Three-Dimensional Structures of Biological Macromolecules 
rubrum. In collaboration with Knalf s group, we 
prepared this complex to high purity and in suffi- 
cient quantity to start crystallization experi- 
ments, which are now in progress. 
DNA Photolyase 
Light energy plays an important role in reac- 
tions other than photosynthesis. An example of 
the use of light energy to drive a chemical reac- 
tion is found in the molecular machinery that en- 
ables cells to repair DNA damaged by ultraviolet 
light. One type of damage frequently caused by 
ultraviolet irradiation is the crosslinking of two 
neighboring thymine bases in a DNA strand. Most 
of these crosslinks are in the form of a cyclobu- 
tane ring connecting four carbon atoms, two from 
each thymine base. An enzyme, DNA photolyase, 
can locate and bind to such lesions and, upon 
input of light of suitable wavelength, cleave the 
carbon-carbon bonds between the bases, repair- 
ing the damage. 
DNA photolyase has been found in prokaryotes, 
eukaryotes, and archaebacteria. The enzyme from 
Escherichia coli has been sequenced, overex- 
pressed, and purified in the laboratory of Aziz 
Sancar at the University of North Carolina, Chapel 
Hill. It consists of a single polypeptide chain of 
471 amino acids and two cofactors — a flavin- 
adenine dinucleotide (FAD) and 5, 10-methenyl- 
tetrahydrofolate. The FAD cofactor fully reduced 
to FADH2 is essential for the enzyme's function; 
the folate's role appears to be that of a light- 
harvesting antenna. 
Despite significant problems with the en- 
zyme's tendency to denature, we were able to 
crystallize DNA photolyase from E. coli. The crys- 
tals, obtained after repeated seeding of crystalli- 
zation solutions, are plates less than 0.05 mm 
thick, but they diffract x-rays to at least 2 .8 A reso- 
lution. That should suffice to construct an atomic 
model of this interesting enzyme after the phase 
problem has been solved. Collection of x-ray dif- 
fraction intensity data and experiments to bind 
heavy atom compounds to the protein in the crys- 
tal are under way. We also are trying to crystallize 
the enzyme in complex with a substrate, a five- 
nucleotide piece of single-stranded DNA contain- 
ing a crosslinked pair of thymine bases. These ex- 
periments have to be done in the dark, or under 
yellow or red light, to prevent the enzyme from 
repairing and releasing the substrate. 
Since light is an essential ingredient of the enzy- 
matic reaction of DNA photolyase, the enzyme- 
substrate complex will be a suitable system in 
which to study the time course of the reaction, 
using the Laue technique. This utilizes the broad 
spectrum of x-ray wavelengths in a synchrotron's 
powerful beam to record within a very short time 
a sufficient fraction of a crystal's diffraction pat- 
tern for structural interpretation. In such an ex- 
periment the crystal is irradiated with a light 
pulse suitable to trigger the enzymatic reaction, 
and diffraction patterns are recorded at different 
times after the pulse. Data from such an experi- 
ment can provide snapshots of the structural rear- 
rangements during the reaction, thus contribut- 
ing to the understanding of the enzyme's 
mechanism. 
Other Projects 
In addition to the two projects described 
above, we are doing purification and crystalliza- 
tion experiments on several other proteins. The 
subjects of these studies include the catalytic do- 
main of human HMG-CoA reductase, a key en- 
zyme in the synthesis of cholesterol and a likely 
target for drugs; human synapsin I, a protein bind- 
ing to synaptic vesicles and mediating their re- 
lease; the small GDP-binding protein smgp25A 
from bovine brain; the DNA-binding protein myo- 
genin; the SecA protein from E. coli, one of the 
key parts of the protein export system; and mam- 
malian phosphofructokinase. 
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