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Exploring the interactions between signal sequences and E. coli SRP by two distinct and complementary crosslinking methods
Author(s) -
Clérico Eugenia M.,
Szymańska Aneta,
Gierasch Lila M.
Publication year - 2009
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21181
Subject(s) - signal peptide , chemistry , benzophenone , peptide , biophysics , signal (programming language) , signal recognition particle , combinatorial chemistry , residue (chemistry) , peptide sequence , biochemistry , stereochemistry , organic chemistry , computer science , biology , gene , programming language
Photoaffinity crosslinking comprises a group of invaluable techniques used to investigate in detail a binding interaction between two polypeptides. As the diverse photo crosslinking techniques available display inherent differences, the method of choice will provide specific information about a particular system under study. We used two complementary crosslinking approaches: photo‐induced crosslinking of unmodified proteins (PICUP) and benzophenone‐mediated (BPM) crosslinking to extensively examine the interaction between the signal recognition particle (SRP) and signal sequences. Signal peptide binding by SRP presents a central puzzle in the protein targeting process because signal sequences must be recognized with fidelity but lack strict primary structural homology. The concurrent use of PICUP and BPM crosslinking to link signal peptides to E. coli SRP allowed us to explore the crosslinking pattern resulting from using different crosslinking chemistries, varying the position of the photoprobe in the hydrophobic core of the signal sequence, and shifting the crosslinking reactive group away from the signal peptide backbone. By PICUP, signal peptides crosslinked exclusively to the NG domain of the SRP protein Ffh, regardless of the position of the reactive residue. Benzophenone‐modified amino acids preferentially crosslinked the signal peptide to the C‐terminal (M) domain of Ffh. We conclude that signal peptide binding is largely mediated by the M domain. Importantly, our data also indicate intimate, at least transient, contacts between the hydrophobic core of the signal peptide and the NG domain. These results reopen the possibility of a direct involvement of the NG domain in signal sequence recognition. © 2009 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 201–211, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com