STRUCTURAL STUDIES ON BIOLOGICAL MACROMOLECULES 
Wayne A. Hendrickson, Ph.D., Investigator 
Dr. Hendrickson's laboratory studies macromo- 
lecular structure, with an aim toward in-depth un- 
derstanding of biological activity. Diffraction analy- 
sis is the primary research tool, but other 
theoretical, physical, and biochemical methods are 
also used. Three broad themes are emphasized: 
crystallographic studies on molecules of immediate 
interest; methodology development; and general 
principles of information, dynamics, and assembly. 
L Oxygen-carrying Proteins. 
The proteins that transport oxygen— hemoglo- 
bins, hemerythrins, and hemocyanins— are subjects 
of long-standing interest for structural studies. Dur- 
ing the past year advances have been made on a 
number of problems in this area. First, in collabora- 
tion with Dr. Emilia Chiancone (La Sapienza Uni- 
versity in Rome), the structure of clam hemoglobin 
from Scapharca inaequivalvis has been analyzed 
o 
at 2-4 A resolution in the carbonmonoxy state, and 
the analysis of crystals in the deoxy state has been 
initiated. This dimeric hemoglobin features a novel 
subunit interface with directly communicating 
hemes apparently responsible for cooperative oxy- 
gen binding. Second, in collaboration with Dr. Ste- 
ven Boxer (Stanford University), the structure of a 
recombinant mutant of human myoglobin has been 
determined and refined at 2.8 A resolution. This 
mutant (arginine for lysine at residue 45) affects the 
kinetics of ligand binding, and the structure shows 
a water-mediated interaction with the distal histi- 
dine. Third, the giant (3 8 million Da) erythro- 
cruorin from the earthworm Lumbricus terrestris 
was shown to have D^ molecular symmetry, and 
structure analysis is proceeding at 5 5 A resolution. 
Finally, excellent crystals of a functional fragment 
obtained after limited proteolysis of octopus hemo- 
cyanin have been produced and characterized. Dif- 
fraction extends to 1.8 A Bragg spacings. 
n. Streptavidin. 
The extraordinarily high binding affinity of avidin 
for biotin has generated considerable interest in its 
relation to biophysical principles of ligand binding 
and the basis it forms for technological applica- 
tions. The bacterial analogue streptavidin has 
proved more suitable than avidin in biotechnology 
and now also in biophysical studies. The structure 
of orthorhombic crystals of core streptavidin 
was first determined as a complex with seleno- 
biotin, which was exploited for phase determina- 
tion in the diffraction analysis. Subsequently 
the streptavidin structure in its uncomplexed state 
was also determined in three crystalline modifica- 
tions: tetragonal and monoclinic forms and the 
original orthorhombic form. In addition, the avidity 
structure has been solved by molecular replace- 
ment from the streptavidin model. Studies are 
under way to explore the structural basis of the 
avidity of streptavidin for biotin, by examining com- 
plexes with biotin analogues and preparing to pro- 
duce site-directed mutants. The orthorhombic crys- 
tal form is especially suitable for the binding 
studies, and additional refined structures are now 
available for complexes with desthiobiotin, 
biocytin, and a carboxybiotin derivative. Candidates 
for directed mutagenesis are being designed on the 
basis of the structural results, and these will be im- 
plemented in an expression system under develop- 
ment in Dr. Charles Cantor's laboratory (Columbia 
University). 
in. New Structural Initiatives. 
Studies are also under way on several other 
structures, most of which have not yet advanced to 
the level of atomic models. Interest centers on mol- 
ecules involved in transmembrane signaling, pro- 
teins of the immune system, and components of ge- 
netic replication and transcription. These projects 
include a complex of DNA with the drug chro- 
momycin, for which a recently obtained model is 
being refined against data extending to 1.8 A spac- 
ings; Escherichia coli ribonuclease H, which is 
o 
being analyzed at 1.7 A resolution (with Dr. Robert 
Crouch, National Institutes of Health); p-bun- 
garotoxin, for which 2.3 A data have been pro- 
cessed (with Dr. Paul B. Sigler, HHMI, Yale Univer- 
sity); and human insulin, for which 1.8 A data have 
been collected. Proteins at early stages of crystallo- 
graphic analysis include soluble recombinant CD4 
(with Dr. Richard Axel, HHMI, Columbia University, 
and Dr. Ray Sweet at Smith Kline & French) and the 
carbohydrate recognition domain of a mannose- 
binding mammalian lectin (with Dr. Kurt Dricka- 
mer, Columbia University). Crystals of several other 
proteins have not yet been fully characterized, and 
still other projects have not yet yielded crystals. 
Continued 
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