Probing the Affinity of SecA for Signal Peptide in Different Environments
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- 作者：Monika Musial-Siwek ; Sharyn L. Rusch ; and Debra A. Kendall
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：October 25, 2005
- 年：2005
- 卷：44
- 期：42
- 页码：13987 - 13996
- 全文大小：288K
- 年卷期：v.44,no.42(October 25, 2005)
- ISSN：1520-4995
文摘
SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with anumber of ligands during export, including signal peptides, membrane phospholipids, and nucleotides.Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) andmutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in thepresence of other transport ligands. We find that residues on the third 
-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueoussolution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of2.4 
M. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signalpeptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has twoconformational states; our results do not support different oligomeric states of SecA. Using native gelelectrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution andlipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffectedby temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP orAMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in anelongated form while inserting the signal peptide into membranes.
-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueoussolution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of2.4 M. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signalpeptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has twoconformational states; our results do not support different oligomeric states of SecA. Using native gelelectrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution andlipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffectedby temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP orAMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in anelongated form while inserting the signal peptide into membranes.