NEWS OF THE WEEK
PROTEIN CRYSTALLIZATION Method may have broad applications A n ingenious new method of assem/ ^ bling two-dimensional arrays of J L J L protein molecules may lead to a general, simple technique for the usually arduous task of protein crystallization, as well as to a new breed of biomolecular "imprints" for materials design. Protein crystallization—which is vital to enable protein structure determination by electron or X-ray diffraction methods—is one of chemistry's most vexing problems. The technique was developed by groups led by California Institute of Technology chemical engineer Frances H. Arnold and by University of Washington bioengineers Viola Vogel and Patrick S. Stayton. In an article to be published in the May 14 issue of the Proceedings of the National Academy of
Sciences, the two groups describe successful 2-D crystallization of the protein streptavidin on a lipid monolayer studded with copper. Streptavidin already has been studied extensively. Researchers not only have created 2-D crystals of streptavidin using biotinylated lipids [Biophys. /., 59, 387 (1991)], but also have prepared 3-D crystal structures. Thus, Vogel notes, this protein was an ideal candidate to test the new method's validity. The method is based on the relatively simple idea that the side chain of the amino acid histidine, found in most proteins, binds to copper atoms attached to lipids. The Caltech group— which included graduate student Daniel W. Pack and postdoctoral researcher Chao-Tsen Chen—developed a lipid with an iminodiacetate-Cu(II) end. The amphiphilic lipid molecules form a single layer with their hydrophobic tails floating on the water surface and their hydrophilic copper heads dangling down. Water-soluble streptavidin molecules under the surface are drawn to and bind with the lipids, then spontaneously crystallize into a 2-D array. The researchers viewed the crystals as they 6
MAY 6,1996 C&EN
In this computer representation of the protein crystallization process, black and yellow ovoids symbolize lipid molecules with long hydrophobic tails (light gray) spread across the water's surface in a layer one molecule thick. Yellow lipids have metal-binding iminodiacetate portion added, with small coppery spheres representing copper ions bound to the iminodiacetate. Large red ovoids represent molecules of the protein streptavidin, which line up in neat rows and crystallize as they bind to copper ions.
were forming with a specially designed Brewster angle microscope developed by Vogel, graduate student William R. Schief Jr., and postdoctoral researcher Wolfgang Frey. Such 2-D arrays "will be of enormous interest in bioscience, and will aid in the structural determination of large assemblies and otherwise difficult-to-crystallize materials,,, says Harold Bright, program officer at the biological science and technology division of the Office of Naval Research in Arlington, Va. Researchers have developed other lipid techniques for crystallization, but they require design of specialized affinity groups for each protein. However, the Caltech/Washington method could be extended to many other proteins. If so, notes Wah Chiu, professor of biochemistry at Baylor College of Medicine in Houston, the method will be of "high interest" to the structural biology
community. "Any recipe one can follow to make 2-D crystals is welcome and exciting," he says. Each monomer of the tetrameric streptavidin contains two histidine units, only one of which binds to the copper. To determine which of the two histidines is involved, Stayton's group created mutant forms of the protein, deleting first one histidine and then the other. They found that the molecules appear to offer their most sterically accessible histidine to the copper, Arnold explains. Additionally, it is fairly easy to attach histidine units to proteins at any location, Stayton points out. This may make it possible to choose which protein face will be upturned in an array. The ability to crystallize a protein in several different conformations also would make its characterization much more rigorous. Some scientists are "very excited" about the possibility of
flipping proteins at will, Stayton notes. The 2-D crystals also could be used to seed growth of 3-D protein crystals, the scientists believe. The group now is looking at different proteins to test the extent of the meth od's applicability. It also is exploring the nature of the biomolecular arrays, which could have many uses in materials sci ence, including as biosensors. Elizabeth Wilson
Two groups explore new allergy treatments Allergy sufferers could be reaching for designed peptides and DNA vaccines for relief, instead of antihistamines and allergy shots, if two recent studies pan out. A team of scientists in Philadelphia and London is using structure-based drug design to develop small peptides that can prevent triggering of the events leading to the allergic response. Another team, composed of Taiwanese and U.S. scientists, is exploring the use of vaccines containing allergen-coding DNA to in duce tolerance to foreign proteins. Nei ther group is close to a drug or a treat ment, but their initial findings give aller gy sufferers something to cheer about. An allergy begins when immuno globulin Ε (IgE) binds to an allergen, such as pollen, and then to IgE's recep tor in so-called mast cells. Histamines released by these cells trigger the in flammatory response—leading to sneez ing spells, itchy eyes, and dripping nos es. Thus, antihistamines are widely used to treat allergies. Alternatives to antihistamines are widely sought, and some researchers are targeting the IgE-receptor binding interaction in their work on drug de sign. For example, Brian J. Sutton, a reader in molecular biophysics at Ran dall Institute, King's College, London, and colleagues there and at Kimmel Cancer Institute of Jefferson Medical College, Philadelphia, have developed two peptides that can bind IgE by mimicking the section of the natural re ceptor that contributes most to binding [Nat. Struct. Biol., 3, 419 (1996)]. One peptide, called CVC1O(L-262), con tains only L-amino acids except for the carboxyl-terminal D-cysteine. The other peptide, called cyclo(rD-262), contains only D-amino acids except for the car
boxyl-terminal L-cysteine. Disulfide bridges between cysteines at the ends cyclize these peptides and restrict their conformational flexibility. Sutton and coworkers have shown through in vitro assays that both pep tides trap IgE and inhibit "degranulation" of mast cells, the process that un leashes allergic symptoms. By binding IgE before it reaches the natural recep tor, these peptides can thus, in princi ple, prevent an allergic response. This approach is not novel, says immunologist and Philadelphia team member Robert Korngold. Other groups have tried to use whole proteins to do the same thing. What's different is use of small peptides: If they work, they are preferable to whole proteins because they are easier to design for better effica cy and easier to produce in large quanti ties, he explains. Another widely used therapy is al lergy shots. Such injections induce tol erance to foreign proteins by exposing the body to small allergen doses and increasing the dose over time. This ap proach protects against allergens such as bee venom and grass pollen, but has
not had consistent success with other allergens. In a twist on this strategy, Kaw-Yan Chua and Kue-Hsiung Hsieh of the Col lege of Medicine of National Taiwan University, Taipei, and coworkers in Tai wan and at Johns Hopkins University School of Medicine are studying use of DNA vaccines to protect against aller gens [Nat. Med., 2,540 (1996)]. Unlike al lergy shots, these vaccines contain aller gen-coding genes rather than allergens. The researchers injected rats with plasmid-borne DNA that codes for an allergen made by house dust mites. The allergen was expressed and persisted in muscle tissues for up to six months without triggering an IgE response. And challenging the vaccinated rats with the allergen produced a much reduced IgE response and histamine release, com pared with untreated rats. The team also found that the re sponse of the treated rats to another al lergen, ovalbumin, is not affected by the dust mite DNA vaccine. Thus, the allergy-suppressing effect is not only long-lasting but also specific. Maureen Rouhi
Dow to spend $1 billion on environment, health Dow Chemical will invest $1 billion over the next 10 years to achieve an ambitious set of environmental and health and safety goals at its plants worldwide. By 2005, it seeks a 90% reduction in work place illnesses and injuries, a 50% cut in overall chemical emissions, and a 50% drop in generation of waste and waste water per pound of production. These goals were set forth late last month at the National Press Club in Washington, D.C., by Dow President and Chief Executive Officer William S. Stavropoulos. "We have known for a long time that pollution is environmentally and eco nomically wasteful," Stavropoulos not ed. "Now we are going to prove it. Compared to what we've spent histori cally, this future investment is not ex traordinary. What is extraordinary is that, overall, we expect a 30 to 40% re turn on this investment." The baseline is 1994, and the goals are to be met by Dec. 31,2005. For example, to cut injuries and illnesses 90%, Dow must reduce incidents reportable to the Occupational Safety & Health Adminis tration from 2.35 per 200,000 hours
worked in 1994 to 0.24 in 2005. And the company seeks a 90% drop in "loss of primary containment incidents" (leaks, breaks, and spills from processing equipment). It will cut emissions of dioxins 90%. And it targets a 75% cut in emissions of "priority compounds," which include
Stavropoulos: pollution is wasteful MAY 6,1996 C&EN
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