Protein behavior might hold the key to synthetic silk

A trans-Atlantic collaboration of scientists has revealed the structure of a key protein of silk and discovered a previously unknown behavior of this protein: to self-organize into tiny fibrils a single molecule in diameter. This discovery sets the stage for the eventual creation of synthetic silk—not just the luxury fabric that’s a product of silkworms, but also the manufacture of ultra-tough spider silk familiar to fans of the Marvel superhero Spider-Man.

Hannes Schniepp cautions that the world’s textile mills aren’t likely to start producing “spidey silk” in the near future, but says his work describing the structure of the silk protein and its self-organizing behavior is an important step in that direction. Schniepp is an assistant professor in the Department of Applied Science at the College of William & Mary. Along with graduate student Minzhen Cai and a set of collaborators at the University of Oxford in the United Kingdom, he has published a paper describing silk at the molecular level.

“Silk is a polymer,” Schniepp explained. “It’s not a synthetically-made polymer, but it’s a polymer made out of proteins.”

Synthetic plastics are polymers, but these macromolecules are common in the natural world, too. Schniepp pointed out that much of the human body—including DNA—is constructed of various polymers. 

“What’s so fascinating about silk is that in terms of its mechanical properties, silk is better than any polymer that we can make synthetically,” he said. “Particularly, certain spider silks are even tougher than Kevlar, the best high-performance polymer we have.”

You can’t farm spiders

For millennia, people have been using the cocoons of silkworms to weave silk cloth. Humans have used spider silk to a much lesser degree, but spiders have proven to be impossible to cultivate: “They start eating each other,” Schniepp says.

Figuring out a process to make synthetic silk has been a sort of Holy Grail of materials science for nearly as long as people have been making silk. After years of scientific study, the exact natures of both the biochemistry and the mechanics of silk creation by silkworms and spiders remain elusive.

“The big question really is how does the spider do it? How does the silkworm do it?,” Schniepp says. “The problem is it’s a tiny animal and it happens really in very small dimensions inside the animal, and it’s really almost impossible to watch what’s going on there.”

He said that most of the scientific study on structure of silk has focused on examination of the product through microscopy and other analytical tools. The study has yielded a fair amount of understanding about the structural nature of silk, but scientists had no idea of what shape an individual silk protein had.

Schniepp and his team at William & Mary took a different approach than most materials scientists, sampling “silk dope,” the gel-like material inside the silkworm that the worm exudes to spin its cocoon.

“A lot of these biomolecules, they’re very sensitive to changes. So the closer you can be to the native state, the more valuable this information is that you get,” he said.

Working with silk dope

Schniepp and his research group examined the silk dope in their McGlothlin-Street Hall laboratory, using an atomic force microscope (AFM), an instrument capable of looking at materials at the nanoscale. Before placing them in the AFM, they prepared their silk dope, diluting the samples with a bit of water, then spun the sample on a plate, so that the silk spread out on the surface.

“When you spin liquid on a plate like this, you shear it. And that does something to these proteins that’s similar to the way that the animals do,” he explained. “They have a gland that produces this material and at the end is something like a nozzle. So they squeeze this material out through the nozzle. To create a similar effect, you shear the solution. By spinning it very quickly, the liquid is forced away, and it is similar to what happens when the animal pushes the silk out.”

A number of curious things happen when the material is sheared. For one thing, the water-soluble silk dope has been transformed into something waterproof. More importantly, the shearing somehow induces individual proteins to “find each other,” as Schniepp describes, and to self-organize into fibrils. One molecule thick, the fibrils are the thinnest possible threads of silk and are precursors to silk fibers.

Seen through AFM magnification, each fibril shows where the individual proteins have conglomerated. The magnification resembles a string of pearls. It’s the first time that the structure of the native silkworm protein has been imaged at such high resolution.

The work on silk is supported by the Jeffress Memorial Trust. Schniepp published his findings in a paper, “Shear-Induced Self-Assembly of Native Silk Proteins into Fibrils Studied by Atomic Force Microscopy” in the journal Biomacromolecules. Fritz Vollrath of Oxford University is a co-author, as is Cai. They are continuing their work on the structure of the material.

“We don’t know what other secrets silk has hidden for us,” Schniepp says.

Provided by The College of William & Mary

Building a beetle antifreeze

Xylomannan was first reported in 2009, and has been shown to be amongst the most active insect antifreezes found to date. Antifreeze compounds, which are also known as thermal hysteresis factors (THFs), protect the insects’ cells from damage as temperatures fall and ice crystals begin to form. THFs seem to work by sticking to the surface of nascent ice crystals and somehow stopping them from growing, protecting nearby cell membranes from being punctured by needles of ice. 

The unusual thing about xylomannan is its constituents. Every natural THF isolated to date is protein based, but xylomannan is a glycan, a long-chain sugar-based compound. “Xylomannan is the first example of a THF biomolecule with little or no protein component,” says Ishiwata. “Its mode of action is not entirely clear, but it should be different to those of common THFs such as antifreeze proteins and glycoproteins.” 

To confirm the proposed structure of xylomannan, so that they can begin to study how it interacts with ice crystals, Ishiwata, Ito and their colleagues synthesized what they thought to be a key component of the compound’s sugar-based backbone. Their structural analysis, using nuclear magnetic resonance techniques and molecular modeling, confirmed that the structure matches that of the natural compound. It also hints at the way that xylomannan might stick to ice crystals: one face of xylomannan is much more polar than the other face, making one face hydrophilic and the other hydrophobic (Fig. 1). 

“We propose that the hydrophilic phase of xylomannan might bind to the ice crystal, exposing the hydrophobic phase on the ice crystal’s surface,” says Ishiwata. This hydrophobic surface should repel water molecules away from the ice crystal, stopping it from growing any further. “However, the binding mode is still not clear from our structural analysis,” he adds. To test the theory further, the team now plans to synthesize longer fragments of xylomannan to examine their ice-binding ability.

More information: Ishiwata, A., et al. Synthetic study and structural analysis of the antifreeze agent xylomannan from Upis ceramboides. Journal of the American Chemical Society 133, 19524–19535 (2011).

Walters, K.R. Jr., et al. .A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides. Proceedings of the National Academy of Sciences of the USA 106, 20210–20215 (2009).

Provided by RIKEN (news : web)

Environmentally friendly cleaning and washing

Detergents are everywhere – in washing powders, dishwashing liquids, household cleaners, skin creams, shower gels, and shampoos. It is the detergent that loosens dirt and fat, makes hair-washing products foam up and allows creams to be absorbed quickly. Up until now, most detergents are manufactured from crude oil – a fossil fuel of which there is only a limited supply. In their search for alternatives, producers are turning increasingly to detergents made from sustainable resources, albeit that these surfactants are usually chemically produced. The problem is that the substances produced via such chemical processes are only suitable for a small number of applications, since they display only limited structural diversity – which is to say that their molecular structure is not very complex. Now researchers at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB are taking a different approach: they are manufacturing surfactants using biotechnological methods, with the assistance of fungi and bacteria. "We produce biosurfactants microbially, based on sustainable resources such as sugar and plant oil," says Suzanne Zibek, a technical biologist and engineer at the IGB in Stuttgart. The scientist and her team use cellobiose lipids (CL) and mannosylerythritol lipids (MEL) because testing has shown these to be promising for industrial application. They are produced in large quantities by certain types of smut fungus, of the kind that can affect corn plants. What is more, CL also has antibacterial properties.

What marks biological surfactants out from their synthetic competitors is their increased structural diversity. In addition, they are biodegradable, are less toxic and are just as good at loosening fats. But despite all this, to date they are used in only a few household products and cosmetics. The reason is that they are costly and difficult to produce, with low yields. One substance that has been successfully brought to market is the sophorose lipid made by Candida bombicola, which is used by a number of manufacturers as an additive in household cleaning products. This biosurfactant is produced by a yeast that is harvested from bumble-bee nectar.

"If we want natural surfactants to conquer the mass market, we need to increase fermentation yields," says Zibek. To this end, the scientists are optimizing the production process in order to bring down manufacturing costs. They cultivate the microorganisms in a bioreactor, where they grow in a continuously stirred culture medium containing sugar, oil, vitamins and minerals salts. The goal is to achieve high concentrations in as short a time as possible, so they need to encourage as many microorganisms as possible to grow. There are numerous factors with a bearing on the outcome, including the oxygen supply, the pH value, the condition of the cells, and the temperature. The composition of the culture medium itself is also crucial. It is not just a question of how much sugar and oil go into the mix, but also the speed at which they are added. "We have already achieved concentrations of 16 grams per liter for CL and as high as 100 grams per liter for MEL – with a high production rate, too," the group manager is happy to report.

The next step is to separate the biosurfactants from the fermentation medium and to characterize them with the help of industrial partners, determining which surfactants are suitable for use in dishwashing liquids, which are more suited to oven cleaning products, and which are ideal for use in cosmetics. The substances can finally be modified or improved at the enzymatic level. "For instance, we managed to increase wa-ter solubility. After all, the biosurfactant shouldn't form an oily film over the surface of the dishwashing liquid," explains Zibek. The experts have even managed to produce biological surfactants using waste products, by obtaining the sugar needed for the culture medium from straw. The researchers will be presenting biosurfactants they have produced themselves at HANNOVER MESSE from April 23 to 27, 2012 (Hall 2, Booth D22).

Provided by Fraunhofer-Gesellschaft (news : web)

Japan scientist makes violin strings from spider silk

Thousands of the tiny strands can be wound together to produce a strong but flexible string that is perfect for the instrument, said Shigeyoshi Osaki, professor of polymer chemistry at Nara Medical University.

Osaki, who has been working with spider silk for 35 years, has previously suggested the material could be used for surgical sutures or for bullet proof vests, but his passion for the violin inspired him to create something with a musical twist.

In the process of weaving the threads, their shape changes from cylindrical to polygonal, which means they fit together much better, Osaki told AFP.

"During the assembly of normal threads there are many spaces between individual fibres," he said.

"What we achieved left no space among the filaments. It made the strings stronger. This can have all sorts of applications in our day-to-day lives," he said, adding 300 female Nephila maculata spiders had provided his raw materials.

The strength and durability of spider silk is not a new discovery, with previous studies showing it can withstand high temperatures and the effects of ultraviolet light.

Osaki once produced a rope spun from spider silk that he said could theoretically support a 600 kilogram (1,300 pound) weight.

Now his latest creation is making waves among musicians, who have praised the sonorous quality of the spider silk violin strings for their "soft and profound timbre".

"Professional violinists have said they can tell the difference" whether the strings are on a Stradivarius or on Osaki's own $1,200 violin, he said.

"It's one thing to create scientifically meaningful items, but I also wanted to produce something that would be socially accepted by ordinary people," he said.

Details of Osaki's research will be published in Physical Review Letters, a journal of the American Physical Society.

(c) 2012 AFP

Squeezing polymers produces chemical energy but raises doubts about implant safety

In a new study, Northwestern University scientists turned to squeezed polymers and free radicals in a search for new energy sources. They found incredible promise but also some real problems. Their report is published by the journal Angewandte Chemie.

The researchers demonstrated that radicals from compressed polymers generate significant amounts of energy that can be used to power chemical reactions in water. This energy has typically been unused but now can be harnessed when polymers are under stress in ordinary circumstances -- as in shoe soles, car tires or when compacting plastic bags.

They also discovered during the study that a silicone polymer commonly used in implants for cosmetic procedures releases a large quantity of harmful free radicals when the polymer is under only a moderate amount of pressure. These findings suggest the safety of certain polymer-based medical implants should be looked at more closely.

"We have established that polymers under stress create free radicals with overall efficiencies of up to 30 percent and shoot the radicals out into the surrounding medium where they can drive chemical reactions," said Bartosz A. Grzybowski, an author of the paper and the Kenneth Burgess Professor of Physical Chemistry and Chemical Systems Engineering. "These radicals can be useful or they can be harmful, depending on the situation."

Grzybowski and his team are the first to use this energy to drive chemical reactions by simply surrounding the compressed polymer with water containing desired reagents.

The radicals created in the polymer migrate toward the polymer/water interface where they produce hydrogen peroxide, which then can drive chemical processes.

"You can get a surprisingly large amount of chemical energy from a polymer under compression," Grzybowski said. "This energy is, in a sense, free for the taking. Under normal circumstances, the energy is virtually never retrieved from deformed polymers, which then age unproductively. But you could recharge a battery from the energy produced by walking or driving a car. And you could capture even more energy when compacting millions of plastic bags."

Grzybowski is also director of Northwestern's Non-Equilibrium Energy Research Center, which is funded by the U.S. Department of Energy.

"We are interested in new sources of chemical energy, and this energy from the simple breaking of polymers' bonds is not being used," he said. "By surrounding the polymer with a medium, such as water, we can produce environmentally friendly chemical energy. One direction we are pursuing is to use this energy to sanitize water in developing countries. This is possible because hydrogen peroxide produced by squeezed polymers kills bacteria."

The researchers confirmed that mechanical deformation -- moderate squeezing -- created free radicals in the polymers. They also determined the number of radicals produced in a polymer under pressure is approximately 1016 (10 to the 16th) radicals per cubic centimeter of polymer -- a substantial amount.

They next filled polymer tubes with water, squeezed the tubes and measured the total number of radicals that migrated into the surrounding solution. They found that nearly 80 percent of the radicals made the trip.

Grzybowski and his team demonstrated they can squeeze a polymer, such as what might be found in a shoe, tire or plastic bag, and get a mechanical-to-chemical energy conversion of up to 30 percent -- approaching the energy efficiency of a car engine.

The hydrogen peroxide produced when a polymer surrounded by water is squeezed can power a variety of chemical reactions, including fluorescence, nanoparticle synthesis and dye bleaching, the researchers showed.

To illustrate the process, they converted a Nike Air LeBron shoe into a "lightning shoe," where the air pockets in the polymeric sole are filled with a solution of a compound that lights up in the presence of radicals. After a person walked in the shoe for 30 minutes or more, enough radicals were created to generate a blue glow visible to the naked eye.

The researchers studied seven different polymers, including a number of particular public interest. Poly(dimethylsiloxane), a silicon-based material commonly used in medical implants, was one of them. In the lab experiments, the medium surrounding the polymer and the amount of pressure exerted on the material were similar to what would be found in the human body, Grzybowski pointed out.

"Our findings are somewhat worrisome since every polymeric implant in the human body experiences mechanical stresses and, as we now know, can produce harmful free radicals and liberate them into surrounding tissues, which may contribute to diseases such as cancer, stroke, myocardial infarction, diabetes and other major disorders," Grzybowski said. "With this knowledge, I am quite happy to have a metal implant in my knee, rather than a polymer implant.

"From a scientific perspective, our work proves yet again that a phenomenon can be useful or harmful depending on how we implement it," he said. "The same polymer can be a useful source of energy when outside of a human body, yet a potential risk hazard when implanted into it."

More information: The title of the paper is "Mechanoradicals Created in 'Polymeric Sponges' Drive Reactions in Aqueous Media." In addition to Grzybowski, other authors of the paper are H. Tarik Baytekin and Bilge Baytekin (who contributed equally).

Provided by Northwestern University (news : web)