PMID:11969393
| Citation |
Hui, D and Ling, V (2002) A combinatorial approach toward analyzing functional elements of the Escherichia coli hemolysin signal sequence. Biochemistry 41:5333-9 |
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| Abstract |
Secretion of hemolysin is directed by a signal sequence located within its C-terminal 60 amino acids. Deletion analyses have indicated that the extreme end of this C-terminus is critical for transport; however, it is not known if this region contains structural features necessary for function. In this study, we have used a combinatorial approach to generate two contiguous 8-residue random libraries (Cterm1 and Cterm2) in the signal sequence to investigate the functional specificity of the last 16 residues. The large number of variants generated had provided us with a rich data set to determine if a restricted subset of sequences was actually required for function in the extreme C-terminus. We observed that over 90% of the random sequences in the Cterm1 region were secreted at close to wild-type level, while the Cterm2 region was more restricted with only 50% of the random sequences supporting wild-type-like transport. It appeared that, in the Cterm2 region, the relative lack of positive charge is favored for function. These findings, along with previous results, allow us to propose a model for recognition and transport of hemolysin that emphasizes secondary structure and general biophysical properties over primary sequence. This model may have implications for understanding the broad substrate specificity common among ATP-binding cassette transporters. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/physiology; Bacterial Proteins/secretion; Biological Transport/genetics; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/physiology; Carrier Proteins/secretion; Combinatorial Chemistry Techniques/methods; Escherichia coli/chemistry; Escherichia coli/genetics; Escherichia coli/physiology; Escherichia coli Proteins; Gene Library; Hemolysin Proteins/chemistry; Hemolysin Proteins/genetics; Hemolysin Proteins/physiology; Hemolysis; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments/chemistry; Peptide Fragments/genetics; Peptide Fragments/physiology; Protein Sorting Signals/genetics; Protein Sorting Signals/physiology; Sequence Deletion |
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