FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein

Abstract

A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall alpha-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage.

FIGURE 1

(A) Emission spectra of M13 coat protein mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C with AEDANS-labeled Cys in DOPC/DOPG vesicles after subtraction of the fluorescence of equimolar wild-type samples. The histogram shows the values of the acceptor emission maxima of the mutants. (B) Experimental excitation spectra obtained for mutant N12C at different titration points of wild-type proteins. The emission was detected at 496 nm. Labels 1–3 correspond to r_ul values of 0.27, 2.16, and 5.63, respectively. The lipid/protein ratios r_LP are 277, 111, and 53, respectively (see Table 1). The sample showing the highest peak at 290 nm (spectrum 3) has the highest protein density (lowest r_LP) and r_ul. Although the efficiency of energy transfer (Fig. 3) for this case is smallest, the overall energy absorbed by the donors in such a system, and therefore the transferred (intermolecular), is higher than for the other values of r_LP and r_ul (3).

FIGURE 2

Schematic drawing of the two-helix protein model with a donor (Trp-26, solid circle, located at a distance l_D from the protein helix axis) and acceptor (AEDANS, shaded circle, located at a distance l_A from the protein helix axis) attached at positions 26 and 9, respectively, in its own protein axis system (x, y, z). The orientation of the x axis is defined by the location of Trp-26, which is used as the reference amino acid residue. The complete set of structural parameters that describes the protein-lipid system is presented in Table 2.

FIGURE 3

Experimental energy transfer efficiencies E (solid circles) and their approximation by the model (dotted and solid lines) after global analysis versus the ratio between unlabeled and labeled proteins r_ul. The mutant names are given in the right top corner of each plot. The dotted line corresponds to initial fit of data for acceptor label positions 7–18. The solid line presents efficiencies obtained after fitting data for acceptor label positions 12–18.

FIGURE 4

(A) Resulting 100 structures obtained from global analysis of experimental FRET data of AEDANS-labeled M13 coat protein mutants in DOPC/DOPG vesicles. The structures are presented in terms of C_α positions that are projected on the plane formed by the OZ axis and the direction of tilt of the transmembrane domain. The protein domain from amino acid residue 1–9 cannot be described by a rigid α-helix and is schematically presented as a “cloud” containing several gray “unstructured” conformations. (B) Final set of 52 structures obtained after fitting of experimental data and filtering using Stokes shift information. The resulting tilt angle of the N-terminal domain ϕ = 5.0 ± 4.7°.

FIGURE 5

Experimental energy transfer efficiencies E (solid circles) and their approximation by the model (solid lines) after introducing an unstructured domain to the protein model between positions 1 and 10. The dotted lines show the previous fits by a model without unstructured domain (see Fig. 3).