Update: Links to the Höcker PNAS abstract and paper are now functional, and The Scientist has posted the full text of comments submitted by Hellinga and Looger (links to their statements posted below among the comments).
Whispers that more of Homme Hellinga’s landmark work could not be replicated began last summer as part of our lengthy discussion of the retraction of Hellinga’s 2004 Science paper (here and earlier here) and then his 2007 JMB paper … and his accusation of misconduct laid against his grad student Mary Dwyer. More recently, observers close to the situation had advised us to watch PNAS for an interesting report on the matter.
Whereas Science was mum on the whole debacle when their article was retracted, Nature has taken a proactive analytic approach to the possibility that Hellinga’s 2003 Nature article (and a 2004 PNAS paper) may need to be retracted based on a report entitledComputational design of ligand binding is not a solved problem in PNAS by Schreier et al. … announced even in advance of e-publication by Nature News.
According to Nature News,
Now Birte Höcker, a former postdoctoral fellow of Hellinga’s, and her team at the Max Planck Institute for Developmental Biology in Tübingen, Germany, have assembled and analysed five of the designed proteins that seemed to work best5. She found that all five were very unstable, and one was too unstable to analyse further.
The group then examined the structure of one of the proteins using crystallography and found that its binding pocket was similar to that predicted by Dezymer — but that it did not bind its intended ligand, which was serotonin. And using three methods to detect the changes in stability, heat and shape that normally occur when proteins bind their ligands, the team found no evidence that the designed proteins were binding their intended ligands.
“All together, our combined analysis of the binding properties of the designs indicates that no specific binding of the target ligands to the respective designs occurs,” Höcker’s team reports.
Hellinga’s lab is studying her re-analysis.
Höcker speculates that she obtained different results from Hellinga because she used different methods to test binding. Her methods included direct measurements of binding, such as isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. Hellinga used an indirect method: he designed the proteins so that they included a fluorophore that emitted a signal when the proteins changed shape, which his team interpreted as an indicator that the proteins had bound their ligands. But Höcker says the assay might have given a false-positive signal if the ligand or the solvent in which it was dissolved interacted with the fluorophore.
Hellinga says if his studies of low concentrations of proteins find that they do not bind their ligands, then he will accept Höcker’s explanation: “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,” he wrote.
More deep regret. Other scientists interviewed considered various angles as to why the results might be so different – but all agreed the controversy needs to be resolved and soon. While one former Hellinga postdoc and Nature article coauthor stands by at least one of the proteins reported, the lead author – a former grad student and co-author on the retracted Science article – is not surprised that his findings have been called into question.
“I am not terribly surprised by [Höcker's] results,” Looger wrote to Nature, adding that he had begun to think “that the designed proteins did not work very well, due to several lines of data” collected in Hellinga’s and other labs.
… In retrospect, Looger says in his opinion that “more attention [should] have been drawn to the aggregation and instability of the proteins, and how that might give rise to artefacts.”
Indeed, Hockner became concerned about questions raised and their impact on her own work:
Höcker says that she had a “good relationship” with Hellinga, and had discussed crystallizing the designed proteins with Hellinga while she was still at Duke. “Since I never heard of the outcome [of the crystallization trials], and the program Dezymer, which I myself was using, was under question, it became more and more important for me to know what it was good at and what not,” she says.
Höcker says her results also have broader importance for the protein-design field. “I think that we need to focus again on binding in order to improve receptor design,” she says, “as well as enzyme design”.
Nature alludes to findings from researchers at the University of Western Ontario (Telmer & Shilton), as does Perplexed Periplasmic in a comment in the prior Hellinga discussion on this blog.
Nature: Another group has analysed the structure of a different set of engineered proteins described by Hellinga in a 2001 PNAS paper6, and found that the proteins behaved differently than Hellinga predicted they would7. The designed proteins did bind their intended ligand, a zinc ion, but did not adopt the ‘closed’ state normally associated with binding.
Perplexed Periplasmic: There has been an attempt to reproduce this [Telmer & Shilton, cited by Nature above]. It is described in a poster published on the internet from a group at Imperial College. Unfortunately it’s not clear if this was ever published elsewhere in any peer-reviewed forum.
And what of the misconduct investigation at Duke?
Hellinga wrote to Nature in July 2008 indicating that Duke had, at his request, opened an enquiry into his own actions in connection with the events surrounding last year’s retractions8. [there was also a grad student petition requesting an investigation] Duke declined to answer questions about the status of the enquiry. [insiders suggest nothing is happening, though one would not expect publicized activity in such an investigation]
Other scientists said that the new developments should spur Duke to complete its analysis of both the previous retractions and the current developments. “I think it should be brought to a conclusion fairly quickly because the scientific community is perplexed by the contradictory results both from this and Richard’s analysis,” says Kirsch.
The Scientist offers their take on the whole matter as well.
