Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

This study focused on determining the location of the preprotein binding site on SecA prior to insertion of the preprotein into the SecYEG channel. To map the binding site, FRET experiments were performed between different regions of the preprotein and three distinct locations on the SecA and SecYEG proteins (Figure 1A–D). From the distances obtained and three-dimensional structures of SecA, SecYEG, and the preprotein, the location of the preprotein binding site was predicted. Rather than employing three separate entities (SecA, SecYEG, and preprotein) to perform these measurements, the PhoA signal sequence was attached to SecA through genetic modification after incorporation of a Gly-Ser linker^2,3. For facile labeling with dyes, Cys residues or amber mutations were introduced at residues 2, 22, 35, and 45 in the PhoA preprotein (Figure 1E).

Identification of sites and labeling
A putative binding site for the signal sequence had been previously identified using similar FRET mapping methods^2,32. These and other studies had identified the two-helix finger (THF) and the preprotein cross-linking domain as possible binding sites of the signal peptide with a suggested orientation in a parallel position to the THF^{33,51,52,53,54} (Figure 1F). Thus, identification of potential labeling sites on the SecA and SecYEG proteins was done based on the location of the putative binding site in the SecA and SecYEG crystal structure (shown in green in Figure 1A–D). Three sites were chosen to triangulate the position of the putative binding site, where essentially the three sites form a triangle around the putative binding site. As shown in Figure 1, the three sites were within the FRET range of the binding site (50-70 Å). The dye pair of Alexa Fluor 488 (AF488) and Alexa Fluor 647 (AF647) was chosen, as the R₀ value of 55.7 ų⁶ corresponds well with the expected distances between the labeled sites and the putative binding site ensuring measurement accuracy.

The three sites chosen for labeling, SecA37, SecA321, and SecY292 (shown as magenta, violet, and cyan spheres in Figure 1A–D) are located throughout the protein complex forming a triangle around the putative binding site. The three sites were separately mutated to Cys residues in a Cys-less mutant to ensure that only the correct position was labeled^2,37. For the SecY292 experiments, the PhoA preprotein sites were labeled with AF647 and SecY residue 292 was labeled with AF488 using maleimide chemistry. In the chimeric protein, sites SecA37 and SecA321 were labeled with AF647 and the preprotein was labeled with AF488. In the SecA-PhoA chimeric protein, residues 2, 22, 37, and 45 of the PhoA preprotein segment were each mutated to an amber codon in individual proteins. The amber codon mutations allowed the introduction of unnatural amino acid, p-azidophenylalanine, at those positions, which were subsequently labeled with the AF488 using click chemistry^39,40. Each mutation was generated and labeled independently to ensure correct, differential labeling of the protein components. The degree of labeling was determined for all proteins and generally needed to be 50% or better in order to proceed with the sample.

Determination of transfer efficiencies and distances
Prior to performing the energy transfer measurements, the donor quantum yield, overlap integral and R₀ values were determined (steps 3.4-3.6). The donor quantum yield was measured relative to the dye, fluorescein, which has a quantum yield of 0.79 in 0.1 M NaOH⁴⁷. Absorption and fluorescence emission spectra were obtained at a series of concentrations to generate a linear plot of absorption relative to fluorescence emission intensity to determine the quantum yield. In these measurements, it is critical to measure the absorption in the linear range (0.1-1.0) and all emission measurements need to be generated with the same slit settings. As these values are used to determine overlap integrals and R₀ values, they should be measured under FRET conditions. Protein local environment profoundly affects dye emission and consequently, donor quantum yields should be measured for each of the sites investigated. We note that sites on SecA and SecYEG influence the R₀ values more strongly than those on the PhoA portion of the chimera. For dye pairs with the same SecA or SecYEG site, the R₀ values are typically within 5 Å of each other; whereas the R₀ values can differ by as much as 20 Å for the two different SecA locations (residues 37 vs. 321), underscoring the importance of determining R₀ values for each dye pair (Table 1).

The calculation of R₀ assumes that the donor and acceptor dyes are freely rotating and the degree to which the dyes do not rotate contributes to the overall uncertainty in the measurement. To appropriately take into account the relative motion of the dyes and their orientations, steady-state fluorescence anisotropy measurements were performed on all the donor and acceptor dyes in the different labeling positions. These values, which were in the 0.10-0.21 range, were used to calculate the error associated with the distance measurements^2,3,48. The relatively high anisotropy values observed for both the donor and acceptor dyes correspond to a reduction in dye rotation, which is inconsistent with the assumption of free rotation. The lack of free rotation generates an error of 19%-25% in the distance calculations. As shown in Table 1, this led to an average uncertainty in the measured distances of at most ± 15 Å. When mapping the FRET distances, these uncertainties in the distance measurements are an important consideration, as discussed below.

The calculated distance between donor-acceptor pairs is based upon the relationship between efficiency and distance, in which a higher efficiency is indicative of donor-acceptor pairs separated by a shorter distance. To determine FRET efficiencies, fluorescence emission spectra excited at the donor excitation wavelength (488 nm) are obtained on donor-only and donor-acceptor samples. Typically, a reduction in donor emission intensity signifies the presence of energy transfer (step 5.1). Figure 1G depicts the donor-acceptor spectra for the SecA 37 residue with either residue 2 or 22 of the PhoA chimera. The SecA37 residue is labeled with AF647 or the acceptor dye, and the PhoA residues are labeled with AF488 or the donor dye. At either position, the donor fluorescence is reduced and a small increase in acceptor fluorescence intensity can be seen in the donor-acceptor samples. Since excitation is done at the donor excitation wavelength of 488 nm, which does not directly excite the acceptor, any acceptor fluorescence observed results from energy transfer. Thus, the decrease in donor intensity and concomitant increase in acceptor intensity results from energy transfer between the two dyes. Significantly, the donor fluorescence intensity is higher for the PhoA2 position (blue) relative to the PhoA22 position (yellow) in the presence of the acceptor. This relative difference in the donor intensity decrease indicates that energy transfer between the PhoA2 residue and the SecA37 residue is weaker than the transfer between the PhoA22 and SecA37 residues, which implies that the PhoA2 residue is located further away from SecA37 than PhoA22. Distances are determined from the relationship between efficiency and R₀ values (step 5.1.4).

Since the steady-state fluorescence measurements could represent an average of two or more distances, we also performed time-resolved fluorescence measurements. For these experiments, the lifetime of the donor dye is measured in the presence and absence of the acceptor (Figure 1G). If there were two distinct energy transfer processes contributing to the measured steady-state fluorescence efficiency, they would be observed as discreet lifetimes, provided they were resolvable within the time resolution of the instrument. To enhance the ability to resolve the lifetimes, 10,000 counts or more should be collected at the peak; however, the peak height or peak channel counts needs to be balanced with the time of acquisition and potential damage to the sample. Time-resolved measurements yielded single lifetimes for each donor-acceptor pair consistent with only one orientation or distance between the dyes. We note that small differences in the distance as observed in the areas revealed by our FRET mapping technique would not lead to resolvable lifetimes in our system. Moreover, the efficiencies as determined by the time-resolved fluorescence measurements were in good agreement with those determined from the steady-state measurements, providing further support that the measured efficiencies arise from only one distance between the dye pairs³.

Mapping the FRET distances onto the 3-dimensional structure
The resonance energy transfer measurements yield sufficient distance information to identify the binding site and orientation of the signal sequence on SecA. The three locations on SecA and SecYEG along with the four positions on the PhoA region of the SecA-PhoA chimera provide the 12 different distances used to map the binding site (Table 1). The twelve distances were mapped onto the three-dimensional X-ray cocrystal structure of the Thermotoga maritima SecA-SecYEG complex (PDBID: 3DIN) to identify the binding site of the signal sequence³³. The structure of the SecA-SecYEG complex is similar to that observed in E. coli as evidenced by an in vivo photocrosslinking study⁵⁵.

We use the beginning (PhoA2) and end (PhoA22) residues of the PhoA signal sequence in the SecA-PhoA chimera to illustrate how residue locations were identified on the SecA-SecYEG complex. As energy transfer can occur in all directions, the FRET distances and associated errors describe a spherical shell, with one of the dye locations from the donor-acceptor pair designated as the center. In this study, the residues SecA37, SecA321, and SecY292 form the centers of three spherical shells that describe the location of the PhoA2 residue of the signal sequence. Visualization of the overlapping regions arising from the three separate locations, SecA37 (magenta), SecA321 (purple), and SecY292 (cyan) are shown in Figure 3. Only a portion of each FRET shell intersects with the protein structure, and the residues and backbone that fall within that shell are highlighted. Thus, the protein regions within the shell defined by the SecA37-PhoA2 distance are shown in magenta (Figure 3A,E), while the regions defined by the SecA321-PhoA2 and SecY292-PhoA2 shells are shown in purple (Figure 3B,F) and cyan (Figure 3C,G), respectively. The putative binding site, consisting mainly of the two-helix finger, is shown in green.

As shown in Figure 3, each of these FRET shells defines a relatively large section of the protein complex. For all three locations, the FRET shell does intersect with the putative binding site; however, for the SecA321 residue, for example, the intersected area is smaller and lies towards the ends of the fingers with significant overlap with the helical scaffold domain. The intersection or the common area of all three FRET shells (Figure 3D,H), defines the location of the PhoA2 residue. This area is considerably smaller than each FRET shell and includes only a small portion of the THF with a large contribution from the helical scaffold. The scripts used for generating the FRET shells and the intersected areas for the molecular visualization program, PyMOL, are given in the Supplemental Information. Portions of the shells are visualized as pink dots on the SecA-SecYEG complex in Supplementary Figures 1–3.

A similar strategy was used to identify the location of the PhoA22 residue. The FRET shells defined by the PhoA22 FRET distances (Figure 4A–C,E–G) describe a smaller area relative to the PhoA2 residue (Figure 4D,H vs. Figure 3D,H). We interpret this difference to suggest that the PhoA2 residue and associated region are more flexible and labile than PhoA22. Significantly, the area ascribed to the PhoA22 residue is located closer to the tip of the THF and the mouth of the SecYEG channel, with regions of SecY identified in the common area (Figure 3D,H). All three dye pairings identify regions along with the putative binding site; however, the intersected common areas center the PhoA22 location (Figure 4D,H) at the opposite end of the THF relative to the PhoA2 location (Figure 3D,H). These findings would suggest that the signal sequence of the preprotein which extends from residues 2-22, lies along the THF in a relatively unstructured state. This result is consistent with earlier studies suggesting the signal sequence binds to the protein along the THF in an extended state and that the C-terminal end of SecA in the B. subtilis crystal structure essentially models the structure of the signal peptide and occupies the same location (shown in red Figure 1F)^2,26. We employed a similar approach to identify two locations in the early mature region (residues 37 and 45) of the SecA-PhoA preprotein chimera to further define the binding and orientation of the signal sequence and early mature region as discussed below. In other studies FRET distances have been used effectively to refine an existing structure or molecular dynamics simiulation-derived model^18,19,56; we were not able to do this, as no structure for the signal sequence bound to SecA exists.

Figure 1: Labeling sites in SecA-SecYEG complex with representative FRET spectra. (A–D) Four different views of the SecA-SecYEG co-crystal structure (PDBID: 3DIN)³³ in which the labeling sites of SecA37, SecA321, and SecY292 are shown as magenta, violet, and cyan spheres, respectively. A-C are side views of the complex and D is a top view. SecA is shown in light grey, SecYEG is shown in dark grey and the putative binding site, the THF, is shown in green. (E) Schematic of the SecA-PhoA chimera construct, which connects the SecA protein to the PhoA preprotein through a Ser-Gly linker (not drawn to scale). The labeling sites on the PhoA portion of the chimera are given in blue, green, yellow, and red, corresponding to residues 2, 22, 37, and 45. (F) Ribbon diagram of the crystal structure of B. subtilis SecA protein (PDBID: 1M6N) colored by domain where nucleotide-binding domains 1 and 2 are shown in blue and light blue, respectively, the preprotein cross-linking domain is shown in gold, the central helix in green, the two-helix finger in cyan, the helical wing domain in dark green and the C-terminal linker in red²⁶. The unstructured C-terminus serves as a model of the bound PhoA signal peptide based on a previous FRET mapping study². (G) Steady-state fluorescence spectra of the donor only and donor-acceptor samples of the SecA37-AF647 and PhoA2-AF488 FRET pair and the SecA37-AF647 and PhoA22-AF488 FRET pair. The reduction in donor intensity for the donor-acceptor sample is indicative of energy transfer. Greater energy transfer occurs from PhoA22 relative to the PhoA2 site based on the decrease in donor intensity. (H) Time-resolved fluorescence donor only (magenta) and donor-acceptor (light magenta) decay spectra of the SecA37-AF647 and PhoA22-AF488 FRET pair. The instrument response function is shown in grey. The donor-acceptor complex gives a shorter decay and consequently a faster lifetime consistent with energy transfer. All molecular structures were generated with the indicated PDB file and PyMOL⁵⁰. Figure 1E-H has been modified from Zhang et al.³. Please click here to view a larger version of this figure.

Figure 2: User interface of the FluorEssence program. (A) The opening window is shown with a circle around the red M. This must be clicked to connect the program with the fluorometer. (B) The experiment set-up window illustrates the different areas (monos, detectors, accessories) where scan relevant parameters are entered. Please click here to view a larger version of this figure.

Figure 3: FRET distance shells determined for the SecA-PhoA2 position. FRET distance shells constructed from the FRET distances and the associated uncertainties (Table 1) are depicted on the SecA-SecYEG complex (PDBID: 3DIN). (A–C) FRET distance shells for the PhoA2 location constructed with SecA37 (magenta), SecA321 (violet), and SecY292 (cyan), respectively at the center position. The shells are colored according to the center residue. (D) The intersection of the three FRET shells defines the location of PhoA2, shown in blue. (E–H) Views are rotated approximately 180° from A-D. All molecular structures were generated with the indicated PDB file and PyMOL⁵⁰. Please click here to view a larger version of this figure.

Figure 4: FRET distance shells determined for the SecA-PhoA22 position. FRET distance shells constructed from the FRET distances and associated uncertainties (Table 1) are depicted on the SecA-SecYEG complex (PDBID: 3DIN). (A–C) FRET distance shells for the PhoA22 location constructed with SecA37 (magenta), SecA321 (violet) and SecY292 (cyan), respectively at the center position. The shells are colored according to the center residue. (D) The intersection of the three FRET shells defines the location of PhoA22, shown in yellow. (E–H) Views are rotated approximately 180° from A-D. All molecular structures were generated with the indicated PDB file and PyMOL⁵⁰. Please click here to view a larger version of this figure.

Figure 5: FRET-mapped locations of PhoA2, PhoA22, PhoA37, and PhoA45 projected onto the B. subtilis SecA –Geobacillus thermodenitrificans SecYE cocrystal structure (PDBID: 5EUL). (A) Coloring of SecA-SecYE as in Figure 1. The OmpA peptide substrate inserted at the end of the THF is shown in pink. FRET-mapped regions were generated in the presence of ATP-γS, with PhoA2 shown in blue, PhoA22 in green, PhoA37 in yellow, and PhoA45 in red. Overlap regions are shown in olive (PhoA22 and PhoA37) and orange (PhoA37 and PhoA45). The peptide substrate (residues 749-791, cyan) was excised from the original structure and modeled into the putative binding region without any alterations to the structure (circled in red). (B) Enlarged view of the modeled peptide substrate. Residues 2 (Lys), 22 (Tyr), and 37 (Gly) of the OmpA peptide substrate are depicted in stick form in blue, green, and yellow, respectively. These residues in the modeled peptides exhibit excellent agreement with the predicted FRET mapped locations. For clarity, the nanobody in the original structure has been omitted. This figure has been modified from Zhang et al.³. Please click here to view a larger version of this figure.

Table 1: Transfer Efficiencies and Distances Determined for the SecA-PhoA-SecYEG complex. FRET efficiencies, distances, and R₀ values are given for the 12 distances used for mapping the preprotein binding site.

Supplementary Figure 1: FRET distance shell, shown in pink dots, determined for the SecA37 residue and the PhoA 37 residue on the SecA-SecYEG complex (PDBID: 3DIN). Sec A is shown in light grey, SecYEG is shown in dark grey and the SecA37 residue is shown in magenta. Please click here to download this File.

Supplementary Figure 2: FRET distance shell, shown in pink dots, determined for the SecA321 residue and the PhoA 37 residue on the SecA-SecYEG complex (PDBID: 3DIN). Sec A is shown in light grey, SecYEG is shown in dark grey and the SecA321 residue is shown in violet. Please click here to download this File.

Supplementary Figure 3: FRET distance shell, shown in pink dots, determined for the SecY292 residue and the PhoA 37 residue on the SecA-SecYEG complex (PDBID: 3DIN). Sec A is shown in light grey, SecYEG is shown in dark grey and the SecY292 residue is shown in cyan. Please click here to download this File.

Supplementary File. Please click here to download this File.