Protein folding observed with unmatched accuracy

For the first time, researchers can visualize the work of folding helper molecules in their natural environment

Using cryo-electron tomography, or cryo-ET for short, cellular structures can be made visible and analyzed in their natural environment. Researchers from the Max Planck Institute of Biochemistry in Martinsried near Munich and the University Medical Center in Göttingen have used this technique to identify protein folding helpers, so-called chaperonin complexes, in the bacterium Escherichia coli The researchers were able to observe both the conformational changes of the chaperonin and its interactions with the target protein in the folding chamber with unprecedented detail.

Proteins are responsible for practically all vital functions in cells. In order to perform their diverse functions, they must have a certain three-dimensional structure, similar to components in machines. Protein folding helpers support newly produced proteins in assuming their functional form.

Ulrich Hartl, Director at the Max Planck Institute of Biochemistry, explains: “Chaperonins occur unchanged in almost all living organisms and are essential for the correct folding of proteins. Incorrectly folded proteins are associated with diseases such as Alzheimer’s and Parkinson’s, for example. The more we know about the structures of chaperonins, the more precisely we can understand their functions and malfunctions and develop new strategies for treating these diseases.” In order to better understand how chaperonins work, Ulrich Hartl has teamed up with Wolfgang Baumeister, Emeritus Director and inventor of cryo-ET at the Max Planck Institute, and Rubén Fernandez Busnadiego from the Institute of Neuropathology at the University Medical Center Göttingen and member of the Cluster of Excellence “Multiscale Bioimaging: From Molecular Machines to Networks of Excitable Cells”.

Chaperonin complex

Chaperonin complexes consist of two different subunits, GroEL and GroES. GroEL consists of two protein rings on top of each other that form a cylinder. GroES acts like a lid for the GroEL cylinder. Newly produced proteins are encapsulated in the nanometer-sized interior of the GroEL and can fold while being shielded from the cellular environment.

The researchers were able to detect two main forms of the GroEL-GroES complex in the cells, which are called “bullet” and “football” (named after the shape of an American football). The two forms differ in their structural symmetry. In the bullet form, a GroES cap is attached to only one side of the GroEL barrel. This form was found primarily in bacteria during normal growth. Football complexes were also detected.

The microscopic images also show that the proteins to be folded are located in the chaperonin barrel. Jonathan Wagner, lead author of the study and scientist in Martinsried and Göttingen, explains: “It is fascinating that cryo-electron microscopy has now advanced to the point where we can follow processes such as protein folding in living cells in such detail.”

“In this study, we combined cryo-ET with single-particle cryo-electron microscopy and quantitative mass spectrometry. This allowed us to observe different conformations of chaperonin complexes in different cellular states and determine their abundance. The ability to visualize these complexes directly in the bacterium and not just in the test tube represents a major advance in the field and has only recently become possible. The chaperonin complex is only 14 nanometers wide. In decades of experiments with purified GroEL/ES complexes, conflicting results have been obtained about how this machinery works. This is probably due to the fact that In vitroExperiments cannot fully replicate the conditions in the cell. We can now eliminate this contradiction through cellular cryo-ET, as we can visualize the complexes in their native environments with high resolution,” explains Rubén Fernandez Busnadiego.

In conclusion, Ulrich Hartl summarizes: “The results indicate that during chaperonin-assisted protein folding, the chaperonins assemble differently and switch between the bullet and football forms in a reaction cycle. In future work, we will focus on clarifying the intermediate states of these cycles to understand how they are regulated by the chemical reactions of ATP binding and hydrolysis.”

Glossary:
ATP (adenosine triphosphate): is a mononucleotide which has bound energy-rich phosphate residues.

ATP serves as the central energy currency in biological processes.
Chaperones: French chaperone; is a Family of proteins that help newly synthesized proteins fold.

Chaperonins: are large barrel-shaped protein complexes that ensure correct, ATP-dependent protein folding.

GroEL: large subunit of the chaperonin complex in the bacterium Escherichia coli

GroES: “Lid” subunit of the chaperonin complex in the bacterium Escherichia coli

kDa: Abbreviation for kilo-Dalton; indicates the molecular mass for proteins. 1 Dalton = 1.66018 x 10^-27 kg. This corresponds to one twelfth of the mass of the carbon isotope ¹²C

Cryo-electron microscopy: Greek: kryos; cold, cold; Biological samples (e.g. purified proteins or cells) are shock frozen in liquid ethane to prevent the formation of water crystals and to enable preservation under near-natural conditions. The samples can then be visualized in high resolution using electron microscopy.

Cryo-electron tomography: This imaging method enables 3D imaging of cryopreserved samples such as cells. The high resolution enables the determination of protein structures in intact cellular environments.