Three-Dimensional Macromolecular 
and Cellular Structure 
David A. Agard, Ph.D. — Associate Investigator 
Dr. Agard is also Associate Professor of Biochemistry and Biophysics at the University of California, San 
Francisco. He did his undergraduate work at Yale University with Fred Richards, Hal Wyckoff, and 
Thomas Steitz. He received his Ph.D. degree in chemical biology from the California Institute of Technol- 
ogy, where he studied with Robert Stroud and began a continuing collaboration with John Sedat. His 
\ postdoctoral work was done on high-resolution electron microscopic crystallography at the MRC Labora- 
tory of Molecular Biology in Cambridge, England, with Richard Henderson. There he also began the clon- 
ji^H ing of the a-lytic protease gene with Sydney Brenner. 
THIS laboratory is primarily concerned with 
exploring the fundamental relationships be- 
tween structure and function at the molecular 
and cellular levels. Four areas of investigation are 
actively pursued: three-dimensional analysis of 
diploid chromosome structure and topology; 
studies on the structural determinants of specific- 
ity, using a-lytic protease as a model system; 
functional and structural analysis of the role of 
the precursor in proper folding of a-lytic pro- 
tease; and determination of the first three-dimen- 
sional crystal structure of an important protein 
in human cholesterol metabolism — apolipopro- 
tein E. 
Three-Dimensional Analysis of 
Chromosome Structure 
We study chromosome structure in close col- 
laboration with John Sedat (HHMI, University of 
California, San Francisco) ; only a subset of these 
studies will be discussed here. Our primary aim 
in this area is to provide a physical basis for un- 
derstanding chromosome behavior and function 
by directly determining the three-dimensional 
structure of eukaryotic chromosomes as a func- 
tion of both transcriptional state and the cell cy- 
cle stage. To accomplish this goal we are attempt- 
ing to understand how fibers of nucleosomes 
are folded into higher-order structures within 
the chromosome and what role specific chro- 
mosomal proteins play in determining these 
structures. 
We have had to develop the necessary technolo- 
gies (hardware and software) to allow us to exam- 
ine complex noncrystalline specimens in three 
dimensions, using electron microscopy (EM) and 
light microscopy. The past year has seen signifi- 
cant software developments for three-dimen- 
sional image reconstruction with both kinds of 
microscopes. Many of the tedious aspects of EM 
tomography have now been automated, greatly 
speeding the task of generating a three-dimen- 
sional reconstruction. 
The Role of Topoisomerase II in 
Chromosome Structures 
Current work focuses on deepening our under- 
standing of chromosomal structure by combining 
three-dimensional observation methods with bio- 
chemical probes in an effort to correlate struc- 
tural aspects with specific macromolecular 
components. Topoisomerase II is a major chromo- 
somal protein that is important for relieving the 
torsional stress of supercoiling. It is also postu- 
lated to play a crucial role in organizing the 
higher-order structure of chromosomes. We have 
begun to investigate the localization and function 
of topoisomerase II in chromosomal samples pre- 
pared by methods that preserve their in vivo 
structure. We have examined prophase, meta- 
phase, and anaphase embryonic cycle 12 and 13 
nuclei, and metaphase and anaphase Kc nuclei. 
Our data show that although topoisomerase II 
is clearly associated with mitotic chromosomes, 
it is concentrated at specific sites along them 
rather than localized to an internal core. These 
sites can be spatially coincident with the chro- 
mosome or adjacent to the chromosome arm. 
This seems incompatible with a purely structural 
role for the enzyme. The nature of the topoiso- 
merase II sites is still unknown, but we hypothe- 
size that those we have recorded are locations of 
the enzyme's activity during chromosome con- 
densation and segregation. We have observed that 
topoisomerase II is specifically localized to re- 
gions of nondisjunction in failed mitoses. 
We are extending this work by examining the 
distribution of the enzyme at high resolution in 
the electron microscope and its dynamics in live 
Drosophila embryos. We have recently devel- 
oped several monoclonal antibodies to topoiso- 
merase II as an important aid to our studies. 
The Structural Basis of Enzyme Specificity 
We had previously chosen a-lytic protease as a 
model for investigating structural and energetic 
aspects of enzyme-substrate specificity, because 
its binding pocket, made of the side chains of 
three amino acids (Met 192, Met 2 1 3 , Val 2 1 7A) , 
provided a large volume that could be experimen- 
tally manipulated. To probe the structural basis 
for specificity, we are combining solution kinetic 
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