Table 3.

Predicted and Measured Distances for SecA-Signal Peptide Binding^a

	experimental FRET		parallel-binding modeld	perpendicular-binding modele
protein with exogenous signal peptide or Sec A chimera	efficiencyb	distance (Å)c	distance (Å)	distance (Å)
bsSecA783 F652 with SP2-IAEDANS	0.60 ± 0.05	24.6 ± 3.7	27	33
bsSecA783 F652 with SP22-IAEDANS	0.61 ± 0.06	24.4 ± 3.6	21	20
bsSecA783 F652-GS(5)-PhoAss Cys2-IAEDANS	0.67 ± 0.03	23.4 ± 4.3	27	33
bsSecA783 F652-GS(5)-PhoAss Cys22-IAEDANS	0.41 ± 0.02	27.9 ± 5.8	21	20
bsSecA783 F652-GS(2.5)-KRRLamBss Cys2-IAEDANS	0.56 ± 0.05	25.3 ± 4.5	23	45
bsSecA783 F652-GS(2.5)-KRRLamBss Cys29-IAEDANS	0.45 ± 0.04	27.2 ± 5.4	27	24

^aDistances between Trp724 of bsSecA or Trp775 of ecSecA protein and the indicated residue on the signal peptide were determined utilizing the indicated model and PyMOL or by experimental measurement utilizing FRET.

^bThe calculated energy transfer efficiency from different samples; each value is the average number from at least three individual trials, and standard deviation is reported.

^cDistances between Trp724 and the indicated IAEDANS-labeled residue on the signal peptide were determined by FRET as described in Experimental Procedures. The calculated error included consideration of the minimal and maximal values of κ² based on anisotropy measurements as described previously.⁴⁴

^dTo model the SecA-bound, 21-residue, PhoA signal peptide, we used the NMR structure of the ecSecA-bound, KRRLamB signal peptide²¹ utilizing the stretch of KRRLamB from Leu11 to Met22 that is the hydrophobic core region. We use the hydrophobic core regions as they are approximately the same length in the two peptides. This 19.4 Å region projected onto the bsSecA X-ray structure corresponds to CTL residues Glu 785 to Gln793. Consequently, in keeping with this alignment, Glu781 and Gln798 correspond to Cys2 and Cys22, respectively, of the bound PhoA signal peptide. Similarly, Met776 and Gln800 correspond to Cys2 and Cys29, respectively, of the bound KRRLamB signal peptide. The predicted distance is measured from the Cγ atom of the identified residue to Cγ of Trp 724.

^eThis model is based on the NMR structure of the ecSecA-bound KRRLamB signal peptide.²¹ To compensate for the shorter PhoA signal peptide, the 20-residue stretch from Arg6 to Gln25 that centers on the hydrophobic core region was utilized. KRRLamB Lys7 or Ala26 was utilized to mimic Cys2 or Cys22, respectively, of the bound PhoA signal peptide. KRRLamB Met2 or the final residue in that peptide (Ala28) was utilized to mimic Cys2 or Cys29, respectively, of the bound KRRLamB signal peptide. The predicted distance is measured from the Cγ atom of the indicated residue to Cγ of Trp775.