Bionanoscience Seminar, 12.03.2015
Speaker: Gunnar von Heijne (Stockholm University)
Author: Edgar Schönfeld
In some situations, the force on a polypeptide chain can be measured with a technique that does not make use of magnetic tweezers and similar stuff. This is for example the case when a polyptide migrates through the inner cell membrane in E. coli via a special channel, the ‘translocon’. In order for this to take place the migration must happen cotranslational, meaning that the ribosome is docked to the translocon while the nascent chain travels through the translocon and does not fold into a protein before it has reached the other side of the inner membrane. I learned about the existence of the translocon for the first time in this seminar, and so did some of the PHD students that attended the seminar with me.
One of the proteins that uses the translocon in the way I described is the protein LEP (“Leader Peptase”). It is a transmembrane protein itself and uses the translocon to reach its position in the membrane. The research team used LEP as a model system to measure the forces it is exposed to during its passage through the translocon. Their unconventional approach to measuring forces was the incooperation of so-called “arrest peptides” (AP’s) into the LEP protein, but before I get into the details of this mechanism I will explain what AP’s do.
An arrest peptide is a sequence of amino acids that induces translational stalling. An example is the sequence HAPIRGSP from the SecM protein of E.Coli. Whenever this sequence is translated by the ribosome the Proline (P) at the end of the sequence will not be incooperated well, which results in a blockage of further elongation of the nascent chain. However, the cell seems to have ways to overcome this blockage. It is believed that it does so by applying a ‘pulling force’ on the nascent chain, in the exact moment when the elongation stalls. In the case of LEP this force is provided by another protein that is translated parallely, and the research team of G.v Heijne measured this force.
In order to prepare LEP for their experiment, they genetically engineerd a 19 amino acid long hydrophobic sequence H and an AP near the C-terminus of the LEP polypeptide. H and AP are thereby a distance L apart. The researchers had reason to speculate that the force depends on L and on the hydrophobity of H. Thus they conducted a large number of experiments in which they varied the distance L and the hydrophibity of H. The engineerd LEP sequences were cloned into a plasmid and introduced into E.coli. Then the proteins where radioactively labelled, the cells were lysed and the protein products were made visible on a gel. Everytime two bands were seen, one representing fully translated LEP and the other incompletely (arrested) LEP. The intensity of these bands varied with different distances L and hydrophobities. At a distance L of circa 30 and 40 aminoacids the intensity of the band representing the fully translated LEP was at a maximum, which indicates strong pulling forces. As we can see, the ratio of fully-translated and arrested LEP fractions gives a good benchmark of the force. Despite the high precision of the force measurements the translation into Newton is not very accurate yet. The forces we are talking about are in the range of circa 10 to 40 pN.
The power of this method of force measurement is that it is relatively simple and precise and can be applied in many other situations. The team of G.v Heijne used it for instance in a later experiment in which they measured the electrostatic force that a peptide chain has to overcome to migrate through a membrane.
Special Thanks to Gunnar von Heijne, who sent me the ppt of his presentation to share it with you.