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.

Site-Directed Fluorescence Labeling of a Membrane Protein with BADAN: Probing Protein Topology and Local Environment

“…For simplicity, we have assumed that the M13 coat protein can be described by a full a-helix. However, previous work has demonstrated that the amino acid residues 1-9 are unstructured (4,5,30). These amino acid positions are located on the N-terminal hydrophilic anchor that is emerging from the headgroup region into the water phase, given by distances from the bilayer center .30 Å in Fig.…”

“…In these studies, we demonstrated that the coat protein is a single membrane spanning almost straight a-helical protein (3-5) that did not differ much from the structure in the intact phage (6). AEDANS has several physicochemical properties that make it a useful label for such investigations: 1), the fluorescent spectrum is a simple line shape of which the position is sensitive to the environ-mental polarity (i.e., it is an environmental probe) (7-10); 2), the AEDANS linker is sufficiently long and flexible to enable the probe to sense the global environment and not local molecular effects; and 3), the linker flexibility enables accurate Förster (or fluorescence) resonance energy transfer experiments to be carried out on the donor-acceptor pair Trp-AEDANS using an averaged orientation factor k 2 ¼ 2/3 (3)(4)(5)11). From the environmental properties of the AEDANS label, the membrane embedment also was found, resulting in a slightly tilted protein topology (10).…”

Profiling of dynamics in protein–lipid–water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths

“…As a result, the depth of protein insertion, orientation and tilt of the almost straight α-helical protein with respect to the bilayer interface are now known with great precision (Nazarov et al 2007; Vos et al 2007). These results are in excellent agreement with recent findings from site-directed spin label ESR experiments on the same system (Stopar et al 2006b).…”

Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy

“…2). For simplicity, we assumed a membrane-embedment of the protein based on a recently published model, using an a-helical protein with a tilt angle of 18°w ith respect to the membrane normal and with membrane crossing points at positions 9 and 47 [19,21,22]. To analyze the effect of protein conformation and membrane-embedment on the simulated free rotational space X, we generated a number of 5000 different helical structures of the protein with dihedral angles / and w uniformly distributed around the values for an a-helix: À57 ± 30°and À47 ± 30°, respectively.…”

“…The red triangles correspond to the experimental values of X. The white triangles indicate the X values related to the original a-helical protein model (u = À57°and w = À47°) as defined in[19,21,22].…”