## 4 Discussion  
The expression of channelrhodopsin-2 for purposes of optogenetic manipulation has been carried out in a variety of different cell types, including cardiac cells and neuronal cells [5; 50]. Importantly, ChR2 undergoes marked conformational changes during photoactivation which are crucial for the gating mechanism and these are likely to affect the interaction with the surrounding membrane environment. Therefore, we undertook here the quantification of an important component of the interaction of ChR2 with a model membrane, employing an approach that combines computational methods that include MD simulations and CTMD analysis [22; 51], with related experimental approaches using electrophysiology [32; 46] and kinetic modeling [13]. We explored the contribution of the surrounding lipid environment on ChR2 function by quantifying the membrane deformation around the TM core and found that the membrane deformation does not fully eliminate the hydrophobic mismatch between the ChR2 protein and the surrounding membrane, as several residues remain exposed to unfavorable environments. The large energy penalty associated with this RHM was located at the cytoplasm-facing membrane-protein interface located between adjacent TMs 1/2 and 6/7. This is especially interesting in the context of ChR2 gating because of the large movements of TMs 2 and 7 after photoexcitation [16; 17; 52]. Analysis of MD simulation trajectories showed that the RHM penalty in WT ChR2 is associated with water penetration into the membrane caused by thinning of the bilayer around the polar residues Q210 and Q73. As a result, V269 becomes exposed to a \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**State Population** & **WT** & **Q210A** & **V269N** & **Q210N** \\ \hline Time to Peak (ms) & 8.0 & 7.7 & 5.3 & 7.3 \\ O1 Peak & 0.46 & 0.55 & 0.52 & 0.38 \\ O2 Peak & 0.09 & 0.10 & 0.11 & 0.09 \\ O1 Stat & 0.13 & 0.19 & 0.16 & 0.10 \\ O2 Stat & 0.65 & 0.61 & 0.67 & 0.65 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: State population comparison. The population of O1 and O2 were determined at specific time points as described in the text. Values are shown as a fraction of the maximum occupancy, i.e., O1 + O2 + C1 + C2 = 1. Figure 6: Four-state photocycle theoretical fits. Theoretical fits were obtained by parameter optimization and objective function minimization as described in the Materials and Methods. Solid black line is the experimental average while the dashed red line is the average theoretical fit. more polar environment. Using experimental electrophysiological approaches, we examined the relation between predicted RHM and measured functional properties to probe the predictive value of the RHM in identifying critical residues in lipid-protein interactions. The simulations were carried out in a generic model of the membrane environment of POPC lipids to enable the evaluation of results in the context of the overwhelming majority of studies of membrane proteins and GPCRs in particular, using this model membrane. These include computational studies by us [19; 53; 54; 55] and many others [56]. The expectation that membrane-determined effects will result from RHM for the ChR was confirmed by the computational results and the corresponding experimental probing. This opens the way for future studies to interrogate cell-specific environments and their role in the use of the ChR tool, in the way demonstrated in our other studies in which the effects of cholesterol, PIP2 and lipids with varying chain lengths and charge properties [34; 35; 57; 58]. The effects of mutating V269 (in TM7) to asparagine illustrate the relation between a change in the mode of interaction of ChR2 with the membrane and the measured electrophysiological properties. As described, the V269N mutation reduces the hydrophobic mismatch (see Figure 2B) and a hydrophobic residue at this position is highly conserved in channelrhodopsins.