Three-Dimensional Macromolecular and Cellular Structure 
Last year we had developed a new method for 
predicting the energetics of protein-substrate in- 
teractions. This approach (based on Ponder- 
Richards rotamers combined with energetics and 
solvation terms) can predict k^^JK^ with stun- 
ning accuracy. We have used this method to de- 
sign a new enzyme with particular properties, 
and so far the results have been remarkable. This 
year we have been working to extend these re- 
sults to other systems and to improve their accu- 
racy. We have been able to predict quantitatively 
100 values of k^^JK^ for subtilisin with equiva- 
lent accuracy. This approach is being expanded 
to allow its use in drug design situations and for 
modeling proteins based on the structure of a ho- 
mologous protein. 
Structural and Biochemical Probes 
of Folding of a-Lytic Protease 
a-Lytic protease is synthesized as a prepro- 
enzyme. Experiments in Escherichia coli have 
demonstrated that the 166-amino acid precursor 
domain is required for the proper folding of the 
198-amino acid protease domain. Furthermore, 
we have shown that the pro region does not have 
to be attached covalently in order to function. 
The development of the in vitro folding system 
has led to the unprecedented ability to trap and 
purify a stable folding intermediate under non- 
denaturing conditions. Although the interme- 
diate is unable to fold by itself, it is rapidly con- 
verted to active enzyme upon addition of the pro 
region. Analysis of the folding kinetics proves 
that the pro region acts essentially as a "foldase" 
to stabilize the transition state for folding, 
thereby speeding up folding by more than 10^. 
Current efforts are focused on characterizing 
the folding intermediate and analyzing the fold- 
ing reaction. We plan to use a combination of 
approaches — including NMR and crystallogra- 
phy — to probe the structure of the intermediate 
and the role of the pro region in the final stage of 
folding. 
Receptor-Ligand Targeting and Human 
Cholesterol Metabolism 
Apolipoprotein E is an important protein in 
cholesterol metabolism in mammals. Responsi- 
ble for targeting chylomicrons, very low density 
lipoprotein (VLDL), and high-density lipoprotein 
(HDL) particles to the low-density lipoprotein 
(LDL) receptor, apolipoprotein E has a major role 
in triglyceride and cholesterol metabolism. The 
protein itself has two distinct structural and func- 
tional domains: the amino-terminal 22-kDa do- 
main contains the receptor-binding functional- 
ity; lipid binding resides primarily with the 
10-kDa carboxyl-terminal domain. In collabora- 
tion with the Mahley group at the Gladstone 
Foundation Laboratories for Cardiovascular Dis- 
ease, we obtained crystals of the 22-kDa recep- 
tor-binding domain. Last year we reported that 
the protein is an unusually elongated four-helix 
bundle. Although the surface is exceptionally 
charged, there is a precise balance between 
groups with a positive charge and groups with a 
negative charge, except in what we believe is the 
receptor-binding region. We have recently fin- 
ished the analysis of a naturally occurring human 
mutant that significantly reduces receptor bind- 
ing and can lead to premature atherosclerosis. 
The structure reveals that the mutant disrupts the 
complex set of surface salt bridges, which then 
causes a key arginine required for binding to be 
recruited into a salt bridge. Current efforts are 
aimed at generating a soluble fragment of the LDL 
receptor-binding domain for crystallographic 
study. 
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