Proteins are basic constituents of cells and they have numerous functions. In order for proteins to be able to fulfil their vital functions in the cell, they have to adopt a unique, correctly folded three-dimensional structure, a process that is susceptible to faults. Proteins are linear polymers synthesised by ribosomes from amino acids. During their synthesis, the proteins fold and adopt a defined three-dimensional structure determined by the amino acid sequence. Protein folding is decisively assisted or actually sometimes even made possible by molecular chaperones that are vital proteins found in all organisms, from bacteria to human beings. Cells always have a network of different chaperones that act together and control and assist proteins in folding correctly, both in spatial and temporal terms.

Prof. Elke Deuerling and her team at the University of Constance focus principally on the investigation of the process of protein folding as well as on the importance and function of chaperones. The biologist from Constance University also envisages that her research will open up possibilities that could lead to application, such as the production of recombinant proteins for example, "since this process is often characterised by the massive misfolding and aggregation of the resulting proteins" and hence "renders biotechnological production processes such as the production of protein therapeutics" difficult. In addition, she also envisages that her basic research will be used in the long term by medically relevant science since faulty protein folding and chaperone functions result in the aggregation of proteins, in human as well as in other cells. "Such protein aggregates are characteristic features of neurodegenerative diseases and are presumably also decisive in neuronal cell death," explains Prof. Deuerling.

Her current work focuses on specialised chaperones that are located at the ribosome and that already interact with the protein chains during their synthesis. Deuerling and her team have for the first time ever been able to show that such chaperones receive the growing protein chains and protect them against external influences during the folding process. The researchers use two different model systems for their investigations, the baker’s yeast Saccharomyces cerevisiae (an eukaryotic cell) and the bacterium Escherichia coli (a prokaryotic cell). E. coli has a single ribosome-associated chaperone, which is known as “trigger factor”. The “trigger factor” chaperone binds to the tunnel exit of the ribosome, thereby interacting with all newly synthesised proteins that leave the ribosome through this tunnel,” reports Deuerling. Cells are perfectly viable without the “trigger factor” chaperone, but when the researchers removed another chaperone, the “trigger factor’s” cooperation partner, the Hsp70 chaperone DnaK, they found that several hundred newly produced cytosolic protein species had aggregated and the cells eventually died.

"In our work with Prof. Nenad Ban (ETH Zurich) and Prof. Bernd Bukau (ZMBH Heidelberg), we have been able to discover what the trigger factor looks like and suggest how a model of its functions would work," said the molecular biologist from Constance. According to this model, trigger factor binds to the L23 protein of the ribosome and bends over the exit of the ribosomal tunnel from which the protein chain emerges during synthesis, thereby creating a protected folding space in which the newly synthesised proteins are protected against protein-degrading proteases and aggregation. This work has uncovered a new basic principle of protein folding and the role of molecular chaperones. This basic principle, for the first time ever shown in bacteria, is also present in higher, eukaryotic cells, where other factors are also involved.

In contrast to bacteria and chloroplasts, eukaryotic cells do not have the trigger factor chaperone for the folding of cytosolic proteins. Therefore, the central question arises as to how this process, which is vital for initial protein folding, happens in such cells. "We already know that higher cells have other ribosome-associated factors, including an Hsp70 chaperone system and the protein complex NAC (nascent polypeptide-associated complex)," said Prof. Deuerling who is now focusing on investigating the functions of these factors and comparing them with the bacterial trigger factor. "For these investigations we use Saccharomyces cerevisiae as a model, because all known factors and chaperones that bind to eukaryotic ribosomes are evolutionarily conserved and therefore also present in yeast," said Deuerling.

Both Saccharomyces cerevisiae and E. coli are of particular importance as model organisms in Prof. Deuerling’s chaperone research. This is not just because they provide the researchers with insights into protein folding in prokaryotes and eukaryotes, but there are also concrete practical reasons. The entire genome of the two organisms has been known for many years and numerous methods to manipulate them are available. In addition, both yeast and bacteria cells grow relatively quickly and are relatively cheap to cultivate.

Deuerling sees a number of advantages in using these two unicellular organisms: “The use of Saccharomyces and E. coli enables us to combine efficient genetic, biochemical and cell biological approaches. The optimal use of these two model systems also provides us with basic insights into the protein folding process in prokaryotic and eukaryotic cells, which can then be transferred to the situation in other organisms,” said Deuerling adding that many genetic screens and the intensive search for substances that inhibit chaperones or have an effect on protein aggregation, can be done quickly and cheaply with yeast.

In order to uncover the functions and mechanisms of chaperones, Prof. Deuerling and her fellow scientists use genetic and microbiological analyses of chaperone and ribosome mutants in yeast and E. coli as well as numerous biochemical and biophysical methods. They particularly use cell-free in vitro translation systems produced from different chaperone mutant cells, which allow the investigation of the synthesis and folding of model proteins in the absence or presence of certain chaperones. In addition, they use spectroscopic methods, chemical cross-linking experiments and amide proton exchange experiments, combined with high-resolution mass spectrometry, in order to analyse the interaction between proteins.

Despite the many similarities between the two model systems, Deuerling and her team use E. coli and Saccharomyces to clarify different questions. E. coli is used to study bacterial processes associated with protein folding and chaperone function and Saccharomyces is used to analyse these processes in higher cells. “Although protein biogenesis and the chaperone functions have numerous principles in common in these model organisms, which we can uncover with analyses and the direct comparison of the two models, there are also fundamental differences between prokaryotes and eukaryotes,” said Deuerling. The one single fact that eukaryotes do not have the bacterial trigger factor chaperone and have developed other chaperones instead, shows the scientists that they can expect completely new aspects in higher cells, which are not found in bacteria. In addition, the chaperone network in eukaryotic cells is bigger and far more complex than in prokaryotes, and there are also some new chaperones that are not present in bacteria.

Therefore, the researchers do not exclude the use of another model organism for their chaperone research: “There are huge differences between lower eukaryotes such as yeast and higher, multicellular organisms such as humans. Therefore, we are envisaging the possibility of using an additional model organism for our research in order to be able to study protein folding processes in more complex organisms,” highlighted Prof. Elke Deuerling.

Despite the huge differences between microorganisms and highly developed eukaryotes, the researcher also partially envisages the possibility of being able to transfer the findings gained with these two microorganisms to other organisms. “On the cellular level, a surprisingly large number of processes are similar in yeast and human cells. The yeast already has all the basic elements and processes of eukaryotic cells,” added Prof. Deuerling. She believes that the insights obtained with the analysis of yeast, can “often be transferred to the situation in higher cells”, for example in the analysis of conserved basic processes such as protein biogenesis and chaperone functions. “Even protein folding defects, which can lead to diseases in human beings, can be investigated in yeast on the cellular level,” said Deuerling.

The researchers envisage that such findings might in the long term lead to medically relevant results, because “besides their importance in the aforementioned protein biogenesis and the role of ribosome-associated chaperones, it was discovered a few years ago, first in yeast and then in E. coli, that there are chaperones that are able to dissolve large protein aggregates and hence reactivate proteins,” explains the microbiologist adding “some molecular details of the Huntingtin protein, which is the cause of the genetic disease Huntington’s, and its toxic effects in cells could be uncovered using yeast.” “However, this does not mean that everything is possible in yeast,” pointed out Deuerling as she sees “clear limits, in particular when the interaction of united cell structures, tissues and organs have to be taken into account or when the specific characteristics of a certain cell type or species are concerned.”

Further information:

Prof. Dr. Elke Deuerling
Department of Molecular Microbiology
University of Constance
Universitätsstrasse 10, Box M 607
78457 Konstanz