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Forschungsarbeit

Single molecular protein folding studies using high-resolving optical tweezers

By Johannes Stigler (22.08.2011)

The autonomous folding of long polypeptide chains into highly complex and functional molecular machines is one of the most fascinating self-assembly processes in nature. Possible misfoldings of proteins are closely related to to-date untreatable diseases, such as Alzheimer’s or Parkinson’s. Consequently, to follow a protein find its native structure has been a dream of structural biology for a long time.

However, there is still no common knowledge of how to determine the structure of a protein from its sequence. Although at first sight, this seems to be an achievable task by simply minimizing the energy of the initially extended polypeptide, this “holy grail” of structural biology is still posing a difficult problem for non-minimal systems. In recent years, computational studies have been able to push the limits of simulation time and all-atom molecular dynamics simulations have been employed to gain insight into the detailed folding process of small proteins. Nevertheless, for increasingly complex systems computational methods become rapidly unfeasible and too costly.

To this end, we employ an ultrastable optical tweezers assay to mechanically unfold single proteins and gain knowledge about the folding and unfolding processes.

In a typical experiment, two small micron-sized glass beads are trapped each in a strongly focused laser beam. We can apply load to specific sites of the protein by fusing two DNA handle molecules of about 170 nm length each to the protein. The ends of these handles are functionalized to bind to the two glass beads. The final construct is a dumbbell configuration in which the protein, separated by DNA linkers, is bound to the glass beads (Fig. 1). We can optically record the deflection of the bead centers from their zero-position, a direct measure of the response exerted by the protein. By moving the beads one can apply load on the protein.

 

Figure 1: A dumbbell configuration for protein unfolding. The protein (depicted by its crystal structure) is bound with DNA handle molecules (green) to functionalized glass beads (gray) caught in strongly focused laser beams (red).[Bildunterschrift / Subline]: Figure 1: A dumbbell configuration for protein unfolding. The protein (depicted by its crystal structure) is bound with DNA handle molecules (green) to functionalized glass beads (gray) caught in strongly focused laser beams (red).

As an example system we investigated the folding and unfolding properties of calmodulin (CaM), a 148 amino-acid two-domain protein which serves as a calcium transducer in the cell and undergoes significant structural changes upon binding of calcium.

At high calcium concentrations, CaM unfolds at about 10 pN in a multi-state process and samples a variety of intermediate states (Fig. 2). We are able to assign structural information to the states by investigating mutants. Furthermore we can determine relevant information about the calcium-dependent stability of the protein and infer the properties of CaM folding.

Figure 2: Typical folding and refolding traces of calmodulin as observed with optical tweezers. The protein samples six resolvable distinct states (colored in rainbow colors from purple (folded state) to red (unfolded state). By applying increasing tension[Bildunterschrift / Subline]: Figure 2: Typical folding and refolding traces of calmodulin as observed with optical tweezers. The protein samples six resolvable distinct states (colored in rainbow colors from purple (folded state) to red (unfolded state). By applying increasing tension to the construct the equilibrium gradually shifts from a primarily folded configuration (top trace) to more and more intermediates populated to a configuration where the protein is in the unfolded state most of the time (bottom trace).

The single molecule approach lets us detect details such as only sparsely occupied intermediates that are unlikely to be found in bulk measurements. With this high resolving assay we are able to show that even in seemingly simple systems there is a surprising complexity in the folding process. In contrast to the expected independence of the two domains we observe cooperative and anti-cooperative effects between the domains and a complex network of intermediate states.

In a more general scope we can show that in the case of CaM, where the single domains are very well investigated, there are intriguing aspects to the folding process that only show when looking at the protein as a whole instead of a sum of its parts.


Johannes Stigler
* 1983

Stationen
  • 2002 - 2008
  • Physics at LMU München
  • 2005 - 2006
  • Physics at Lund University
  • 2007 - 2008
  • Diploma thesis at University College Dublin
  • 2008
  • Visiting scientist at Lund University
  • Since 2008
  • PhD program at TU München in Biophysics
  • Since 2010
  • Member of CompInt

Veröffentlichungen
  • * Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proceedings of the National Academy of Sciences (2008) vol. 105 (38)
  • * Stigler J, Lundqvist M, Cedervall T, Dawson KA, Lynch I. Protein Interactions with Microballoons: Consequences for Biocompatibility and Application as Contrast Agents. Ultrasound Contrast Agents (2010) pp. 53-66
  • * Johannes Stigler, Fabian Ziegler, J. Christof M. Gebhardt, Matthias Rief: The Complex Folding Network of Single Calmodulin Molecules. Science, Oct. 28, 2011, pp. 512-516. DOI: 10.1126/ science. 1207598.