Study protein folding and conformational dynamics at the nanoscale
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Why Dynamic Single-Molecule?
A new view on the structure-function relationship of proteins
Studying how proteins fold correctly and undergo conformational changes to accomplish their biological function is crucial to understanding the underlying biological mechanism. Single-molecule Force Spectroscopy (SMFS) represents an ideal tool to study these molecular phenomena because of its unique capability to isolate individual biomolecules and observe conformational transitions and unfolding processes as they happen in real-time. Correlating SMFS data (molecular extension vs force) with single-molecule fluorescence readouts adds another dimension of information and enables the characterization of more complex relations, e.g. between conformation and binding kinetics, or between local (domain) and global (whole molecule) conformational dynamics.
Overcome these challenges with Dynamic Single-Molecule technology through:
Explore how structural motifs determine protein folding and function
The function of proteins strongly depends on their structureand correct folding of the polypeptide chain. Different substructures likesheets and coiled coils contribute to the folding pathway and at the same timeprovide the flexibility required to carry out various dynamic functions.
Case study
The “elbow” structure in SMC complexes is critical for proper folding and ensures functionality
A study in the lab of Johannes Stigler (LMU Munich) investigatesthe role of coiled-coil elements in the structure of structural maintenance ofchromosomes (SMC) complexes. By quantifying the conformational dynamics,folding pathways and stability of wild type and mutated constructs, the lab wasable to reveal that
· The coiled-coil region unfolds through threeobligatory intermediates
· Misalignments of the wild-type CC strands arerarely observed
· Replacing the so-called elbow domain inducesfrequently appearing metastable and non-productive misfolding states
Further reading:
Freitag et al.Biophysical Journal (2022)
Explore further
https://lumicks.com/knowledge/dsm-publications/
Uncover unknown mechanisms that modulate protein folding
Regulatory mechanisms that support proper folding ofpolypeptide chains into functional proteins, prevent or dissolve misfolding andaggregation are fundamental in maintaining proper cellular function and inavoiding misfolding-induced disorders like Alzheimer’s or Parkinson’s disease,Amyloidoses and others.
Thus, characterizing the mechanistic details of theseprocesses is crucial if we want to better understand related diseases anddevelop efficient therapeutic strategies. The C-Trap’s correlativeforce-spectroscopy and fluorescence assays provide a powerful tool tocharacterize sub-steps and intermediates, and to shed light on factorsinfluencing protein folding pathways.
Case study
Hidden in bulk data: Chaperonins accelerate folding by strengthening chain collapse
By investigating protein folding modulated by the chaperoninGroEL-ES with C-Trap single-molecule assays, Sander Tans (AMOLF Amsterdam) andhis team were able to differentiate two seemingly counter-acting mechanisms thatwere obscured in bulk biochemistry assays. They detected distinct GroEL-ESinduced conformational transitions on the time scale of seconds and foundindications that:
- GroEL apical domain interactions delay folding
- GroEL-ES can accelerate the folding of proteinsby strengthening their collapse
- This collapse modulation is distinct from otherproposed GroEl-ES folding acceleration mechanisms
These findings significantly contribute to our understandingof chaperonin-assisted protein folding and highlight the role of collapsemodulation as a generally relevant mechanism within the protein quality controlmachinery.
Further reading:
Avellaneda et al. Nature (2020)
Naqvi et al. Science Advances (2020)
Conformational dynamics of membrane proteins: the key to deciphering cellular signaling and transport
Membrane proteins which constitute about 30-40% of allproteins encoded in the human genome play a pivotal role in cellular transportand signaling and many of them are thus considered key drug targets for relateddisorders. The fact that most of these proteins only resume their proper foldedshape (and thus function) when embedded in a lipid (bi-)layer environment makesit particularly challenging to characterize them in comparison to solubleproteins.
Regardless of those difficulties, quantifying how molecularinteractions modulate membrane protein conformation and vice versa is criticalto a full understanding of signaling, transport and cellular interactionmechanisms. Innovative sample protocols combining single-molecule forcespectroscopy with membrane nano-discs have enabled researchers to investigateexactly those structure-function relations for membrane proteins.
Case study
Innovative single-molecule assays enable novel insights on ABC transporter dynamics and function
Kasia Tych and her group developed a novel assay thatenables single-molecule force spectroscopy of membrane-embedded proteins withthe C-Trap. Membrane nanodiscs accommodating one protein each provide the lipidenvironment that is required to observe native structure and function. In theirproof-of-concept study, they examine the mechanical properties and potentialinteractions of the substrate-binding domains of the ATB-binding cassette (ABC)transporter OpuA.
The high spatial and temporal resolution of the C-Trap allowedthem to
- Investigate the influence of ionic strength onthe conformational dynamics of the protein’s substrate binding domains (SBDs)
- Compare the contribution of SBDs andtrans-membrane domains (TMDs) to the overall conformational change of thetransporter
- Quantify interactions between the SBDs in thehomo-dimeric arrangement of OpuA
The innovative sample design in combination with the C-Trapstop of the line sensitivity and stability enables a new class of proteins to becharacterized via single-molecule force spectroscopy. Eventually, this willlead to a more complete picture of the structure-function relationship ofmembrane proteins like channels and transporters, and improved developmentstrategies for drugs targeting related signaling and transport mechanisms.
“We will be able to use microfluidics to studyfunction-related dynamics of a single molecule under different experimentalconditions. Combined with the power of having a simultaneous mechanical and fluorescent readout, we will be able to uncover previously unexplored detailsof the conformational cycles of our model proteins.” - Kasia Tych PhD, Principal Investigator, Rijksuniversiteit Groningen
Solutions
C-Trap
Biomolecular interactions re-imagined
The C-Trap® provides the world’s first dynamic single-molecule microscope to allow simultaneous manipulation and visualization of single-molecule interactions in real time.
Technical note:
These cards are NOT components because they use the finsweet nested collection logic. To pull in the posible multiple people wo worked on it.
This type of 1-many relation is not supported native in Webflow.
Also this section is hidden when emtpy. To keep everything visible here, that is being done outside the webflow designer from within Slater.
These cards are NOT components because they use the finsweet nested collection logic. To pull in the posible multiple people wo worked on it.
This type of 1-many relation is not supported native in Webflow.
Also this section is hidden when emtpy. To keep everything visible here, that is being done outside the webflow designer from within Slater.
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