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 the Yale University School 
of Medicine. He received his B.S. degree in biochemistry from the University of Pittsburgh and his M.Phil, 
and Ph.D. degrees in molecular biophysics and biochemistry from Yale University while working with 
Frederic Richards in the area of x-ray crystallography. He carried out postdoctoral studies at Yale University 
in protein engineering with Nigel Grindley and studied protein folding using NMR spectroscopy 
with Christopher Dobson at Oxford University 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 using staphylococcal 
nuclease as a model protein system to understand 
how the amino acid sequence of a secondary 
structural element dictates its detailed conforma- 
tion in the context of a folded protein molecule. 
We combine a number of methodologies in these 
studies, including x-ray crystallography, NMR 
spectroscopy, and molecular biology. 
Mapping Structure in the Unfolded State 
of Proteins 
Protein molecules in the unfolded and molten 
globule 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. 
This approach is being used to investigate staphy- 
lococcal nuclease variants, nuclease fragments, 
and the molten globule state of myoglobin. A po- 
lar chelator has been designed and synthesized 
that can be specifically attached to a cysteine resi- 
due engineered into the protein chain. When this 
chelator is loaded with iron and the reaction is 
initiated with a reducing agent, hydroxyl radicals 
and other reactive oxygen species are generated; 
these in turn cleave peptide bonds at positions in 
the protein chain in proximity to the chelator. 
The cleavage sites can be determined by peptide 
mapping and protein sequencing. The reagent 
cleaves native proteins at a number of solvent- 
accessible sites close to the site of attachment. 
The reagent has also been used to map the prox- 
imity of several residues of 76 resolvase to its 
DNA-binding sites. 
Analysis of Protein Folding Using NMR 
Spectroscopy 
Protein molecules are generally thought to 
adopt a final tertiary structure where all back- 
bone and side chain conformations and tertiary 
contacts are within local energy minima. Several 
well-refined protein molecules display excep- 
tions to this view, where significant strain is in- 
duced at a particular point in the structure, 
usually involving a residue in the enzyme ac- 
tive site. Examples include c/s-peptide bonds, 
eclipsed side chain rotamers, and energetically 
unfavorable van der Waals contacts. How a pro- 
tein imposes the stress needed to favor locally 
strained conformations remains unclear. 
Staphylococcal nuclease provides an excellent 
system to examine the relationship between 
stress and strain in a globular protein. Residues 
115-118 adopt a type Via /3-turn, containing a 
c/5-peptide bond, forming one wall of the nu- 
cleotide-binding pocket of the active site. Al- 
though the conformation of the Lysl l6-Prol 17 
peptide bond of this jS-turn is predominantly in 
the c/5-configuration, the equilibrium between 
cis- and ^ra«5-conformers can be monitored by 
NMR spectroscopy. Site-directed mutants within 
this type Via ;8-turn have been examined by NMR 
spectroscopy and x-ray crystallography. Limiting 
the backbone conformational space available to 
the residue preceding the cw-proline contributes 
to the stress favoring the strained c/5-peptide 
bond conformation. We have begun a collabora- 
tion with Axel Brunger (HHMl, Yale University) 
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