Biophysical Genetics of Protein Structure and Folding 
structure of globular proteins and to define de- 
sign principles for protein engineering. 
We have developed a genetic system to deter- 
mine which amino acid sequences are consistent 
with a particular jS-turn structure in staphylococ- 
cal nuclease. Each member of our gene library 
contains a unique sequence at this ^-turn. Only a 
small fraction of the sequences examined are 
consistent with an enzymatically active and 
stable protein in Escherichia coli. The i8-turn 
under consideration is well removed from the ac- 
tive site, suggesting that the modulation in the 
observed enzyme activity is due to changes in the 
stability of the protein. There are strong biases in 
the amino acids occurring at each position in the 
i8-tum. Recently, a statistical analysis of the ge- 
netic data has led to a predictive model for this 
j8-turn type in all globular proteins. This ap- 
proach may be of general use in defining other 
sequence-secondary structure relationships. 
Crystal structure determinations of several 
mutants derived from a genetic analysis of this 
i8-turn site indicate that substitutions can be made 
without greatly influencing the tertiary structure. 
In contrast, crystallographic analyses of several 
point mutants in another (8-turn near the active 
site demonstrate that single-amino acid substitu- 
tions can result in a change from one ;8-turn type 
to another, causing a significant rearrangement in 
the local protein structure. We are currently 
working to understand the physical bases for the 
selections observed in the genetic experiment 
and the conformational alteration they impart to 
the protein. Results of this and related experi- 
ments should provide insight into the relation- 
ship between amino acid sequence and structure 
required for the rational design and engineering 
of protein molecules. 
Structural Basis of Immunoglobulin 
Maturation 
The sequence diversity found in immunoglobu- 
lin molecules is generated at several different lev- 
els. As B cells develop, combinatorial variability 
arises from the rearrangement of germline V, D, 
and J gene segments. When a B cell recognizes an 
antigen, somatic mutation of the variable region 
of the immunoglobulin genes is stimulated, add- 
ing further diversity to the immune response. Al- 
though these mechanisms have been well charac- 
terized, the structural basis by which sequence 
differences modify antibody affinities remains rel- 
atively unexplored. 
Harden McConnell and his laboratory at Stan- 
ford University have prepared a panel of 1 2 mono- 
clonal antibodies to a particular hapten (a small 
molecule that interacts with antibody but does 
not itself elicit an immune response) and have 
sequenced the heavy- and light-chain cDNAs. The 
antibody panel provides an opportunity to inves- 
tigate the structural role of such rearrangements 
in determining antibody-hapten affinity. 
We have crystallized a fragment of one of these 
monoclonal antibodies, with and without bound 
hapten, and have solved the structure of the com- 
plex, in collaboration with Axel Briinger (HHMI, 
Yale University). The crystal structure provides 
an opportunity to assess the effect of antigen bind- 
ing on antibody structure and will serve as a basis 
for understanding the influence on hapten affin- 
ity of sequence variants resulting from gene rear- 
rangement or somatic mutation. 
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