MUTANTS POINT TO PACKING’S ROLE IN PROTEIN EVOLUTION AND STABILITY

FAYETTEVILLE, Ark. - University of Arkansas researchers have created mutants in a model protein and demonstrated the importance of protein core packing in stabilizing proteins and influencing their evolution. These findings may help researchers create more stable proteins for use in medicine and industry.

Welsey Stites, associate professor of chemistry and biochemistry, and graduate student Junmei Chen reported their findings in a recent issue of the journal Biochemistry.

"The genome can give you a protein sequence, but not its structure," Stites said. "It is structure that determines a proteins’ function."

Researchers believe that packing, or the way the protein’s side chains fit together like a three-dimensional jigsaw puzzle, influences the molecule’s stability. They also believe that proteins may not be packed for optimal stability, due to other considerations within a cell.

Many industries that use enzymes could benefit from increasing protein efficiency, literally sweetening the deal in the case of soft drink companies, Stites said. Take the case of the enzyme glucose isomerase, used to make high-fructose corn syrup, the most common sweetener used in the United States today; the enzyme beds used to produce this sweetener eventually wear out. An increase in protein stability that decreases the need for this enzyme could save companies millions of dollars a year.

Stites focuses on the protein’s core, an area found on the interior of a protein that is thought to influence its structure, because small mutations may cause big energy changes.

The researchers sought to determine the energetic stability of the protein core, and to see if cores from similar proteins pack themselves in similar ways. They targeted the protein staphylococcal nuclease, comparing the sequence of amino acid side chains in the core of this protein and 42 other proteins from the same family.

They determined the frequency with which different amino acids occurred at specific positions in the protein cores. In the laboratory, they created about 200 mutated cores by substituting between one and four amino acids at six specific sites in the staphylococcal nuclease core.

They then compared the energies of the most common sequences in the protein family with the energies of the core mutants that shared those sequences. They found that these were not necessarily the most stable proteins, but that they had best interaction energies between atoms within the core. In other words, even large-scale mutations mattered most in terms of their local interactions.

"If you change one side chain, you change its interactions with all of the side chains," Stites said. "This research shows that the side chains interact with their immediate neighbors. Energy optimization is a local problem."

The chemists found that packing interaction energies proved to better predict side-chain identity than protein stability.

"This further supports the idea that the importance of packing has been under-appreciated relative to other factors that influence the stability of proteins," Stites and Chen said in the paper.

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