Caltech Researchers Create New Proteins by Recombining the Pieces of Existing Proteins

PASADENA, Calif.—An ongoing challenge in biochemistry is getting a handle on protein folding-that is, the way that DNA sequences determine the unique structure and functions of proteins, which then act as "biology's workhorses." Gaining mastery over the construction of proteins will someday lead to breakthroughs in medicine and pharmaceuticals.

One method for studying the determinants of a protein's structure and function is to analyze numerous proteins with similar structure and function-a protein family-as a group. By studying families of natural proteins, researchers can tease out many of the fundamental interactions responsible for a given property.

A team of chemical engineers, chemists, and biochemists at the California Institute of Technology have now managed to create a large number of proteins that are very different in sequence yet retain similar structures. The scientists use computational tools to analyze protein structures and pinpoint locations at which they can break them apart and then reassemble them, like Lego pieces. Each new construction is a protein with new functions and new potential enzyme actions.

Reporting in the April 10 issue of the Public Library of Science Biology, Caltech graduate student Christopher Otey and his colleagues show that they have successfully taken three proteins from nature, broken them each into eight pieces, and successfully reconstructed the pieces to form many new proteins. According to Otey, the potential number of new proteins from just three proteins is three raised to the eighth power, or 6,561, assuming that each protein is divided into eight segments. "The result is an artificial protein family," Otey explains. "In this single experiment, we've been able to make about 3,000 new proteins."

About half of the 6,561 proteins are viable, having an average of about 72 sequence changes. "The benefit is that you can use the new proteins and new sequence information to learn new things about the original proteins," Otey adds. "For example, if a certain protein function depends on one amino acid that never changes, then the protein apparently must have that particular amino acid."

The proteins the team has been using are called cytochromes P450, which play critical roles in drug metabolism, hormone synthesis, and the biodegradation of many chemicals. Using computational techniques, the researchers predict how to break up this roughly 460-amino-acid protein into individual blocks of about 60 to 70 amino acids.

Otey says that this is an important result when considering the old-fashioned way of obtaining protein sequences. Whereas, over the past 40 years, researchers have fully determined 4,500 natural P450 sequences, the Caltech team required only a few months to create 3,000 additional new sequences.

"Our goal in the lab is to be able to create a bunch of proteins very quickly," Otey says, "but the overall benefit is an understanding of what makes a protein do what it does and potentially the production of new pharmaceuticals, new antibiotics, and such.

"During evolution, nature conserves protein structure, which we do with the computational tools, while changing protein sequence which can lead to proteins with new functions," he says. "And new functions can ultimately result in new treatments."

In addition to Otey, the other authors of the paper are Frances Arnold (the corresponding author), who is Dickinson Professor of Chemical Engineering and Biochemistry at Caltech, and Otey's supervising professor; Marco Landwehr, a postdoctoral scholar in biochemistry; Jeffrey B. Endelman, a recently graduated Caltech graduate student in bioengineering; Jesse Bloom, a graduate student in chemistry; and Kaori Hiraga, a Caltech postdoctoral scholar who is now at the New York State Department of Health.

The title of the article is "Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450."