Exploring the interactions between signal sequences and E. coli SRP by two distinct and complementary crosslinking methods.
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.
Clérico
Eugenia M
EM
Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst MA 01003, U.S.A.
Szymańska
Aneta
A
Gierasch
Lila M
LM
D000595:Amino Acid Sequence; D001577:Benzophenones / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D021382:Protein Sorting Signals; D018271:Signal Recognition Particle / Q000737:chemistry*
D001577:Benzophenones; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D018271:Signal Recognition Particle; C047723:benzophenone