On the Origin of the Polar Order of T4 Lysozyme on Planar Model Surfaces

Abstract

Site directed spin labeling is used to investigate the origin of the macroscopic alignment of T4 lysozyme vectorially tethered to planar biomimetic surfaces. T4 lysozyme was adsorbed to a quartz-supported dioleoylphosphatidylcholine (DOPC) bilayer by selective binding of the histidine-tagged protein to functionalized headgroups (1,2-dioleoyl-sn-glycero-3-[[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl], DOGS NTA) of the bilayer. This results in a polar oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance (EPR) spectra. In order to reveal the mechanism of the protein alignment, the influence of protein coverage on the order of the molecules was addressed. Along the lines described previously for a full monolayer (Jacobsen, et al. Biophys. J. 2005, 88, 4351), the polar orientation of the molecules was inferred from an analysis of the EPR line shape using the stochastic Liouville equation (SLE) approach developed by Freed and co-workers. The simulations reveal that the orientation of the protein is strongly determined by lateral protein-protein interactions. In comparison to the lipid bilayer, a fusion protein of T4 lysozyme (T4L) with Annexin XII was investigated, where the two-dimensional crystallization of Annexin XII on a dioleoylphosphatidylserine (DOPS) bilayer provides a surface layer of regularly anchored T4L molecules. For this system, it is found that the interaction between T4L and Annexin plays a more important role for understanding the structure in the adsorbed state.

Direct evidence of overdamped Peierls-coupled modes in the temperature-induced phase transition in tetrathiafulvalene-chloranil

“…In this spectral range, a resolution of 2 cm −1 was chosen. The reflectivity values agree well with available literature data, 25 which extend from 3000 to 14 000 cm −1 and thus cover the chargetransfer transition. The extension to high wave numbers is needed to obtain reliable Kramers-Kronig transformation ͑KKT͒ of the data.…”

“…Figure 2 shows the room-temperature TTF-CA reflectivity along the stack axis in the experimentally accessible spectral range of 5 -6000 cm −1 up to 14 000 cm −1 taken from Ref. 25. The reflectivity is rather low, as expected for a nonmetallic compound.…”

“…͑8͒, where f CT , ⍀ CT , and ⌫ CT are derived directly from experiment. 25 Finally, we set ⑀ ϱ = 2.5 to account for the highenergy contributions, ⌫ j 0 =2 cm −1 , for all the modes and k = 0.5 cm.…”

Stationary-Phase EPR for Exploring Protein Structure, Conformation, and Dynamics in Spin-Labeled Proteins

“…Tethering proteins to solid surfaces is fundamental for the development of biochips and optical biosensors for high-throughput analysis of molecular interactions based on surface plasmon resonance and bio-layer interferometry. − In addition, tethering of spin-labeled proteins on a solid support suitable for EPR spectroscopy offers a number of potential advantages in site-directed spin labeling (SDSL) studies of protein structure and dynamics. − For example, if a protein or complex can be rotationally immobilized on a solid support, then existing CW and time-domain methods in SDSL-EPR can be implemented to reveal internal dynamic modes of the protein on the nano- to microsecond time scale, free from contributions due to overall rotary diffusion, ,, and for determination of interspin distances at room temperature using dipolar spectroscopy. ,,, Moreover, high local concentrations needed for time-domain applications such as saturation recovery can be achieved without concern for protein aggregation, and a continuous flow system could be designed that provides for EPR-based rapid screening of binding partners, including small ligands. In anticipation of this latter application, we refer to the general strategy using tethered proteins in SDSL as stationary-phase SDSL-EPR.…”