Biophysical Genetics of Protein Structure and Folding 
to simulate this system using molecular dynamics 
techniques. 
A structural analysis of early protein-folding in- 
termediates has been difficult because of their 
transient nature. Amide hydrogen exchange has 
been used by others to examine the appearance of 
hydrogen-bonded protein-folding intermediates 
for a number of proteins. We have begun to use 
this technique to examine early folding interme- 
diates of a staphylococcal nuclease variant. Par- 
tial protection of amides occurs early in the fold- 
ing process, despite the long overall folding time 
for nuclease. 
Genetic Analysis of a /S-Turn 
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 j8-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 /3-turn types to better understand the detailed 
structure of globular proteins and to define de- 
sign principles for protein engineering. 
We have developed a genetic approach to de- 
termine which amino acid sequences are consis- 
tent with a particular |S-turn structure in staphylo- 
coccal nuclease. Each member of our gene 
library contains a unique sequence at this jS-turn. 
Only a small fraction of the sequences examined 
are consistent with an enzymatically active and 
stable protein in Escherichia coli. The |8-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-turn. Recently, a statistical analysis of these data 
has led to a predictive model for this /3-turn type 
in all globular proteins. This approach may be 
useful in defining other sequence-secondary 
structure relationships. Results of this and related 
experiments should provide insight into the rela- 
tionship between amino acid sequence and struc- 
ture required for the rational design and engineer- 
ing of protein molecules. 
Structural Studies of Trypanosome 
Calmodulin 
Calmodulin serves as a calcium-dependent reg- 
ulatory subunit of a variety of cytoskeletal pro- 
teins and cytoplasmic enzymes, including a num- 
ber of protein kinases. The novel structure of rat 
calmodulin was determined in the laboratory of 
Charles Bugg. The protein is composed of two 
largely helical domains, each with two calcium- 
binding sites, separated by an extended solvent- 
exposed a-helix. We have obtained crystals of 
calmodulin from Trypanosoma brucei rhode- 
siense, in collaboration with Curtis Patton (Yale 
University). The crystal structure in progress 
should serve as the basis for rational drug design 
against this and related organisms. 
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