LBL Scientists Develop New Techique for Studying Protein Stability

August 21, 1992

By Lynn Yarris,

A team of scientists at LBL's Center for Advanced Materials (CAM), led by organic chemist Peter Schultz, winner of this year's E.O. Lawrence Award, has developed a new technique for studying protein stability using unnatural amino acids. The technique should benefit the efforts now underway at CAM and elsewhere to develop new proteins with properties not restricted by the 20 naturally occurring amino acids.

In addition to Schultz, who is also a professor of chemistry at UC Berkeley, other members of this team included David Mendel, Jonathan Ellman, and Zhiyuh Chang of CAM and UCB, plus David Veenstra and Peter Kollman, of the UC Med Center in San Francisco.

Stability, like other protein properties, is largely determined by structure, which in turn, is a product of the amino acids that link together into the long folding chains that make up all proteins. To measure protein stability and to look for ways of increasing that stability, scientists have been using a technique called "site-directed mutagenesis." In this technique, one of the protein's amino acids at a select target site is replaced with a different type of amino acid and the property changes that result are observed. A major problem with this technique is that in replacing one natural amino acid with another, often more than one of the protein's properties are affected.

This drawback can be eliminated by replacing one or more of the 20 natural amino acids with an unnatural amino acid -- one that was created in the laboratory. During the past year, Shultz and his colleagues have effectively expanded the genetic code to include over 60 unnatural amino acids with novel backbone and side chain structures.

Says Schultz, "The ability to site-specifically incorporate unnatural amino acids into proteins makes possible more precise changes in their properties. This dramatically increases our ability to probe protein structure-function relationships by allowing us to tailor amino acid substitutions to probe specific structural or mechanistic questions."

Schultz and his colleagues recently used unnatural amino acids to study the stability of lysozyme, an enzyme found in human tears that breaks down the cell walls of bacteria. Scientists believe lysozyme could be used to synthesize novel polymers along the lines of chitin, the main ingredient in the hard outer shells of crustaceans. For the Schultz team, lysozyme was a good model because its crystal structure has been well characterized.

The team designed their unnatural amino acids with the help of molecular modeling and computer simulations. They then substituted their artificial amino acids for several natural amino acids along lysozyme's active site (where catalysis takes place). This site is a side chain featuring a hydrophobic (water-repelling) core surrounded by a cavity. With their unnatural amino acids in place, the team was able to do such things as increase the hydrophobic surface area; enlarge the surrounding cavity; alter the hydrophobicity of the core; and alter the configurational entropy of the side chain.

Says Schultz. "We were able to demonstrate that hydrophobicity, packing, cavity formation, and side chain conformation entropy all play important roles in determining lysozyme's stability and must be considered in any effort to increase that stability."

Schultz says this technique can also be applied to studying the forces that determine protein folding, including hydrogen bonding, electrostatic interactions, and main amino acid chain effects. For the future, expanding upon the 20 amino acids created by Nature with laboratory-designed amino acids should open the door to virtually limitless possibilities in the modification of existing proteins and the development of brand new varieties.

Schultz won the Lawrence Award for his research into the creation and application of unnatural amino acids and for his contributions to understanding the mechanisms behind molecular recognition and catalysis in biological systems, particularly his development of catalytic antibodies. He is scheduled to receive his award at a ceremony in Washington, D.C., on September 16. The Ernest Orlando Lawrence Memorial Award, given by DOE each year to outstanding young scientists and engineers, consists of a citation, a gold medal, and $10,000.