Diffusion coefficients and dissociation constants of enhanced green fluorescent protein binding to free standing membranes

Recently, a new and versatile assay to determine the partitioning coefficient KP as a measure for the affinity of peripheral membrane proteins for lipid bilayers was presented in the research article entitled, “Introducing a fluorescence-based standard to quantify protein partitioning into membranes” [1]. Here, the well-characterized binding of hexahistidine-tag (His6) to NTA(Ni) was utilized. Complementarily, this data article reports the average diffusion coefficient D of His6-tagged enhanced green fluorescent protein (eGFP-His6) and the fluorescent lipid analog ATTO‐647N‐DOPE in giant unilamellar vesicles (GUVs) containing different amounts of NTA(Ni) lipids. In addition, dissociation constants Kd of the NTA(Ni)/eGFP-His6 system are reported. Further, a conversion between Kd and KP is provided.


Subject area Biophysics
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Molecular Biophysics
Type of data We provide a conversion between K d and K P for the His 6 -NTA(Ni) system, which can be extended to any protein-lipid interaction with a known 1:1 stoichiometry.
Protein diffusion coefficients could be used as an indicator of crowding effects. As for DOPC/DGS-NTA(Ni) the lipid dynamics is independent of increasing protein concentrations, the ATTO-647N-DOPE diffusion coefficient could serve as a standard.

Experimental design, materials and methods
The materials, the preparation of eGFP-His 6 and GUVs, the optical setup used and the FCS data acquisition/analysis were described elsewhere [1].

Determination of average diffusion coefficients
We determined the average diffusion coefficients D of eGFP-His 6 attached to DGS-NTA(Ni) in the lipid bilayer and of ATTO-647N-DOPE (Table 1 and Fig. 1) by applying the following equation: The average focal waist w 0 obtained from a calibration with Alexa488 and with ATTO-655, were

K d for eGFP-His 6 DGS-NTA(Ni) system
Only in cases where the protein-lipid binding is purely stoichiometric and if the stoichiometry is known, the protein affinity for the lipid membrane can be expressed by the dissociation constant K d . In equilibrium, an identical number of molecules P will dissociate from and associate to the lipid phase L per area and time P þ nL-nPL. For 1:1 binding stoichiometry ðn ¼ 1Þ, K d is defined as: where P f Â Ã is the freely diffusing species in solution, PL ½ ¼ P m ½ the membrane associated fraction and L f Â Ã ¼ L ½ À½L m with the total accessible lipid concentration L ½ c L m ½ . Thus, L ½ is constant in a given sample and can be expressed by: Here, A is the total accessible lipid area, A L the area per lipid, N A the Avogadro's constant and V the volume of the sample chamber. P f Â Ã and P m ½ can be determined by FCS [1]. In particular, P m ½ is obtained by: where P 2D ½ is the surface concentration on the top pole of a GUV. A rearrangement of Eq. (3) gives: Combining Eq. (6) with Eqs. (4) and (5) gives the following main equation (A and V cancel out): When a set of P f Â Ã and P 2D ½ is plotted and fitted with a linear equation passing through the origin of the axis, K d can be calculated from the slope a: Comparing Eq. (8) with Eq. (7) in Thomas et al. [1] leads to the following conversion between K d and partition coefficient K P : with the water concentration W ½ being constant with W ½ ¼ W ¼ 55:5 M. Assuming that the binding stoichiometry for the NTA(Ni)/eGFP-His 6 system is 1:1 [2,7], we could calculate the dissociation constant K d from the reported partitioning coefficient K P [1] with Eq. (9) or directly from the slope a with Eq. (8). In Table 2 and Fig. 2 the values of the dissociation constant K d are given for the different content of DGS-NTA(Ni). They correspond to the upper range of values reported in the literature, which vary from 10 nM to 10 μM [7][8][9]. Table 2 K d determined by GUV-FCS assay. Calculated dissociation constants by fitting all data points for increasing amounts of DGS-NTA(Ni) via the GUV-FCS method (mean 7 combined s.e.m.).