BIOPHYSICAL GENETICS OF PROTEIN STRUCTURE AND FOLDING 
Robert O. Fox, Ph.D., Assistant Investigator 
Dr. Fox is investigating the role of the amino acid 
sequence in determining the folding pathways and 
the final detailed three-dimensional structure of 
globular protein molecules. One current focus is 
the role of sequence in determining turn and loop 
structures at the combining sites of immunoglobu- 
lin molecules and the active sites of enzymes such 
as staphylococcal nuclease. These investigations 
should shed light on the structural basis of immu- 
noglobulin maturation and provide guidelines for 
the engineering of new protein molecules. 
I. Structural Basis of Immunoglobulin Maturation. 
The sequence diversity found in immunoglobulin 
molecules is generated at several different levels. 
Combinatorial variability arises from the rearrange- 
ment of germline y D, and J gene segments as 
B cells develop. Somatic mutation of the variable 
region of the immunoglobulin genes is stimulat- 
ed when a B cell recognizes an antigen, adding 
further diversity to the immune response. Although 
the generation of antibody sequence diversity by 
these mechanisms has been well characterized, the 
structural basis by which sequence differences 
modify antibody affinities remains relatively unex- 
plored. 
A panel of 12 monoclonal antibodies to a 
TEMPO-dinitrophenyl hapten have been prepared, 
and the heavy- and light-chain cDNAs have been se- 
quenced by Dr. Harden McConnell's laboratory 
(Stanford University). Three members of this panel, 
ANOl, AN02, and AN03, use the same or a similar 
set of germline genes, although they are the result 
of different VJ and VDJ rearrangements. The AN02 
Fab binds the hapten more tightly than the ANOl or 
AN03 Fab molecules. Sequence analysis of the 
AN02 germline genes for AN02 (at Stanford) indi- 
cates several changes occurred in the variable re- 
gions of the heavy and light chains as the immune 
response matured. Expression of the germline se- 
quences, and the AN01-AN12 panel of monoclonal 
antibodies will provide an opportunity to investi- 
gate the structural role of VJ and VDJ rearrange- 
ment and somatic mutation in determining anti- 
body-hapten affinity. 
The Fab fragment of the monoclonal antibody 
AN02 has been crystallized, with and without 
bound hapten. The Fab-hapten complex crystal 
structure has been solved by molecular replace- 
ment in collaboration with Dr. Axel T. Briinger 
(HHMI, Yale University), using the PC-refinement 
methods developed in his laboratory. The structure 
o 
has been refined to 2.8 A resolution, revealing the 
Fab-hapten interactions in atomic detail. Refine- 
ment at the diffraction limit of this crystal form 
o 
(2.0 A) will provide a more detailed view of the Fab- 
hapten complex. Analysis of the sequence differ- 
ences between the germline and mature AN02 
genes in light of the crystal structure suggests sev- 
eral structural mechanisms by which Fab-hapten af- 
finity may be modified. 
II. Constraining (S-Turn Conformation in Model 
Immunogens. 
Antipeptide antibodies raised against a short seg- 
ment of a known protein sequence have proven in 
many cases to be highly specific biological probes. 
A limitation of the technique is that the resulting 
antibody displays a low binding affinity for the pro- 
tein antigen, which precludes its use in applica- 
tions that require a strong antigen-antibody interac- 
tion. The low affinity is thought to be due to the 
conformationally unconstrained peptide im- 
munogen, which can stimulate B cell clones to pro- 
duce antibodies against nonnative peptide struc- 
tures. To improve epitope presentation, exposed 
turn sequences have been selected from known 
protein structures and the conformation of the se- 
quence constrained by substitution into a turn site 
of a host protein. Incorporation into a stable pro- 
tein should restrict the guest turn sequence to a 
limited conformational range that better mimics the 
structure of the parent molecule. 
The first sequence selected is an exposed five-res- 
idue loop from concanavalin A. The sequence was 
substituted for four turn residues in staphylococcal 
nuclease. The resulting hybrid protein adopts a sta- 
ble folded conformation and retains nuclease enzy- 
matic activity. The crystal structure of the hybrid 
o 
has been determined to 1.8 A resolution, revealing 
that the conformation of the sequence in con- 
canavalin A has been preserved when introduced 
into a foreign turn site. The modest stability of the 
original hybrid protein can be returned to near 
wild-type levels by limiting the substitutions to sur- 
face residues. 
Attempts to raise monoclonal antibodies to the 
hybrid protein or a linear peptide containing the 
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