CALCIUM-DEPENDENT CONFORMATIONS OF SP-A

Project: Research project

Description

The aims of the research proposed are to understand in molecular detail
and rearrangements that occur amongst the components of pulmonary
surfactant as it is metabolized in the alveolar space. Previous studies
by the applicant suggest that calcium-induced conformation transitions
(C+ conformation) in SP-A may be a critical step in the lamellar body to
tubular myelin transformation. Additional evidence suggests that at
least part of the conformation transition occurs in the carbohydrate
recognition domain of SP-A. The nature of the C= conformation in SP-A
will be studied. Whether the conformation transition includes the
exposure of a hydrophobic domain will be determined. Use will be made
of hydrophobic interaction chromatography analysis of SP-A in the
presence of calcium and its chelator EDTA and fluorescent hydrophobic
probes labelling of the exposed domain. This domain of SP-A will be
covalently labelled using carbene-generating photoreactive hydrophobic
reagents in an attempt at determining its precise location. Existing
antibodies specific for the C_ conformation in SP-A will be characterized
by immunoblot analysis and ELISA. The ability of antibodies to inhibit
calcium-dependent properties of SP-A, membrane aggregation and fusion,
will be tested. The antibody epitope will be mapped on the C+
conformation of SP-A, using limited proteolysis and matching of the
epitope against random peptides generated in a peptide library. The
domain of SP-A defined by hydrophobic and epitope mapping will be tested
for its functional importance by site-directed mutagenesis of the
carbohydrate recognition domain (CRD) and full length SP-A cDNAs, using
oligonucleotide-mediated techniques. The modified proteins will be
tested for their ability to undergo calcium-induced conformation
transitions, and promote membrane aggregation and fusion.
StatusFinished
Effective start/end date1/1/9312/31/97

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

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Membrane Fusion
Calcium
Epitope Mapping
Peptide Library
Conformations
Antibodies
Myelin Sheath
Site-Directed Mutagenesis
Hydrophobic and Hydrophilic Interactions
Edetic Acid
Proteolysis
Chromatography
Epitopes
Complementary DNA
Enzyme-Linked Immunosorbent Assay
Peptides
Research
Proteins
Fusion reactions
Agglomeration

ASJC

  • Medicine(all)