Biophysical Genetics of Protein Structure 
and Folding 
Robert O. Fox, Ph.D. — Associate Investigator 
Dr. Fox is also Associate Professor of Molecular Biophysics and Biochemistry at Yale University School of 
Medicine. He received his B.S. degree in biochemistry from the University of Pittsburgh and M.Phil, and 
Ph.D. degrees in molecular biophysics and biochemistry from Yale University, in the area of x-ray crys- 
tallography. He carried out postdoctoral studies at Yale University in protein engineering and at Oxford 
University on the NMR spectroscopy of protein folding as a Fellow of the Jane Coffin Childs Memorial Fund 
for Medical Research. Before moving to Yale, Dr. Fox was Assistant Professor in the Department of Cell 
Biology at Stanford University Medical School. 
ALTHOUGH the information that directs the 
folding of a protein molecule into a defined 
three-dimensional structure is genetically en- 
coded, the mechanisms and pathways of the fold- 
ing process are poorly understood. One approach 
to this problem is an analysis of partially struc- 
tured folding intermediates, combined with a 
mutational analysis. We use nuclear magnetic res- 
onance (NMR) spectroscopy and chemical meth- 
ods to probe for structural and kinetic interme- 
diates in the folding process. 
Many polypeptide sequences adopt a common 
folded motif, but they frequently differ in the de- 
tailed arrangement or conformation of structural 
elements in ways that are functionally significant. 
Certain loops of the immunoglobulins (antibod- 
ies) are examples. We are working to understand 
the manner in which the amino acid sequence of 
a secondary structural element dictates its de- 
tailed conformation in the context of a folded 
protein molecule, using staphylococcal nuclease 
as a model protein system. We plan to test the 
generality of our observations and conclusions by 
examining the relationship between the amino 
acid sequence of immunoglobulin loops and 
their structure and the resulting ligand affinity. 
We combine a number of methodologies to ad- 
dress these aspects of protein structure and fold- 
ing, including x-ray crystallography, NMR spec- 
troscopy, and molecular biology. 
are used as a test system. A polar chelator has been 
designed and synthesized that can be specifically 
attached to a cysteine residue engineered into the 
protein chain. When this chelator is loaded with 
iron it can be used to generate hydroxyl radicals, 
which in turn cleave peptide bonds at positions 
in the protein chains in proximity to the chelator. 
The cleavage sites can be determined by peptide 
mapping and protein sequencing. Experiments 
designed to characterize the system and map dis- 
tances in the unfolded chain are in progress. 
Analysis of Protein Folding Using 
NMR Spectroscopy 
In collaboration with Christopher Dobson's 
laboratory in Oxford, we have developed a series 
of NMR experiments to characterize the equilib- 
rium-folding kinetics of staphylococcal nuclease. 
These methods have been used to investigate mul- 
tiple folded and unfolded states of the protein. 
Applied to nuclease variants that differ in thermal 
stability, the methods have allowed us to discrimi- 
nate between mutants that influence the protein 
folding pathway and those that modify the stabil- 
ity of the tertiary structure. By combining site- 
directed mutagenesis with NMR spectroscopy, 
we have been able to quantitate slow intramolec- 
ular equilibria, which should serve as a basis for 
the experimental quantitation of the forces that 
stabilize protein molecules. 
Mapping Structure in the Unfolded 
State of Proteins 
Protein molecules in the unfolded and molten 
globular states are often more compact than 
would be expected for a true random coil confor- 
mation. If this conformational bias is toward that 
of the folded structure, it may explain the rapid 
rate at which proteins fold. We have developed a 
chemical approach to map close contacts be- 
tween a variable reporter residue site and all 
other residues of a protein chain in these states. 
Staphylococcal nuclease variants and fragments 
Genetic Analysis of a |S-Tum 
A sharp change in the trajectory of a polypep- 
tide chain between secondary structure elements 
in a globular protein has been defined as a reverse 
turn or /3-turn. These structures occur in a num- 
ber of defined geometric types and frequently 
contribute side chains to the active site of the 
enzymes, such as staphylococcal nuclease or the 
combining site of binding proteins such as the 
immunoglobulins. We wish to determine the se- 
quence requirements for the formation of differ- 
ent (8-turn types to understand better the detailed 
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