Designed proteins debunked?

Duke biochemist linkurl:Homme Hellinga;http://www.biochem.duke.edu/faculty/homme-hellinga is facing another potential blow to his troubled career in protein and enzyme design.Hellinga has been linkurl:under investigation;http://www.the-scientist.com/blog/display/54889/ for possible research misconduct, following the linkurl:retraction;http://www.the-scientist.com/blog/display/54502/ of a Science paper on computational design of enzymes in February 2008. This week in the Proceedings of the National Academy of Sciences, Hellinga's former postdoc Birte Höcker and colleagues at the Max Planck Institute for Developmental Biology in Germany linkurl:dispute the conclusions;http://www.pnas.org/cgi/doi/10.1073/pnas.0907950106 of his studies on ligand-binding proteins, which appeared in linkurl:Nature in 2003;http://www.ncbi.nlm.nih.gov/pubmed/12736688 and linkurl:PNAS in 2004.;http://www.ncbi.nlm.nih.gov/pubmed/15148405 The interactions between ligands and their receptor are central to many biological processes and can potentially be tweaked to design novel biosensors and enzymes.The original papers from Hellinga's group purported to show that his team modified several proteins from E. coli in order to accept, for instance, the explosive TNT, instead of the sugars to which they normally cinch themselves around. "We applied our algorithms, took the plunge, and built the predicted mutations," Hellinga told The Scientist in an interview last year. "That was very exciting."Hellinga's group had relied on their in-house fluorescent probe to indirectly report successful binding, but Höcker's group has now used direct measurements based on nuclear magnetic resonance imaging and other techniques to probe five of Hellinga's designed proteins. They found that none of the designed proteins were bound to ligands as expected. The proteins are supposed to change their three-dimensional shape when the ligand binds to their receptor, but the one protein for which they solved the structure remained in its open conformation. Höcker also told The Scientist that her unpublished experiments with commercially available fluorophores did not show any binding, but she did not replicate Hellinga's studies with his custom fluorophores. In general, she notes, the fluorophore assay may be prone to false positives.Hellinga did not respond to specific questions about the newest paper but provided a statement to the The Scientist, describing several possible reasons for the discrepancy that his group would investigate. "If these studies also show that our original interpretations are in error, we deeply regret that our reports of these designed receptors do not live up to closer scrutiny. In this case, we offer our sincere apologies to researchers whose work was negatively impacted by these reports," according to the statement. (The 2003 Nature paper has been cited nearly 300 times.) linkurl:Loren Looger,;http://www.hhmi.org/research/groupleaders/looger_bio.html a coauthor on the disputed papers and now a researcher at the Howard Hughes Medical Institute Janelia Farm campus, made it clear that Hellinga was not speaking for the group and considered the fluorophore assay a dead-end. "I do not dispute anything in Dr. Höcker's work; in fact, I strongly encouraged her to pursue this line of research," he said. He explained that he had encountered problems with the stability of the proteins and the robustness of the assay during his time at Duke and with additional research in his postdoctoral laboratory. He did point out, however, that TNT-binding proteins cloned into bacteria were still able to perform their function in a signal transduction cascade that depended on ligand binding. "To me, this remains the biggest unanswered question of the affair," he said.This is not the first time that a structural study has contradicted Hellinga's ligand-binding assay. In 2005, Patrick Telmer and linkurl:Brian Shilton;http://www.biochem.uwo.ca/fac/shilton/Shilton_Biochem/Home.html of the University of Western Ontario in London linkurl:published a paper;http://www.ncbi.nlm.nih.gov/pubmed/16288781 in the Journal of Molecular Biology showing that Hellinga's zinc biosensor -- a modified maltose-binding protein described in a linkurl:2001 PNAS paper;http://www.ncbi.nlm.nih.gov/pubmed/11320244 -- was not changing conformation as Hellinga's group had claimed, suggesting the protein was not functioning properly. "All of our results mesh with the behavior [Hellinga's group] describes," Shilton told The Scientist, "but then we actually looked at the structure it wasn't closing up the way it was designed to."And while no one has alleged misconduct with respect to these three papers, questions linger about the earlier retractions. Hellinga was the senior author of a linkurl:celebrated Science paper;http://www.ncbi.nlm.nih.gov/pubmed/15218149 in 2004, claiming his team had been the first to design and synthesize a novel enzyme, called novoTIM. It was a scientific first, but after an independent researcher was unable to replicate his results, Hellinga retracted it along with a linkurl:follow-up paper;http://www.ncbi.nlm.nih.gov/pubmed/17196220 in the Journal of Molecular Biology. Initially, he directed the blame at his student, lead author Mary Dwyer, who Duke investigated and cleared of research misconduct. Then, as his own behavior came under scrutiny from his critics, he publicly invited Duke to begin an investigation of his role in the disputed work. Duke representatives said that the investigation is still ongoing. "It would be inappropriate for Duke to comment on any specific proceedings due to confidentiality and other restrictions," Doug Stokke said. Hellinga told The Scientist that the Institute for Biological Structure and Design that Duke had established for him and his wife, Lorena Beese, in 2005 no longer exists. linkurl:David Baker;http://depts.washington.edu/bakerpg/ of the University of Washington at Seattle has since laid claim to the first designed enzyme, but it has a much lower binding affinity than natural enzymes, which is what inspired Höcker to take a closer look at the problem. "Ligand-binding design has not been attempted [since] the [Hellinga] publications in Nature and PNAS," Höcker said. "We need to focus again on binding in order to improve receptor design as well as enzyme design."Correction (October 14): The original version of the article incorrectly stated that the zinc biosensor did not bind to its ligand. In fact binding was observed, but it did not adopt the predicted shape change, suggesting it was not functioning properly. The Scientist regrets the error.
**__Related stories:__***linkurl:Duke investigating misconduct?;http://www.the-scientist.com/blog/display/54889/
[25th July 2008]*linkurl:Rumblings over Science retractions;http://www.the-scientist.com/blog/display/54502/
[26th March 2008]*linkurl:Proteins by design;http://www.the-scientist.com/2006/7/1/26/1/
[July 2006]*linkurl:Making enzymes from proteins;http://www.the-scientist.com/article/display/22250/
[25th June 2004]

Designed proteins debunked?

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