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Max Planck Scientists Develop New Way For Analyzing Protein-Protein Interactions

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Researchers at the Max Planck Institute have developed a new method for observing protein-protein interactions by incorporating specially synthesized photo-reactive amino acids into proteins.

The trick to the new method is that two modified, photo-reactive amino acids — photo-leucine and photo-methionine — irreversibly cross-link to other nearby proteins when exposed to ultraviolet light. Researchers can then capture their protein of interest using an antibody, and run a Western blot to see if the protein cross-linked to any other proteins.

There are other chemical-crosslinking methods available for “freezing” protein interactions in time, but they involve the use of large molecules such as paraformaldehyde or DSS, which cross-link to a much wider, less-specific range of proteins, said Monika Suchanek, the first author of the new study, which appears in the April issue of Nature Methods.

“The disadvantage of those other methods is that the range of cross-linking is much bigger, and it is not so specific,” said Suchanek, who is a PhD student in Cristophe Thiele’s laboratory at the Max Planck Institute. “Our [modified] amino acids will not cross-link things that are not close to each other in cellular context.”

Thiele said that he is in principle interested in finding a partner to commercialize the amino acids, but that he has not contacted any companies so far. The Max-Planck Society has submitted a patent application that covers the amino acids and their use in detection of protein-protein interactions, he said.

Irwin Chen, a graduate student in Alice Ting’s laboratory at the Massachusetts Institute of Technology, who recently developed a new method for labeling proteins using small molecules (see PM, 1/28/2005), said that Thiele’s photo-amino acid method is a “novel twist” in that it uses photo-amino acids for analyzing protein-protein interactions, rather than for labeling proteins. The method is advantageous in that the cross-linking of proteins is covalent, and therefore stable for processing and analysis after linking, he said.

However, one potential problem with the method is that the non-site-specific incorporation of the photo amino acids may result in active sites being disrupted, Chen said.

“When you introduce this unnatural amino acid into an active site, where there was an important methionine or leucine, that could screw up the function of the enzyme, and thereby the interaction of the protein with other proteins,” he said.

Chen added that the new technique serves a different purpose than the numerous labeling technologies that are used to observe protein-protein interactions in vivo.

“I see this method as complementing or replacing co-immunoprecipitation,” said Chen. “It’s a method for proteomic analysis, whereas the other methods, such as fluorescence, are used for observing interactions.”

Suchanek said that one limitation of the new technique is that the modified amino acids are hydrophobic, and may not be suitable for observing interactions between non-hydrophobic proteins. The technique was designed especially to serve as a tool for studying membrane proteins, Suchanek explained. Leucine and methionine were chosen to be modified because they are hydrophobic, and they are small.

“In terms of proteins, there are the least tools for studying membrane proteins, so that’s why we chose those amino acids from the beginning,” Suchanek said. “The major limitation is that because they are incorporated into this hydrophobic interaction, it’s good for transmembrane proteins, but it may not be good for hydrosolic proteins.”

Thiele, a chemist by training, synthesized the photo-amino acids based upon the same chemistry that he used to synthesize a photo-cholesterol molecule over five years ago. The photo-cholesterol molecule was used to study interactions between cholesterol and proteins.

Before putting the photo amino acids to use for the first time about two years ago, Suchanek was studying protein-protein interactions by doing co-immunoprecipitation reactions. The downside of doing these reactions was that proteins that were present in the same organelle could co-immunoprecipitate, even if they were not reacting with each other.

“Usually [with co-immunoprecipitation], there was a pretty high background, so I was not sure if the interaction was specific,” said Suchanek. “You could increase the number of washes to increase specificity, but then you would risk losing some of the interaction partners.”

Suchanek said she managed to get Theile’s photo-amino acids to incorporate into a number of different proteins the first time that she tried.

“The big question was whether or not the cells would take up the [modified] amino acids,” said Suchanek. “It’s not so easy to trick the cell machinery. But [the modified amino acids] are small, and very similar to natural methionine and leucine, so they incorporated easily from the beginning.”

David Hill, a functional genomics researcher at the Dana Farber Cancer Institute, noted that getting modified amino acids to incorporate into proteins with reasonable efficiency without causing unwanted effects on cellular metabolism is not an easy task.

“The major advantage of this approach over traditional methods of metabolic labeling with ‘unnatural’ amino acids is that there appears to be no deleterious effects on cellular metabolism,” he said.

Once Suchanek got the modified amino acids to incorporate and cross-link properly in response to UV light, she used the new method to continue studying protein regulators of lipid metabolism.

In previous experiments using co-immunoprecipitation, Suchanek had found that a membrane protein called Insig-1 that is a key regulator of cholesterol homeostasis interacts with two other membrane proteins called SCAP and PGRMC1. However, because of the non-specificity of the co-immunoprecipitation technique, she was not sure how specific the interaction was.

Suchanek decided to confirm the Insig-1 interaction using the photo-amino acid cross linking method. Using the new technique, she found that Insig-1 did indeed cross-link with PGRMC1, which cross-linked with SCAP.

“We were able to confirm this protein interaction previously identified by co-immunoprecipitation,” said Suchanek.

The next step is to use the new photo-amino acid technique to identify novel protein interaction partners, Suchanek said. Her plan is to overexpress a protein of interest, and then to use the photo-amino acids to determine what other proteins it cross-links to.

To study proteins that are non-hydrophobic, a modified non-hydrophobic amino acid would be useful, Suchanek noted. However, it is unclear at this time if the laboratory will work on synthesizing another type of photo-amino acid, she said.

— TSL

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