plex. The TCR crystals are small (~50 mhi'), and 
can only be obtained from glycosidase-treated prepa- 
rations of protein. 
The seven potential N-linked glycosylation sites 
in 2B4 have been altered by site-directed mutagene- 
sis in an attempt to produce homogeneous protein 
and eventually improve the crystals. COS-7 cells 
transfected with the modified cDNAs show cell sur- 
face expression of each mutant chain , and stable Chi- 
nese hamster ovary (CHO) lines expressing the 
aglycosylated proteins are being established. The 
lE'^-peptide crystals diffract to ~4.0 A. To improve 
the data obtained from these crystals, the use of 
synchrotron radiation and cryopreservation, two 
well-established methods for enhancing data qual- 
ity, is being explored. 
Stability studies of MHC molecules with and 
without peptide. A soluble form of the class I MHC 
molecule H-2K'' has been expressed in CHO cells at 
levels up to 100 mg/1. After removal of endogenous 
peptide, K'' was renatured from separated heavy and 
light chains. The resulting empty K** heterodimer 
was immunologically reactive and structurally simi- 
lar to a heterodimer renatured in the presence of an 
appropriate restricted peptide. Overall yields of 
reassembled empty and peptide-filled K'' are 45- 
50%, allowing sufficient material for crystallization. 
Thermal stability profiles indicate that the two 
forms of heterodimer differ in their resistance to de- 
naturation by heat, but that a significant portion of 
the empty class I molecule has a native conforma- 
tion at physiological temperatures. These data are 
consistent with the possible existence of empty 
MHC molecules in vivo, which may play a role in 
immunological processes such as the positive selec- 
tion of T cells during maturation in the thymus. 
Free energies were calculated from a thermody- 
namic analysis of the stability curves, giving a direct 
measure of the stabilization of the class I molecule 
due to peptide binding. The stability assay em- 
ployed is a novel way of evaluating the binding of 
peptide to purified MHC molecules and can be used 
to compare the degree of stabilization conferred by 
different peptides. Future plans include use of ther- 
mal stability measurements to assess the effects of 
peptide length and composition on stabilization of 
class I heterodimers. (A grant from the National In- 
stitutes of Health provided support for the work de- 
scribed above.) 
Expression and Crystallization 
of an Fc Receptor Related to Class I 
MHC Molecules 
Fc receptors expressed in the gut of newborn ro- 
dents bind to maternal immunoglobulin in milk at 
pH 6.5 and transport it to the bloodstream of the 
neonate, where it dissociates at pH 7.4. The intes- 
tinal Fc receptor (FcRn) was recently observed to 
employ the same light chain as class I MHC mole- 
cules and to have a heavy chain with significant se- 
quence similarity to heavy chains of MHC. Although 
FcRn is predicted to share the same type of groove 
that serves as the MHC peptide-binding site, the im- 
munoglobulin ligand of FcRn is not a peptide but a 
macromolecule. The structural similarity between 
these molecules that function so differently in the 
immune system affords an opportunity to study how 
evolution has utilized this structural motif for dif- 
ferent purposes. 
A secreted version of the rat Fc receptor has been 
expressed in CHO cells, and soluble Fc receptor has 
been purified with high yields (~40 mg/1) from 
cell supernatants. In contrast to the structurally re- 
lated class I MHC molecules, purified FcRn shows 
upon analysis no endogenously bound peptides. 
Crystals of soluble FcRn diffract to 2.7 A at room 
temperature, and cryopreserved crystals diffract to 
2.2 A. Complexes of FcRn and Fc have been pre- 
pared, and the stoichiometry of binding has been 
determined to be two FcRn molecules per Fc. Crys- 
tals of the FcRn-Fc complex have also been ob- 
tained, and an analysis is in progress. (This work is 
supported by the National Institutes of Health.) 
Structural Studies of Surface Recognition 
Molecules in the Nervous System 
A number of the molecules found to contain im- 
munoglobulin homology units mediate cell-cell in- 
teractions in the nervous system as well as the im- 
mune system. In collaboration with Dr. Allan Bieber 
(Purdue University), a project to crystallize Dro- 
sopbila neuroglian is under way. Neuroglian is a 
cell adhesion molecule with an extracellular por- 
tion consisting of six immunoglobulin-like domains 
followed by five fibronectin type III repeats. From 
an expressed version of these repeats, a stable pro- 
teolytic fragment corresponding to three repeats has 
been isolated and crystallized. Native and derivative 
data from cryopreserved crystals have been col- 
lected, and a structure determination is in progress. 
Dr. Bjorkman is also Assistant Professor of Biol- 
ogy at the California Institute of Technology and 
Adjunct Professor of Biochemistry at the Univer- 
sity of Southern California School of Medicine, 
Los Angeles. 
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