Pinching a chaperone

We can’t live without chaperones. They are vital to the proper functioning of proteins in our cells. A chaperone – we are of course talking biology here – is a protein that helps another, newly formed protein to attain or maintain the correct three-dimensional structure.

Proteins are long chains of amino acids. After protein synthesis, the chain is folded into a complicated 3D structure, which is essential for its function. In cells, the folding process is assisted by a host of what are known as chaperones. These are themselves proteins which bind to unfolded proteins and help them to attain or keep the correct configuration.

Some chaperones actually prevent folding, for instance in proteins that need to be transported through a membrane. ‘Five years ago, we were the first to show this function of a chaperone at the single molecule level, in our case the chaperone SecB’, says University of Groningen molecular microbiologist Prof. Arnold Driessen. Now he is co-author of a paper published in Nature , describing the role of another chaperone, TF1 (Trigger Factor 1).

‘In this paper, we show how TF1 stimulates folding and stabilizes the folded domains of a protein during production’, Driessen explains. Again, it is the first time these effects have been measured directly, using a system Driessen and colleagues developed for their SecB study. They use optical tweezers to measure the strength required to stretch a folded protein.

Using the optical tweezers, Driessen and his colleagues pulled at a model protein called MBP (maltose binding protein). While pulling at one end, the force required to keep the other end of the protein in a fixed position is measured. As long as the protein resists unfolding, this force will increase. Once the unfolding starts, there will be a steep drop in the force. Thus, a force pattern typical for the protein appears.

‘What we observed is that it takes a much greater force to pull the MBP apart when TF1 is present’, says Driessen. ‘This means that TF1 stabilizes the protein.’ In a second experiment, the scientists pulled at four fused MBP proteins, first without TF1. After unfolding this longer protein, it was left to refold. ‘When we tried to unfold it again, we couldn’t. The protein had folded itself up into a tangle.’

Repeating this experiment in the presence of TF1 gave a different outcome. After unfolding and refolding, the second unfolding went similarly to the first. The logical conclusion is that TF1 actually helps the protein to fold correctly and prevents tangling.

‘Up till now, there were two schools of thought on the function of TF1’, Driessen explains. ‘The first said that TF1 helps the protein chain to stay unfolded. The second proposed that TF1 actually facilitates folding. Our experiments show that the latter is indeed the case.’

Protein folding is not just an obscure hobby for biochemists – misfolding plays a role in many diseases. Protein tangles, the result of misfolding, are for example implicated in Alzheimer’s disease. But Driessen’s interest is not in diseases: ‘We want to know how proteins are transported across membranes in bacteria. Chaperones play an important role in keeping these proteins in the right configuration for transport.’

SecB keeps the proteins meant to be excreted unfolded, so they can be excreted through a pore in the membrane of a bacterial cell. TF1 binds to proteins while they are being formed at the ribosome and helps them to fold. However, TF1 then passes the protein on to another chaperone to complete the folding process.

Using optical tweezers to study chaperones has become the gold standard since the first paper by Driessen and colleagues, some five years ago. The optical tweezers are provided by AMOLF , one of the research laboratories of the Foundation for Fundamental Research on Matter ( FOM ). ‘They will continue to investigate the dynamics of chaperones, while we will focus on the excretion systems of bacterial cells.’ This process is important if you want to use bacterial cells to produce useful proteins .

Reference

Alireza Mashaghi¹, Günter Kramer², Philipp Bechtluft¹, Beate Zachmann-Brand², Arnold J. M. Driessen³, Bernd Bukau², and Sander J. Tans^1: Reshaping of a protein's conformational search by the chaperone Trigger Factor. Nature, DOI 10.1038/nature12293, online publicatie 7 juli 2013

¹ FOM Institute AMOLF, Science Park 104, 1098 XG Amsterdam, the Netherlands
² Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, Heidelberg 69120, Germany
³ Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and the Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9749 AG Groningen, the Netherlands