Protein Folding and Macromolecular Recognition 
Peter S. Kim, Ph.D. — Assistant Investigator 
Dr. Kim is also Associate Member of the Whitehead Institute for Biomedical Research, Assistant Professor 
of Biology at the Massachusetts Institute of Technology, and Assistant Molecular Biologist at the Massa- 
chusetts General Hospital, Boston. His undergraduate degree in chemistry was obtained at Cornell Uni- 
versity, where he studied with George Hess. After receiving the Ph.D. degree in biochemistry from Stanford 
University, where he studied with Robert Baldwin, Dr. Kim moved to the Whitehead Institute for Biomed- 
ical Research as a Whitehead Fellow. 
INFORMATION transfer in biology generally 
proceeds from DNA to RNA (transcription) and 
then from RNA to protein (translation) . The lin- 
ear, unfolded protein chains made during transla- 
tion must fold into a three-dimensional shape to 
be functional. Although the basic mechanisms of 
transcription and translation are understood, at 
least in outline, the transfer of information from 
one to three dimensions — i.e., protein folding — 
remains a major unsolved problem in molecular 
biology. To understand protein folding is a prime 
objective of this laboratory. 
A second effort is aimed at understanding the 
principles of macromolecular recognition: spe- 
cific protein-protein interactions and interac- 
tions between protein molecules and DNA. These 
interactions are central to much of molecular 
physiology and developmental biology. We have 
focused on a structural motif called the leucine 
zipper, v^hich occurs in several different DNA- 
binding proteins, including the products of some 
nuclear oncogenes. 
Protein Folding 
Much of our work in this area is centered on 
bovine pancreatic trypsin inhibitor (BPTI) , argu- 
ably the protein most thoroughly characterized 
in biophysical terms. In particular, nuclear mag- 
netic resonance (NMR) assignments have been 
made for essentially every proton in BPTI by Kurt 
Wiithrich and co-workers (Eidgenossische Tech- 
nische Hochschule, Switzerland), and several 
high-resolution crystal structures of the protein 
are available. In addition, Thomas Creighton (Eu- 
ropean Molecular Biology Organization, Ger- 
many) has characterized the folding of BPTI in 
terms of disulfide bond formation. 
It is difficult to determine the structures of 
protein folding intermediates because protein 
folding is a cooperative process. Indeed, a high- 
resolution structure has not yet been determined 
for any protein folding intermediate. Trapped di- 
sulfide-bonded intermediates, such as those iden- 
tified in the early folding steps of BPTI, are often 
rather insoluble; this hinders detailed structural 
characterization by NMR. We have developed a 
peptide model approach that circumvents the 
problem of cooperativiry and improves solubil- 
ity, so that the structures contained within pro- 
tein folding intermediates can be characterized 
in detail. 
Peptide models that simulate two crucial early 
intermediates in the folding of BPTI have been 
designed and synthesized chemically. By using 
two-dimensional NMR, we find that the struc- 
tures contained within these peptide models are 
remarkably native-like, corresponding to subdo- 
mains of BPTI. It appears that much of the folding 
pathway of BPTI can be explained by the forma- 
tion of native-like subdomains in these two inter- 
mediates. If our results are general, and native- 
like subdomains turn out to be key determinants 
of protein folding, then solving the protein fold- 
ing problem might be reduced in large part to 
identifying and understanding subdomains of na- 
tive proteins. 
In addition to peptide models, we are studying 
recombinant BPTI molecules that contain a sub- 
set of the native cysteine residues. We are testing 
our understanding of the folding pathway by 
making mutant BPTI molecules that should alter 
it and are developing new methodologies for 
studying it. The overall goal is to learn in detail 
how this small protein folds, in structural, ther- 
modynamic, and kinetic terms. 
Theoretical attempts to model the protein fold- 
ing process from first principles are severely hin- 
dered because of the enormous number of inter- 
actions to consider and because the calculations 
must necessarily be very accurate (the stability of 
most proteins is determined by a tiny difference 
between large energies favoring and opposing 
folding). One of the major complications is sol- 
vent: interactions between water and protein mol- 
ecules, and between water molecules them- 
selves, are numerous and difficult to calculate 
accurately. We are therefore trying to study pep- 
tides in the gas phase. Using a relatively new 
method called laser desorption, we are putting 
peptides into the gas phase for study by optical 
measurements — e.g., fluorescence and circular 
dichroism. Experimental studies of the structure 
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