C3 - Gerwert / Ollesch (2011-2014)  

In project C3, von Willebrand-Factor (VWF) structural plasticity under induced and controlled shear flow is analysed with FTIR spectroscopy. The approach is based on the multidisciplinary design of a surface acoustic wave (SAW) driven shear flow cell. This enables the detailed structural analysis of the VWF reaction to shear in a most native environment. Both polymeric full length, multidomain VWF complexes and single, functional domains will be analyzed. At least one surface of the flow device will be functionalizable. Thus, a specific immobilization of a VWF domain or a binding partner within adjustable shear flow will be used for binding studies. Further analysis of the enzymatic VWF cleavage with ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) will give structural information about hemostasis regulation.

Fourier transform infrared (FTIR) spectroscopy is a versatile technique to analyze molecular vibrations. Using time resolved difference techniques, protein reactions were monitored at atomic level with nanosecond time resolution [1].

Figure 1: The amide I band (1700-1600 cm-1) is the strongest band in the mid-IR extinction spectrum of a protein. Caused by the C'=O stretching vibration (inset in A), the amide I band shape depends on the secondary structure composition. Exemplified on a calculated, ideal amide I band (A), the workflow for structure analysis is i) measurement of the protein sample extinction, ii) amide I band decomposition, iii) integration of the calculated components (B). The component area fraction of the total band integral specifies the fraction of the secondary structure element, assigned by band position.

Particularly protein structure plasticity can be excellently assessed. The medium infrared spectral range is highly sensitive to the strong stretching vibration of the protein backbone's C'=O group, which is conformationally sensitive. The so called amide I band is analyzed without the need for additional marker substances or fixation of the sample (fig. 1). Structure dependent band shifts are extracted by numerical amide I band decomposition for secondary structure determinations. For the analysis of the alpha-beta-transition of prion protein, which is involved in the pathogenicity of Creutzfeld-Jacob disease, this technique proved extremely useful [2].

But prion protein is a natively membrane attached protein, scarcely occupying the cell membrane of neurons. To comply with the need for most native reaction conditions, a surface tethering setup was designed for the conformational analysis of membrane bound prion protein in flowing physiologically buffered solution. Overcrowding effects were seen sufficient to induce, again, non-native alpha-beta-refolding (fig. 2) [3].

Figure 2: Fully glycosylated prion protein exhibited the expected native conformation when attaching to a raft-like membrane via its GPI anchor (A, schematical view B). Overcrowding the membrane causes drastic refolding (C, D). A drastic increase of a low frequency band (1619 cm-1) assigned to intermolecular beta structure indicates oligomerization.

These results were only possible with spectrometer coupled fluidics and the surface sensitive attenuated total reflection (ATR) setup, in which an infrared beam is coupled into an internal reflection element (IRE) of high optical density. The IRE, in this case Germanium, was covered with a raft-like lipid bilayer that served as accepting matrix for the prion protein's glycosyl phosphatidyl inositol (GPI) membrane anchor. Notably, the protein was kept in buffer during the whole experiment, since the IRE was mounted in a flow cuvette. Surface tethering not only enables previously unmatched physiological conditions [4]. Improved further, even subtle signals from small molecule binding can be detected. In the current approach, surface tethered VWF molecules will be analysed for binding kinetics of ligands and cleavage efficiency with ADAMTS13.

Of special relevance to the proposal is the applicability of microfluidic chips as sample carriers. In a pioneering work, E. Kauffmann designed a mixing microchip for protein folding analysis to use in an FTIR microscope [5]. In continuous flow operation, this device surpassed the then fastest stop-flow UV-circular dichroism systems with a reduced experimental dead time of 0.4 ms.

Consequently, we now develop a SAW driven cell for the structural VWF analysis in controlled shear.


[1] Garczarek F, Gerwert K. Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy. Nature. 2006;439:109―12.
[2] Ollesch J, Künnemann E, Glockshuber R, Gerwert K. Prion protein alpha-to-beta transition monitored by time-resolved Fourier transform infrared spectroscopy. Appl Spectrosc. 2007;61:1025―31.
[3] Elfrink K, Ollesch J, Stöhr J, Willbold D, Riesner D, Gerwert K. Structural changes of membrane-anchored native PrP(C). Proc Natl Acad Sci U S A. 2008;105:10815―9.[4] Güldenhaupt J, Adigüzel Y, Kuhlmann J, Waldmann H, Kötting C, Gerwert K. Secondary structure of lipidated Ras bound to a lipid bilayer. FEBS J. 2008;275:5910–8.
[5] Kauffmann E, Darnton NC, Austin RH, Batt C, Gerwert K. Lifetimes of intermediates in the beta-sheet to alpha-helix transition of beta-lactoglobulin by using a diffusional IR mixer. Proc Natl Acad Sci U S A. 2001;98:6646―9.

Web: http://www.pure.rub.de/projekte/for1543.html.de (Lab)

  C3 - Gerwert / Ollesch (2011-2014)