OWLS APPLICATION NOTES NO-013

OPTICAL ANISOTROPY OF FLAGELLIN LAYERS: IN SITU AND LABEL-FREE MEASUREMENT OF ADSORBED PROTEIN ORIENTATION
Using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection
Polymeric flagellin consists of four linearly connected domains labeled D0, D1, D2, and D3, which are arranged from the inside to the outside of the filament. The refractive index and thickness of the adsorbed ( a ) f l a g e l l i n , ( b ) F 4 0 , a n d ( c ) D 3 _ F l i C f i l m s we r e calculated from the experimental data with An i s o t r o p i c Ad l a ye r M o d e l o f Ad s o r b e d P r o t e i n 1-3 L a ye r s .

The disordered terminal regions are involved in D0 and partly in D1, forming long helical bundles, and their direct interaction is responsible for stabilizing the filament structure. A central segment from Tyr 190 to Val 283 makes up domain D3, which is exposed on the surface of flagellar filaments. D3 is a structurally independent part of flagellin; it is not essential for filament formation The D3 domain is a good target for genetic engineering studies. It can be readily replaced by appropriate peptide sequences or foreign protein domains having specific molecular recognition properties or enzymatic activities without disturbing the polymerization ability. The flagellin domains have rather different characteristics in terms of their surface hydrophobicities, which is in close connection with the mechanism of flagellin assembly into flagellar filam ents. This m akes flagelli n an ideal candidate for studying the roles of the various regions in the protein adsorption process because of the ease of systematically removing one or other of the protein d o m a i n s . T h i s p o s s i b i l i t y i s e xp l o r e d i n t h e p r e s e n t work by investigating the adsorption of flagellin and its truncated variants on an artificial hydrophobic surface using label-free optical waveguide lightmode spectroscopy (OWLS)1. An anisotoropic adlayer can be characterized by three independent parameters: its thickness (dA) and its ordinary and extraordinary refractive indices (no and ne). For the adsorbing proteins in the plane o f t h e wa v e g u i d e t h e r e c a n b e n o p r e f e r r e d direction, hence nx = ny = no and nz = ne. The anisotropic modeling revealed a significant positive birefringence in the layer, suggesting oriented protein adsorption. The adsorbed flagellin orientation was evidenced by monitoring the surface adsorption of truncated flagellin variants, in which the terminal protein regions or the central (D3) d o m a i n we r e r e m o v e d . 1 The previously generally applied homogeneous and isotropic modeling of the protein layers resulted in overestimated thickness and underestimated refractive index values; a clear indication of positive birefringence in the adlayer.2 This is in contrast to glycoprotein films self-assembled on a hydrophobic OW LS sensor and taking up a negatively anisotropic, spread conformation.3 References 1. N. K ovacs, D . Patko, N . Orgovan, S . K urunczi , J. J. 2. 3.
Ramsden, F.Vonderviszt, R. Horvath, Anal. Chem. 2013, 85, 5382-5389. Horvath R, Ramsden JJ, Langmuir 2007, 23, 93309334. Horvath, R.; McColl, J.; Yakubov, G. E.; Ramsden, J. J. J. Chem .P hys. 2008, 129, 071102.

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