random mutagenesis phage display to introduce mutations into the truncated binding domain. In this technique, genes are modified randomly and in serted into phages (bacterial viruses), Taking a key step in efforts to shrink which express the corresponding pro proteins to the size of small organic mol teins on their surfaces. Each protein ecules, scientists at Genentech, South that binds to a target with high affinity San Francisco, have removed almost half is isolated and identified by sequenc a protein's binding domain and then re ing the gene from its associated phage. Braisted and Wells modified the stored the domain's structural stability and binding capacity. The research may protein A domain in three steps—first have implications for drug design and revising an area that became exposed to solvent when the helix was re aid protein-folding studies. "A folded structural unit of a protein moved, next stabilizing the interface can be put on a serious diet and re between the two remaining helices, duced in size without sacrificing bind- and then optimizing residues in the an tibody-binding site. δ The final 33-residue, I two-helix structure ^ bound to antibody I with about one| fourth the affinity of ^ the natural protein I domain. S Genentech scien| tist Brian Cunning§" ham, Wells, and co§ workers used a simI ilar strategy last §. year to reduce the | hormone atrial na| triuretic peptide | from 28 residues to 1-15 while retaining high potency. Other groups have also devised miniprotein Binding domain of protein A (purple) in complex with antibodydomains in other fragment (blue). Braisted and Wells restored domain's structural ways. integnty and binding after deleting helix 3. "The paper by Braisted and Wells mg," says staff scientist James A. Wells represents another particularly excit of Genentech's department of protein ing demonstration that protein do engineering. He and postdoctoral fel mains need not be as large as has low Andrew C. Braisted minimized the been traditionally thought," says a protein domain using mutagenesis and commentary in PNAS by professor rational design techniques [Proc. Natl. William F. DeGrado and research as Acad. Sei. USA, 93, 5688 (1996)]. sistant professor Tobin R. Sosnick of Braisted and Wells removed a pure the department of biochemistry and ly structural α-helix from the 59-resi- biophysics at the University of Penn due antibody-binding domain of pro sylvania School of Medicine. tein A, a popular reagent for purifying The work may be applicable to antibodies. The domain's two other drug design. "Today, there is no good α-helices form its binding site. The de way to be able to take a protein phar letion made the protein domain fall maceutical and rationally reduce it to apart, destroying its distinct structure a small molecule," Wells says. "What and its ability to bind antibody. we've done is to take a protein and The researchers reinstated the do bring it to a realm where it's amena main's structural integrity and a sub ble to manipulation by chemical syn stantial degree of its binding affinity by thesis, which greatly expands the di using a combinatorial technique called versity that can be introduced." He
Minimized protein still binds antibody
hopes to reduce the size of protein binding domains further and to use the minimized domains as models to de sign small organic molecule mimics. Protein minimization may also aid studies of protein folding. "Folding has been difficult to study in part because the large size of proteins has hampered both computational and experimental work," write DeGrado and Sosnick. "This 33-residue structured protein should serve as an attractive starting point for the systematic study of both kinetics and thermodynamics of pro tein folding in a much more fine-tuned manner than has been possible with site-directed mutagenesis," a biological method of modifying proteins. Stu Borman
DuPont loses Benlate birth defect trial A Florida jury has awarded $4 million to the family of a six-year-old boy who was born without eyes—a condition the family claims was caused by prena tal exposure to DuPont's Benlate DF fungicide. The four-week trial was held in the 11th Circuit Court in Dade County. The lawsuit is the first to come to trial linking birth defects to Benlate. For more than three years, DuPont has been wag ing a legal battle against farmers claim ing the fungicide damaged or killed crops. The plaintiffs' lead attorney, James L. Ferraro, says the verdict could pave the way for lawsuits in 40 other cases of deformed or missing eyes that victims blame on Benlate. Stacey J. Mobley, DuPont senior vice president for external affairs, calls the verdict "a blow to science and to our jury system. Birth defects are a human tragedy. The jury in this case was none theless faced with the challenge of looking beyond that tragedy to deter mine whether DuPont's product, Ben late, bears any connection with John Castillo's birth defect. In this instance, the challenge was not met." DuPont plans to appeal the verdict. During her pregnancy, the boy's mother took frequent walks along the edge of Pine Island Farms in Miami, which used the fungicide. At one time, Ferraro says, she was sprayed by a "misty cloud" that covered more than 50% of her body. JUNE 17,1996 C&EN
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