Cell growth protein forms a "pair" on the cell membrane

The RUB team combined the use of three biophysical methods - infrared spectroscopy, computer simulations and fluorescence measurements - and came to the surprising conclusion that two Ras molecules form a pair to take an upright position on the membrane.

It was previously assumed, based on computer simulations, that the protein is located horizontally on the membrane as single molecule.

Ras is the central “switch” for cell growth, and malfunction of this protein is an important factor in the development of cancer. “These results put a completely new light on the nano-cluster formation of Ras at the membrane,” said Professor Dr. Klaus Gerwert from the RUB Faculty of Biology and Biotechnology. The study was chosen as the cover story for the Biophysical Journal.

Orientation affects protein interactions

The orientation of a protein affects its possible interactions with other proteins. “This is similar to comparing the situations of a guest being welcomed with open arms and the host lying on the couch during the greeting,” illustrates Dr. Jörn Güldenhaupt, who conducted the orientation measurements.

Few biophysical methods allow the protein orientation to be determined. ATR-FTIR spectroscopy, which has been established by the Chair for Biophysics, is one of them.

Ras molecules are mutually supportive

The false assumption that Ras lies on the membrane was based on earlier computer simulations. Till Rudack from the Bochum research team also took a virtual look at Ras.

The result: A single upright Ras molecule very quickly falls over and seems to lie on the membrane. “Something must have supported the Ras in our measurements,” said Till Rudack. “And that could only be another Ras molecule that was not present in the simulation.”

In fact, further computer simulations of two mutually supportive Ras molecules yielded a stable upright orientation - which would fit the experimental results.

Fluorescence resonance energy transfer: a molecular yardstick

The team confirmed the results with another piece of experimental evidence using “FRET” (fluorescence resonance energy transfer).

This is currently the best method for detecting interactions between two proteins. Here, researchers mark the Ras proteins with two different dyes. If the proteins interact, they are very close together so that energy is transferred from the one dye to the other.

As with a yardstick, the distance between the proteins can be measured from the ratio of the transferred energy. For the Ras-Ras interaction, the biophysicists determined a distance of 4.6 nanometres, or millionths of a millimetre. This corresponded exactly to the distance they had predicted with their computer simulations for a “double-Ras”.