Beta-barrel protein activates fluorescence made from scratch for the first time

For the first time, scientists have completely created a protein that can bind to a small target molecule. Researchers at the University of Washington School of Medicine Report Progress in the September 12 issue of the journal Nature.

Previously, such small molecule binding proteins have been made by modifying proteins already existing in nature. This approach has considerably limited the possibilities. The ability to make such proteins from scratch, or de novo, opens the way for scientists to create proteins unlike those found in nature. These proteins can be custom designed with high precision and affinity to bind and act on specific small molecule targets.

The main authors of the study are Jiayi Dou and Anastassia A. Vorobieva, both fellows from the lab of lead author David Baker, professor of biochemistry at the UW School of Medicine and director of the Institute of Protein Design at UW Medicine . Baker is also an investigator at the Howard Hughes Medical Institute.

The technique should have wide application in research, medicine, and industry, according to Baker.

"The successful de novo design of custom proteins with small molecule binding activity paves the way for the creation of increasingly sophisticated binding proteins that will not have the limitations of proteins designed by altering existing protein structures." ". .

To make the protein, the researchers had to make another first: to create from scratch a cylinder-shaped protein called beta-barrel. The beta barrel structure was ideal because one end of the cylinder could be designed to stabilize the protein, while the other end could be used to create a cavity that could serve as a binding site for the target molecule.

Proteins consist of long chains of amino acids. Once synthesized, these chains fold into precise forms allowing proteins to perform their functions. The shapes that these chains take are usually incredibly complicated, but two features are often present: the alpha helices, which form when the chain of sections is bypassed by a central axis, and the sheet-like structures, called beta sheets.

The beta sheets are formed when two or more sections of different parts of the amino acid chain, due to folding, move side by side in 3D space. These sections are "joined" by hydrogen bonds, creating a sheet-like structure. These beta sheets, in turn, can assemble into barrel-type structures, called beta barrels. In nature, beta-barrel proteins bind to a wide range of small molecules.

To design the new protein, Dou and Vorobieva used a software platform, developed in Baker's lab called Rosetta. It can predict the shape that a particular amino acid chain will take after synthesis and can indicate how changing the individual amino acids along the chain can alter that shape. This predictive power makes it possible to test different combinations of amino acids to design a protein having the desired shape and function.

To create the cavity, the researchers used a new powerful docking algorithm, called the "Rotamer Interaction Field" (RIF), developed by William Sheffler, principal investigator at Baker Lab. GIR quickly identifies all potential cavities that meet the prerequisites for binding specific molecules.

Featuring new RIF mooring methods, Dou, Vorobieva and Sheffler designed the beta barrels to bind a compound called DFHBI, a component similar to that contained in the green fluorescent protein, which fluoresces when it is exposed to certain light frequencies. Green fluorescent protein is commonly used in biological research to locate molecules and structures in a living organism and to track their movement.

In their article, the researcher demonstrates that their personalized protein has eagerly linked and activated the DFHBI compound.

"It worked in bacterial cells, yeast and mammals," Dou said, "and half the size of green fluorescent protein should be very useful for researchers."

Baker said the approach will allow researchers to explore an effectively unlimited set of basic structures with custom shapes to link the molecule of interest.

"Just as important," he added, "it greatly advances our understanding of the determinants of protein folding and binding beyond what we have learned from describing existing protein structures."

Explore more:
Study Reveals Ways to Design Key Protein Binding Structures

More information:
Jiayi Dou et al, De novo design of a β-barrel of fluorescence activation, Nature (2018). DOI: 10.1038 / s41586-018-0509-0