the free-energy contribution of amino acid side 
chains to the relative stabiHzation of these native 
structures. 
The goal of this line of investigation is to generate 
an accurate body of thermodynamic and structural 
data for this system, using NMR spectroscopy and 
x-ray crystallography, for use in testing and calibrat- 
ing molecular energy simulations of proteins. The 
computational aspect of this project is carried out in 
collaboration with Dr. Axel Briinger (HHMI, Yale 
University). This year Dr. Fox and his colleagues 
have identified several amino acid substitutions that 
perturb the cis- trans equilibrium and have derived 
a structural hypothesis for these observations based 
on high-resolution crystal structure determinations. 
(This work also is supported by a grant from the 
National Institutes of Health.) 
Toward Structural Studies of the Glycine 
Receptor Channel 
Despite intensive biochemical investigations of 
many voltage- and ligand-gated ion channels, little is 
known about their molecular structure and mecha- 
nisms of gating or ion selectivity. The glycine recep- 
tor is an inhibitory chloride ion channel found in 
the spine, hindbrain, and visual system that is gated 
by glycine and targeted by strychnine action. While 
the natural channel is a pentameric assembly of a 
(48-kDa) and (58-kDa) subunits associated with a 
93-kDa peripheral protein, channels can be formed 
from the a chain alone that exhibit the conductance 
and pharmacological properties of the natural 
channel. 
The human glycine receptor al chain has been 
overexpressed in insect cells with a recombinant ba- 
culovirus vector. The expression level is high, with 
little heterogeneity in apparent molecular weight. 
Whole-cell recordings of infected cells display a 
large conductance when the cells are exposed to 
glycine, but not if they have been pretreated with 
strychnine, indicating that function channels have 
assembled in the plasma membrane. Purification of 
sufficient protein for three-dimensional crystalli- 
zation and crystal structure determination is in 
progress. 
Dr. Fox is also Associate Professor of Molecular 
Biophysics and Biochemistry at Yale University. 
Articles 
Antonio, L.C., Kautz, R.A., Nakano, T., Fox, R.O., 
and Fink, A.L. 1991. Cold denaturation and ^H20 
stabilization of a staphylococcal nuclease mutant. 
Proc Natl Acad Sci USA 88:7715-7718. 
Ermacora, M.R., Delfino, J.M., Cuenoud, B., Sche- 
partz. A., and Fox, R.O. 1992. Conformation- 
dependent cleavage of staphylococcal nuclease 
with a disulfide-linked iron chelate. Proc Natl 
Acad Sci USA 89:6383-6387. 
Sanders, S.K., Fox, R.O., and Kavathas, P. 1991. Mu- 
tations in CD8 that affect interactions with HLA 
class I and monoclonal anti-CD8 antibodies. /^jcp 
Med 174:371-379. 
STRUCTURAL STUDIES OF VIRUSES, RECEPTORS, AND TRANSCRIPTIONAL 
CONTROL PROTEINS 
Stephen C. Harrison, Ph.D., Investigator 
Structural studies of macromolecular complexes 
are aimed at discovering basic molecular mecha- 
nisms in cell organization. Dr. Harrison's laboratory 
has focused on three broad areas for x-ray crystallo- 
graphic analysis of assembly and recognition: vi- 
ruses and their interactions with cells, cell-surface 
receptors, and complexes of transcriptional regula- 
tory proteins with their DNA-binding sites. 
Viruses and Viral Proteins 
Building on the successful completion of struc- 
tures for SV40 (simian virus 40) and polyomavirus, 
using x-ray crystallography, the laboratory has been 
working on ways to improve the accuracy and reso- 
lution of these structures and on methods and pro- 
grams for recording good diffraction data from crys- 
tals with even larger unit cells. As a result, data to 
~3.2-A resolution have been collected from SV40 
by image plate recording at the CHESS Fl synchro- 
tron beam line. These data will be used to generate a 
refined structure in the near future. 
Projects for which the new data collection proce- 
dures will be especially important are structural 
analyses of rotavirus single-shelled particles (SSPs) 
and reovirus cores. These are the (probably homolo- 
gous) internal structures derived from double- 
strand RNA (dsRNA) viruses. They are transcription- 
ally active assemblies that synthesize, modify, and 
STRUCTURAL BIOLOGY 471 
