found to be synthesized as a prepro-enzyme. Subse- 
quent experiments in E. coli have demonstrated 
that the 166-amino acid pro domain is required for 
the proper folding of the 198-amino acid protease 
domain. Recent experiments indicate that covalent 
attachment of the pro and protease regions is not 
required— coexpression of separate pro and prote- 
ase domains leads to active protease. This folding in 
trans can also be accomplished by mixing cellular 
extracts that separately contain the precursor and 
mature domains. The current thinking is that the 
temperature stability and protease resistance of the 
mature protease are generated at the expense of 
ease of folding. That is, there is a high-energy bar- 
rier between the folded and unfolded states that is 
too high for the mature protease to cross by itself 
The precursor acts as a "foldase" to stabilize the 
transition state for folding; it essentially acts as a 
template on which the mature enzyme finds its ac- 
tive conformation. 
Current work aims at genetically dissecting the 
precursor and purifying sufficient misfolded mature 
domain and pro-enzyme for crystallization. Pro-en- 
zyme has been partially purified under denaturing 
conditions and can be refolded in vitro. This pro- 
vides the unique opportunity to study the detailed 
structure of a folding intermediate and the "en- 
zyme" that catalyzes the transition to the final 
folded state. 
IV Structure of Apolipoprotein E. 
ApoE is an important protein in cholesterol me- 
tabolism in mammals. It is a component of several 
PUBLICATIONS 
classes of circulating plasma lipoprotein complexes, 
being especially abundant in chylomicrons, very 
low density lipoproteins (VLDL), and certain high- 
density lipoproteins (HDL). These particles func- 
tion in intravascular lipid transport, which involves 
cellular uptake of lipoproteins via specific, 
apolipoprotein-mediated binding to a cell surface 
receptor (the LDL or apoB-E receptor). ApoE is 
one of two proteins that can bind to the LDL recep- 
tor and thus has a major role in triglyceride and 
cholesterol metabolism. The protein itself has two 
distinct structural and functional domains: the 
amino-terminal 22 kDa domain contains the recep- 
tor binding functionality, whereas lipid binding re- 
sides primarily with the 10 kDa carboxyl-terminal 
domain. 
In collaboration with the Mahley group (Glad- 
stone Foundation Laboratories for Cardiovascular 
Disease), Dr. Agard has obtained crystals of the 22 
kDa receptor-binding domain that are suitable for 
high-resolution x-ray analysis, P2 2 2 which dif- 
o 111 
fract beyond 2.5 A. They have collected low-resolu- 
tion native data and are currently screening for de- 
rivatives. High-resolution data have been obtained 
at -150°C and are being processed. Future work 
will involve structural analysis of mutants of this 
protein that are known to cause serious human 
cholesterol disorders. 
Dr. Agard is also Associate Professor in the De- 
partments of Biochemistry and Pharmaceutical 
Chemistry at the University of California at San 
Francisco. 
Books and Chapters of Books 
Chen, H., Sedat, J.W, and Agard, D.A. 1989. Manipulation, display, and analysis of three-dimensional biologi- 
cal images. In The Handbook of Biological Confocal Microscopy . Madison, Wisconsin: IMR Press. 
Articles 
Agard, D.A., Hiraoka, Y, Shaw, P., and Sedat, J.W 1989. Fluoresence microscopy in three dimensions. Methods 
Ce// 5/0/30:353-377. 
Aikens, R.S., Agard, D.A., and Sedat, J.W 1989. Solid-state imagers for microscopy. Methods Cell Biol 29:291- 
313. 
Belmont, A., Braunfeld, M.B., Sedat, J.W, and Agard, D.A. 1989. Large-scale chromatin structural domains 
within mitotic and interphase chromosomes in vivo and in vitro. Chromosoma 98:129-143. 
Bone, R. , Silen, J.L. , and Agard, D.A. 1989. Structural plasticity broadens the specificity of an engineered pro- 
tease. Nature 339:191-195. 
Kettner, C.A., Bone, R., Agard, D.A., and Bachovchin, WW 1988. Kinetic properties of the binding of a-lytic 
protease to peptide boronic acids. Biochemistry 27:7682-7688. 
Continued 
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