Scientists design responsive self-assembling helical protein fibers, which are v

2024-05-22

Recently, the research group led by Professor David Baker from the University of Washington in the United States has, for the first time, designed protein fibers with acid-base responsiveness from scratch and obtained high-resolution structural validation.

Through structural design, a very precise response and control of acidity and alkalinity have been achieved: assembly and disassembly can be realized based on slight changes in pH, and the pH threshold can also be controlled through design.

This design only requires the selection of monomer core structural sites in the computational design and the introduction of amino acids that respond to acid and base. Therefore, this method is universal and can widely produce more acid-base responsive materials.

This de novo designed environmentally responsive, structurally controllable self-assembling protein fiber is expected to develop into a variety of responsive biomaterials applications.

For example, responsive hydrogels, environmentally responsive biomaterials, and drug release for specific pH environments.

The reviewer commented on the study: "This study is an important breakthrough in the rational design of responsive materials. The success rate of the authors' design is obvious, and reliable protein assembly design, especially helical fibers, is very difficult, and only this team can do it at present."Recently, a related paper titled "De novo design of pH-responsive self-assembling helical protein filaments" was published in Nature Nanotechnology [1].

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Hao Shen, a postdoctoral researcher at the University of Washington, is the first author and co-corresponding author, and Professor David Baker serves as the co-corresponding author.

Nature has evolved many nanomaterials that are responsive to environmental factors (such as pH) to achieve precise and dynamic self-assembly.

For example, spider silk proteins assemble under acidic conditions; CTP (cytidine triphosphate) synthetase assembles into fibrous structures under acidic conditions to maintain homeostasis in yeast cells during periods of fasting.

Ribosomal proteins inside bacteria undergo conformational changes under acidic conditions to assemble into long tubular structures to generate power. These materials have inspired many bioengineers to design pH-responsive protein materials.Previous acid-base responsive nanomaterials, some imitated silk fibroin, and some introduced histidine, which changes with the acid-base level, into the peptide chain stacking.

However, designing acid-base responsive fibers with precise structures and adjustable acid-base thresholds from scratch is still an "unsolved problem."

Based on this, the team imagined that they might be able to design a self-assembling helical protein fiber that can control the folding of monomers through the acid-base level.

"We speculate that its monomer core is composed of histidine: the protonation state of histidine is based on the change of the solution's acid-base level. The protein fiber assembled from thousands of such monomers will be very sensitive and coordinated in its acid-base response," said Shen Hao.

The research group has designed an acid-base responsive protein trimer, and by using the author's previous method of designing monomers into helical fibers, the monomer was designed into an acid-base responsive protein self-assembling fiber [2].In this study, researchers first derived various possible conformations of helices for the monomers with acid-base adaptability from computer design.

Then, using the Rosetta computational method developed in this laboratory, the protein interface was designed, and the designed protein sequences were reverse-transcribed into DNA sequences.

Shen Hao explained, "This allows the synthetic gene to express proteins inside bacteria, purify the proteins by biochemical means, and characterize the structure by electron microscopy. Then, find the design that can form micrometer-scale fibers, and resolve the fiber structure by cryo-electron microscopy."

On the other hand, the research group adopted various methods to characterize the acid-base responsiveness of the designed fibers.

Specifically, based on electron microscopy, the equilibrium length of the fibers under different acid-base conditions can be observed. Then, researchers used fluorescence microscopy to observe the dynamic assembly and disassembly of fibers under different acid-base conditions in real-time.To enhance resolution, they also employed atomic force microscopy to be able to observe in real-time the structural changes of fibers under different pH levels with precision. The design of different pH thresholds and the revision of structures went through several iterations and upgrades.

In the initial fiber structure resolved by cryo-electron microscopy (codenamed DpHF18), researchers found that the primary first design interface was in line with the computational design, but the secondary second interface exhibited variations.

For the solubility of protein fibers and computational complexity, the second interface is relatively small. Previously, the fibers designed by Shen Hao also had a second interface that conformed to the computational design, but this time the second interface showed a two-fold symmetry.

Specifically, the original AB interface is now alternated in sequence as AA, BB. This is also an unexpected change that often occurs in experimental science, and two-fold symmetry has its own characteristics and uses.

From this, Shen Hao speculated: "I have not encountered such fibers that produce two-fold symmetry before, possibly because the monomers responsive to pH are derived from trimers, which have their own symmetry. Therefore, the stacking produced by them also has more possible spatial conformations for assembly into fibers."The helical symmetry of self-assembled fibers is quite complex, and slight changes may lead to different helical symmetries or the inability to embed into fibers.

The first time they encountered this situation, although not confident, researchers still tried to disrupt the two-fold symmetry of the second interface by point mutations of some amino acids.

After successfully disrupting it, they then attempted to introduce some complementary charged amino acids to stabilize the interface that originally had pure helical symmetry. Subsequently, the researchers were pleasantly surprised to find that the fibers could grow back.

However, because the monomers themselves have symmetry, and low-resolution fibers are all similar, it was not until the high-resolution cryo-electron microscopy structure of the fibers was resolved that the team confirmed the "new" fiber (code name DpHF19) indeed matched all the interfaces of the helical symmetry calculated in the initial design.

From this, two similar but different fibers are expected to be used for some studies related to chirality.Shen Hao explained, "Due to the two-way symmetry of DpHF18, it has a bi-directional homogeneity (chirality cancels each other out), while DpHF19 has pure helical symmetry, possessing the inherent polarity of the protein."

Shen Hao graduated from the Department of Biology at Tsinghua University with a bachelor's degree, and then went to the University of Washington to pursue a doctorate in Molecular Engineering, under the guidance of American Academy of Sciences member, Professor of the University of Washington, and pioneer in the field of computational protein design, David Baker.

Currently, Shen Hao is engaged in postdoctoral research at the Protein Design Center of the University of Washington.

It is understood that when the team was designing self-assembling helical protein fibers, the AI in the field of protein had not yet emerged, so they mainly used the Rosetta software based on physical energy and statistical energy calculation methods.

Shen Hao pointed out that currently, AI's prediction of helical symmetry is not accurate, and AI methods will be introduced into different aspects of the subject in the future.The next step for this research group is to study the material properties of fibers assembled in response to pH levels. For instance, they will examine the effects on the viscosity of the solution, as well as the physical and chemical properties produced by the combination of biomaterial.

Additionally, fibers can be used to encapsulate drugs through related preparation methods to achieve release at specific pH levels. Currently, they are also exploring more applications of pH-responsive materials, and interested parties are welcome to contact Dr. Shen Hao.

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