Working without the Energy: B CUBE Scientists Reveal Thermodynamics of Chaperoning in the Periplasmic Space

Chaperones prevent complex proteins from folding prematurely. To function, most of them need energy in form of ATP. However, some chaperones have to work without ATP. A team of scientists at the B CUBE - Center for Molecular Bioengineering at the TU Dresden used single-molecule FRET and FCS to show how chaperone-protein complexes are thermodynamically fine-tuned to prevent bacterial outer membrane proteins from folding and aggregating in an ATP-free environment. The results were published in the journal PNAS.

Proteins perform very specific functions inside cells. Each protein has a unique three-dimensional structure that supports its function. The longer the protein chain is, or the more individual subunits the protein has, the harder it is to fold it in a correct three-dimensional structure. A special class of proteins, known as chaperones, exists to help proteins fold correctly and at their dedicated place inside the cell. For example, proteins that are supposed to be embedded in membranes can only fold once they arrive at the membrane and they have to be prevented from misfolding inside the cell.

A Place Without Additional Energy
The interaction of chaperones and proteins requires energy and is typically fueled by energy molecules known as ATP. However, some chaperones have to interact with proteins without ATP. For example, in the special space between the inner and outer bacterial membrane, the so-called periplasmic space, chaperones are working and keeping proteins unfolded and soluble without any additional energy available. Scientists from B CUBE developed a strategy to analyze the interaction of OmpX, a bacterial outer membrane protein, and two key chaperones, Skp and SurA. They discovered how these chaperones prevent OmpX from misfolding and aggregating.

Outer membrane proteins, like OmpX, are crucial for bacterial survival. They take care of adhesion, nutrient uptake, sensing the environment, or interactions with chemicals and toxins. As such, they are also one of the targets of antibacterial drugs. “These outer membrane proteins are very sticky. They are prone to bind to one another and aggregate. However, they have to be kept unbound and unfolded until they can be inserted into the bacterial outer membrane,” explains Prof. Michael Schlierf, research group leader at the B CUBE who led the study.

smFRET as a Tool for Dynamic Structural Biology
To characterize the dynamic structure of OmpX and its chaperones Prof. Schlierf’s team used single-molecule Förster resonance energy transfer (smFRET). It uses fluorescent markers to measure the distance between parts of a single molecule at a nanometer scale with up to nanosecond time resolution. Using smFRET, the team could observe how binding to chaperones changes the dynamics and the structure of OmpX. “Setting up experiments with outer membrane proteins is tricky. We carefully optimized many parameters for both, the chaperones and OmpX, until we could perform these experiments,” adds Dr. Neharika Chamachi, a lead author of the study.  

Preventing Folding by Expansion
The team has discovered that the two chaperones keep OmpX from aggregating by expanding the unstructured OmpX in various ways. Neither Skp nor SurA uses the energy from ATP, thus the binding with chaperones takes advantage of different thermodynamic properties. Skp bound the protein in a specific fashion along the chain. The interaction with the SurA, on the other hand, was exceptionally dynamic. It was continually binding and unbinding the protein in a seemingly unspecific manner.

“These different binding modes could also be quantified with enthalpic and entropic contributions, which we were able to determine by temperature-dependent measurements” explains Dr. Andreas Hartmann, one of the lead authors of the study. By fine-tuning enthalpy and entropy, the two chaperones are able to keep OmpX expanded, and prevent its aggregation in an environment that lacks the cellular energy molecules. At the same time, OmpX remains dynamic and can unbind for the next folding step.

Switching Gears Under Stress
The team also looked at the protein-chaperone interaction during stress conditions. Generally, when cells are exposed to challenging conditions, e.g., changes in temperature or damaging chemicals, the pathway responsible for the folding of outer membrane proteins is also affected. Unstructured OmpX molecules are no longer chaperoned and tend to bind to each other and form large aggregates. The Dresden group showed that in such conditions, both Skp and SurA switched their attention from the single OmpX molecules to the aggregated OmpX proteins, and started to disassemble the aggregates. Even more, Skp and SurA acted synergistically under these conditions.

“Our smFRET assay allows us to characterize the interactions between chaperones and membrane proteins. We believe that it can be useful to other groups who aim to characterize similar transient interactions that can only be measured at very low protein concentrations,” concludes Prof. Schlierf.

Publication
Neharika Chamachi, Andreas Hartmann, Mai Quynh Ma, Anna Svirina, Georg Krainer, Michael Schlierf: Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates. PNAS (February 2022)
Link: https://doi.org/10.1073/pnas.2118919119

Scientific Contact
Prof. Michael Schlierf
Research Group Leader
B CUBE - Center for Molecular Bioengineering
+49 351 463 43050