This allowed them to predictably engineer the nanosheet structure, Zuckermann said.
"To have so much control at the atomic scale in soft materials was completely unexpected," said Balsara, because it was assumed that only proteins could form defined shapes when you have a specific sequence of monomers - in their case, amino acids.
A team approach to new materials
For close to four decades, Berkeley Lab has pushed the boundaries of electron microscopy into fields of science once considered impossible to explore with an electron beam. Pioneering work by scientists at Berkeley Lab also played a key role in the 2017 Nobel Prize in chemistry, which honored the development of cryo-EM.
"Most people would say it's not possible to develop a technique that can position and see individual atoms in a soft material," said Balsara. "The only way to solve hard problems like this is to team up with experts across scientific disciplines. At Berkeley Lab, we work as a team."
Zuckermann added that the current study proves that the cryo-EM technique could be applied to a wide range of common polymers and other industrial soft materials, and could lead to a new class of soft nanomaterials that fold into protein-like structures with protein-like functions.
"This work sets the stage for materials scientists to tackle the challenge of making artificial proteins a reality," he said, adding that their study also positions the team to work on solving a diversity of exciting problems, and to "raise people's awareness that they, too, can begin to look at the atomic structure of their soft materials using these cryo-EM techniques."
ITALIANO
Le molecole simili a proteine chiamate "polipeptoidi" (o "peptoidi", in breve) presentanp una grande promessa come blocchi di precisione per la creazione di una varietà di nanomateriali di design, come fogli di materiale flessibile - ultrasottili, materiali 2D su scala atomica. Potrebbero far progredire una serie di applicazioni - come sintetici, anticorpi specifici per malattie e membrane o tessuti autoriparanti - a basso costo.
Per capire come rendere queste applicazioni una realtà, tuttavia, gli scienziati hanno bisogno di un modo per ingrandire la struttura atomica di un peptoide. Nel campo della scienza dei materiali, i ricercatori in genere usano i microscopi elettronici per raggiungere la risoluzione atomica, ma i materiali morbidi come i peptoidi si disintegrerebbero sotto il duro riflesso di un fascio di elettroni.
Ora, gli scienziati del Lawrence Berkeley National Laboratory (Berkeley Lab) del Dipartimento dell'Energia degli Stati Uniti hanno adattato una tecnica che ravviva il potere degli elettroni per visualizzare la struttura atomica di un materiale morbido mantenendo intatto.
Il loro studio, pubblicato sulla rivista Proceedings of National Academy of Sciences, dimostra per la prima volta come cryo-EM (microscopia elettronica criogenica), una tecnica vincitrice del premio Nobel originariamente progettata per l'immagine di proteine in soluzione, può essere utilizzata per l'immagine atomica cambiamenti in un materiale sintetico morbido. I loro risultati hanno implicazioni per la sintesi di materiali 2D per un'ampia varietà di applicazioni.
"Tutti i materiali che tocchiamo funzionano a causa del modo in cui gli atomi sono disposti nel materiale. Ma non abbiamo questa conoscenza per i peptoidi perché a differenza delle proteine, la struttura atomica di molti materiali sintetici morbidi è disordinata e difficile da prevedere", ha detto Nitash Balsara , uno scienziato della facoltà della Divisione Scienze dei materiali del Berkeley Lab e professore di ingegneria chimica presso l'UC Berkeley, che ha co-condotto lo studio. "E se non sai dove sono gli atomi, stai volando alla cieca. Il nostro uso di cryo-EM per l'imaging dei peptoidi creerà un percorso chiaro per la progettazione e la sintesi di materiali morbidi su scala atomica."
Dai uno sguardo duro ai materiali morbidi
Negli ultimi 13 anni, Balsara ha condotto uno sforzo per l'immagine di materiali morbidi su scala atomica attraverso il programma di microscopia elettronica a materia soffice di Berkeley Lab. Per lo studio attuale, ha unito le forze con Ronald Zuckermann, uno scienziato senior della Fonderia molecolare di Berkeley Lab che ha scoperto per la prima volta peptoidi quasi 30 anni fa nella sua ricerca di nuovi polimeri - materiali costituiti da catene lunghe e ripetute di piccole unità molecolari chiamate "monomeri" - per terapie farmacologiche mirate.
"Questo studio nasce da molti anni di ricerca qui al Berkeley Lab. Realizzare un materiale e vedere gli atomi - è il sogno della mia carriera", ha dichiarato Zuckermann, che ha co-condotto lo studio con Balsara.
A differenza della maggior parte dei polimeri sintetici, i peptoidi possono essere fatti per avere una sequenza precisa di unità monomeriche, un tratto comune nei polimeri biologici, come proteine e DNA.
E come le proteine naturali, i peptoidi possono crescere o autoassemblarsi in forme distinte per funzioni specifiche - come eliche, fibre, nanotubi o nanoschede sottili e piatte.
Ma a differenza delle proteine, la struttura molecolare dei peptoidi è in genere amorfa e imprevedibile, come un mucchio di noodles bagnati. E districare una struttura così imprevedibile è stato a lungo un ostacolo per gli scienziati dei materiali.
Appuntare i peptoidi con cryo-EM
Quindi i ricercatori si sono rivolti a cryo-EM, che congela i peptoidi a una temperatura di circa 80 kelvin (o meno 316 gradi Fahrenheit) in microsecondi. La temperatura ultrafredda di cryo-EM si blocca nella struttura del foglio e impedisce anche agli elettroni di distruggere il campione.
Per proteggere i materiali morbidi, cryo-EM utilizza meno elettroni rispetto alla microscopia elettronica tradizionale, ottenendo immagini in bianco e nero spettrali. Per documentare meglio cosa sta succedendo a livello atomico, vengono prese centinaia di queste immagini. Strumenti matematici sofisticati combinano queste immagini per creare immagini in scala atomica più dettagliate.
Per lo studio, i ricercatori hanno fabbricato nanoschede in soluzione da brevi polimeri peptoidi costituiti da una catena di sei monomeri idrofobici noti come "aromatici", collegati a quattro monomeri polietere idrofili. I monomeri idrofili o "amanti dell'acqua" sono attratti dall'acqua nella soluzione, mentre i monomeri idrofobici o "che odiano l'acqua" evitano l'acqua, orientando le molecole a formare nanoschede cristalline che sono spesse di una sola molecola (circa 3 nanometri o 3 miliardesimi di metro).
L'autore principale Sunting Xuan, un ricercatore post-dottorato nella divisione Scienza dei materiali, ha sintetizzato i peptidi nanosheets e ha usato le tecniche di diffusione dei raggi X presso Advanced Light Source (ALS) di Berkeley Lab per caratterizzare la loro struttura molecolare. L'ALS produce luce in una varietà di lunghezze d'onda per consentire studi sulla struttura e la chimica della nanoscala dei campioni, tra le altre proprietà.
Xi Jiang, uno scienziato del progetto nella divisione Scienza dei materiali, ha acquisito immagini di alta qualità e sviluppato gli algoritmi necessari per ottenere la risoluzione atomica nell'imaging peptoide.
David Prendergast, scienziato dello staff senior e direttore ad interim della Molecular Foundry, ha modellato le sostituzioni atomiche nei peptoidi e Nan Li, un ricercatore post-dottorato presso la Molecular Foundry, ha eseguito simulazioni di dinamica molecolare per stabilire un modello su scala atomica del nanosheet.
Da:
Commenti
Posta un commento