Annexins form a group of homologous cytosolic proteins that bind phospholipid membranes in a calcium-dependent manner. They have been implicated in a variety of cell functions, including secretion, fusion, and signal transduction. Annexins, which typically have molecular weights of 35kDa or 68kDa, exhibit a common canonical sequence of about 70 residues, repeated four or eight times. These repeats do not fit the pattern predicted for """"""""E-F hand"""""""" domains characteristic of the calmodulin superfamily of calcium-binding proteins, which do not bind phospholipids. As such, annexins represent a novel group of calcium-mediating proteins. The long-term goal of this research is to develop a molecular mechanistic model that may be generally applicable to annexins and other calcium- dependent phospholipid-binding proteins.
the specific aims addressed in this proposal are the structural characterization of annexin V, a representative annexin family member, and its interactions with calcium and phospholipid ligands through X-ray crystallography and Raman spectroscopy. The crystal structure(s) will provide a detailed molecular model of the protein, while the spectroscopic studies will probe the effects of the protein-phospholipid binding in solution. Raman spectroscopy will provide a bridge between crystal and solution experiments, where samples can be in either form. Large single crystals of Ca2+-bound annexin V have been grown that are suitable for X-ray crystallographic structure determination. They diffract to at least 2.2A resolution and belong to the R3 space group, with hexagonal unit cell dimensions of a=b=156.8A and c=36.9A. To solve the phase problem, a multiple isomorphous replacement (MIR) or iterative single isomorphous replacement (ISIR) approach will be used. Attempts will be used. Attempts will be made to grow and analyze annexin V crystals in other liganded states, such as the Ca2+-free form or with a soluble phospholipid ligand bound. Raman spectroscopy provides detailed information on protein and lipid conformation, and will be used to study ligand-induced conformational changes in annexin V. A high-quality preliminary Raman spectrum of annexin V has been obtained. This spectroscopic technique will be used to evaluate the effects of annexin-phospholipid interactions on the protein structure, and the bound phospholipid, if a suitable reconstitution system can be developed. Peak assignments will be facilitated by the use of phospholipids with fully deuterated hydrocarbon tails.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM044554-02
Application #
3468144
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Boston University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Concha, N O; Head, J F; Kaetzel, M A et al. (1993) Rat annexin V crystal structure: Ca(2+)-induced conformational changes. Science 261:1321-4
Concha, N O; Head, J F; Kaetzel, M A et al. (1992) Annexin V forms calcium-dependent trimeric units on phospholipid vesicles. FEBS Lett 314:159-62