The signal peptide of alkaline phosphatase mediates transport of the enzyme to the E. coli periplasm. We will examine the structural features of this peptide which facilitate the export process. Mutants will be produced which are identical to wild type alkaline phosphatase except in the amino acid sequence of the signal peptide core region. For each mutant, this region will contain a defined amino acid composition which is optimal for formation of a particular structural element. These structural units will be designed to test the role of conformation, length, and hydrophobicity in signal peptide function. The rapid development of mutants, each containing multiple residue substitutions, will be accomplished by creating unique restriction sites flanking the core region. New signal sequences will be generated by replacing the wild type DA sequence with synthetic oligonucleotides coding for the amino acid sequence of the structural model to be tested. The ability of these mutants to support the secretion process will be evaluated through in vivo transport studies. To further distinguish the role of secondary structure and primary sequence specificity in signal peptide function, additional mutants will be made which contain the core region from signal sequences of other secreted proteins. The structural character of each mutant sequence will be substantiated through analysis of the corresponding chemically-synthesized signal peptides. Together these studies will elucidate the interrelationship between the structural properties of each signal peptide and in vivo function. The information which evolves regarding conformations which complement membrane interactions and the interchangeability of these structural units will then be applied to the redesign of the mature protein. Regions of the mature protein will be replaced with new structural segments designed to determine the requirements for membrane stabilization and transformation of alkaline phosphatase into a transmembrane protein with predicted orientation. This approach will elucidate the guidelines for redesigning structural domains in proteins in general. The structural features of signal peptides which enhance protein translocation can be applied to the design of vehicles to transport other proteins, including eukaryotic proteins, into the E. coli periplasm for subsequent isolation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM037639-01A1
Application #
3293067
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1987-07-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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Bhanu, Meera K; Kendall, Debra A (2014) Fluorescence spectroscopy of soluble E. coli SPase I ?2-75 reveals conformational changes in response to ligand binding. Proteins 82:596-606
Bhanu, Meera K; Zhao, Ping; Kendall, Debra A (2013) Mapping of the SecA signal peptide binding site and dimeric interface by using the substituted cysteine accessibility method. J Bacteriol 195:4709-15
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De Bona, Paolo; Deshmukh, Lalit; Gorbatyuk, Vitaliy et al. (2012) Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: the stabilizing effect of membrane-mimetics on the acquired fold. Proteins 80:807-17
Wowor, Andy J; Yu, Dongmei; Kendall, Debra A et al. (2011) Energetics of SecA dimerization. J Mol Biol 408:87-98
Auclair, Sarah M; Moses, Julia P; Musial-Siwek, Monika et al. (2010) Mapping of the signal peptide-binding domain of Escherichia coli SecA using Förster resonance energy transfer. Biochemistry 49:782-92
Musial-Siwek, Monika; Yeagle, Philip L; Kendall, Debra A (2008) A small subset of signal peptidase residues are perturbed by signal peptide binding. Chem Biol Drug Des 72:140-6
Musial-Siwek, Monika; Kendall, Debra A; Yeagle, Philip L (2008) Solution NMR of signal peptidase, a membrane protein. Biochim Biophys Acta 1778:937-44
Musial-Siwek, Monika; Rusch, Sharyn L; Kendall, Debra A (2007) Selective photoaffinity labeling identifies the signal peptide binding domain on SecA. J Mol Biol 365:637-48

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