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 [Formula: see text] 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 [Formula: see text] 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 [Formula: see text] of the NTA(Ni)/eGFP-His6 system are reported. Further, a conversion between [Formula: see text] and [Formula: see text] is provided.

Specifications table

Value of the data

We provide the first valuable characterization of the eGFP-His

The eGFP-His

We provide a conversion between

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.

Data

Hexahistidine-tag (His6) binding to Nickel (Ni) chelated with nitrilotriacetic acid (NTA) is a well-characterized process [2], [3] and it is extensively used to reconstitute protein systems in giant unilamellar vesicles (GUVs) [4], [5], [6]. We made GUVs consisting of 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholin (DOPC) and 2, 3, 4 or 5 mol% 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] nickel salt (DGS-NTA(Ni)), labeled with 0.05 mol% ATTO-647N-DOPE. These GUVs were incubated with increasing amounts of His6-tagged enhanced green fluorescent protein (eGFP-His6) and point fluorescence correlation spectroscopy (FCS) was performed both at the top pole of the GUVs and in solution. From the obtained FCS auto-correlation functions the diffusion coefficient of both eGFP-His6 and ATTO-647N-DOPE as well as the dissociation constant of the NTA(Ni)/eGFP-His6 system were calculated.

Experimental design, materials and methods

The materials, the preparation of eGFP-His6 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 of eGFP-His6 attached to DGS-NTA(Ni) in the lipid bilayer and of ATTO‐647N‐DOPE (Table 1 and Fig. 1) by applying the following equation:

Table 1

Diffusion coefficient D determined by GUV-FCS assay. Calculated diffusion coefficients by averaging all data points for increasing amounts of DGS-NTA(Ni) via the GUV method (mean ± combined s.e.m.).

DGS-NTA(Ni)	eGFP-His6 D in µm2/s	ATTO‐647N‐DOPE D in µm2/s
2%	4.36 ± 1.12 (n=548)	10.03 ± 0.68 (n=549)
3%	3.20 ± 0.75 (n=775)	9.74 ± 0.66 (n=900)
4%	3.14 ± 0.94 (n=740)	9.67 ± 0.76 (n=969)
5%	1.90 ± 1.01 (n=593)	9.72 ± 0.52 (n=705)

Fig. 1

Diffusion coefficients determined by GUV-FCS assay. for eGFP-His6 coordinated to NTA(Ni) (filled squares) and the membrane dye ATTO‐647N‐DOPE (circles) with increasing amounts of DGS-NTA(Ni). Error bars represent the combined standard error of mean. The of ATTO‐647N‐DOPE shows no significant differences, whereas the of eGFP-His6 decreases with increasing amounts of DGS-NTA(Ni).

The average focal waist obtained from a calibration with Alexa488 and with ATTO-655, were (mean ± s.e.m, n=19) and (mean ± s.e.m, n=19), respectively. The diffusion times were determined fitting the auto-correlation curves with a weighted 2D−3D+T model function. The values were averaged and the significance of their deviation was tested using a one-way analysis of variance (ANOVA) in SigmaPlot 12.3 (Systat Software, Inc., San Jose, CA). This statistical analysis indicated a significance of deviation for the average diffusion coefficients of eGFP-His6 in presence of different DGS-NTA(Ni) concentrations (F(3,78)=19.48, p<0.001). With increasing amount of DGS-NTA(Ni), the eGFP-His6 average diffusion coefficients decreases from (mean ± combined s.e.m., n=548) to (mean ± combined s.e.m., n=593). In contrast, the average diffusion coefficient of ATTO-647N DOPE for all concentrations DGS-NTA(Ni) was (mean ± combined s.e.m., n=3123) and did not show any statistical significant difference (F(3,86)=3.24, p=0.026).

K for eGFP-His6 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 . In equilibrium, an identical number of molecules will dissociate from and associate to the lipid phase per area and time . For 1:1 binding stoichiometry , is defined as:where is the freely diffusing species in solution, the membrane associated fraction and with the total accessible lipid concentration . Thus,

is constant in a given sample and can be expressed by:

Here, is the total accessible lipid area, the area per lipid, the Avogadro׳s constant and the volume of the sample chamber. and can be determined by FCS [1]. In particular, is obtained by:where 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 ( and cancel out):

When a set of and is plotted and fitted with a linear equation passing through the origin of the axis, can be calculated from the slope :

Comparing Eq. (8) with Eq. (7) in Thomas et al. [1] leads to the following conversion between and partition coefficient :with the water concentration being constant with .

Assuming that the binding stoichiometry for the NTA(Ni)/eGFP-His6 system is 1:1 [2], [7], we could calculate the dissociation constant from the reported partitioning coefficient [1] with Eq. (9) or directly from the slope with Eq. (8). In Table 2 and Fig. 2 the values of the dissociation constant 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

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 ± combined s.e.m.).

DGS-NTA(Ni)	K_din M
2%	2.18 ± 0.23 · 10−5
3%	1.28 ± 0.26 · 10−5
4%	3.60 ± 0.27 · 10−6
5%	1.15 ± 0.27 · 10−6

Fig. 2

Graphic presentation of dissociation constant obtained by GUV-FCS assay. Error bars represent the combined standard error of mean.

Subject area	Biophysics
More specific subject area	Molecular Biophysics
Type of data	Table, figure
How data was acquired	Fluorescence Correlation Spectroscopy, Confocal Microscopy using a LSM 780 with a ConfoCor 3 unit (Zeiss, Jena, Germany)
Data format	Analyzed
Experimental factors	GUVs consisting of DOPC and 2, 3, 4 or 5 mol% DGS-NTA(Ni), labeled with 0.05 mol% ATTO-647N-DOPE
Experimental features	Titration of eGFP-His6to the GUVs
Data source location	Max Planck Institute of Biochemistry, Martinsried, Germany
Data accessibility	The data are provided within this article