The directed cooperative assembly of proteorhodopsin into 2D and 3D polarized arrays

Liang et al. 10.1073/pnas.0702336104.

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SI Figure 6
SI Materials and Methods

SI Figure 6

Fig. 6. Estimate the average number of DDM molecules associated with each PR from dilution-induced PR precipitation. (A) A typical normalized UV-Vis spectra of DDM-solubilized PR solution diluted by solution without DDM (red trace) and the control PR solution diluted similarly by solution with 0.05% DDM (black trace). The reduced chromophore absorption is used to quantitatively determine the precipitated PR. (B) Pictures of PR solution diluted by solution without DDM (left) and the control PR solution diluted similarly by solution with 0.05% DDM (right). The dilution-induced PR precipitation is clearly seen when PR solution is diluted by solution without DDM.

SI Materials and Methods

Estimate the Average Number of DDM Molecules Associated with Each PR (#DDM/PR) and the Number of PR Monomers in DDM-solubilized PR Fractions. Critical micelle concentration (CMC) is the maximum concentration of free detergent (oil) in water before its micelle formation. It is defined by Boltzman distribution based on the free-energy difference between oil/water and oil/oil interface. We assume the free energy difference between DDM in micelles and that associated with PR hydrophobic domains is negligible. The concentration of free DDM in equilibrium with DDM-solubilized PR is regarded as its CMC, which is 1.8 ´ 10^-4 M under room temperature (1).

The DDM-solubilized PR solution was dialyzed to the point where PR was precipitated out. At this point the solution had negligible DDM micelles. After centrifugation to remove the precipitated PR, the solution was subjected to a series of dilution experiments. Because dilution decreased the free-DDM concentration in solution, the DDM molecules originally associated with PR hydrophobic domains were released to replenish the free-DDM concentration toward its CMC as defined by the Boltzman distribution. Consequently, PR that lost DDM was precipitated out upon centrifugation. We assume the precipitated PR has negligible detergents associated with them. Because the number of free detergent molecules released to recover CMC (n = V×CMC, V is the volume of dilution solution) was known, and the number of precipitated PR (n) can be determined quantitatively by UV-Vis based on the solution absorption change due to PR precipitation, the number DDM/PR can be estimated (N/n). A typical UV-Vis measurement of dilution-induced PR precipitation is shown as SI Fig. 6.

We found that the number DDM/PR is ~150 ± 20. Interestingly, this number is roughly the same as the aggregation number of DDM in its micelle form (1), which indicates, on average, that one micelle of DDM molecules are involved to solubilize each PR. This phenomenon was also observed for a couple of other membrane proteins (MPs) solubilized by detergents (2), although the detergent molecules solubilizing MPs aggregate much differently from its micelle form.

Gel filtration measurements of DDM-solubilized PR solution found a dominant monodispersed fraction of ~300 kDa (Fig. 2B). The number of PR monomer in that solubilized fraction can be calculated as: MW_fraction/(MW_PR+MW_DDM×N/n), where MW_fraction, MW_PR, and MW_DDM is the molar mass of solubilized PR fraction, PR by itself (~29 kDa) and DDM (~510), respectively. Because the number DDM/PR is ~150, we estimated the DDM-solubilized PR is a trimer form. The improved stability of solubilized PR over bR might be attributed to its trimeric form, where the integrity of PR molecule is better maintained after release from membrane embedment, whereas detergent-solubilized bR is known to be a monomer (~24 kDa) (2) and the bR solution shows progressive chromophore loss within a few days (3). The reason that DDM-solubilized PR exists as a stable trimeric form might lie with the glycine zipper motif (GYFTGYLMG) existed in helix F [ref. 4 and Fernando Valle (Genencor International), personal communication), which is absent in bR.