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RESEARCH INTERESTS
Mutational and chemical studies of protein folding; NMR spectroscopy
Protein folding, dynamics and function
Effects of mutations on proteins
NMR spectroscopy
PUBLICATIONS
The long-range goal of research in my laboratory is to
understand the mechanisms by which polypeptide chains
fold into stable biologically active structures. For some
time, it has been appreciated that the ability of a protein
molecule to interact specifically with other molecules
depends on its unique three-dimensional structure and
that the three-dimensional structure is specified by the
amino acid sequence. At present, however, the
relationship between primary structure and three-
dimensional structure is understood only in general
qualitative terms, and it is not yet possible to predict a
protein's folded conformation from its sequence alone.
To learn how the three-dimensional structures of
proteins are specified, we are using a combination of
biophysical, biochemical and genetic methods to study
the folding mechanisms of small disulfide-bonded
proteins. Disulfide-bonded proteins are particularly
attractive subjects for studying protein folding because
the interconversion between the native structure and an
unfolded state can be easily manipulated by adjusting the
thiol-disulfide redox state of the protein: The native
protein can unfolded by reducing the disulfides and the
resulting reduced and unfolded protein can be refolded
upon the addition of an appropriate oxidant.
Intermediates in the refolding process can be chemically
trapped, physically isolated and characterized
individually. In this way, a detailed folding pathway,
with intermediates characterized by both the disulfides
they contain and their conformational properties, can be
elucidated.
The primary protein with which we work is bovine
pancreatic trypsin inhibitor (BPTI), one of the first
proteins for which the pathway of disulfide-coupled
folding was studied. In order to study the roles of
individual amino acid side chains in determining this
pathway, we have examined the effects of amino acid
replacements on the conformations, stabilities and
kinetic properties of the native protein and folding
intermediates. These studies have demonstrated that
substitutions at different sites in the protein can have
very different effects on the relative stabilities of the
various folding intermediates. Some of our current
studies with BPTI are directed towards understanding
how the folding process is influenced by the properties
of the polypeptide backbone. To address this question,
we are studying circularly permuted variants, as well as
mutants in which various residues have been replaced
with proline or glycine.
Another major goal of our current work is to learn how
protein flexibility influences both folding and function.
Towards this end, we are using high-resolution NMR
spectroscopy to examine the effects of mutations on the
dynamics of both native BPTI and the final intermediate
in the folding pathway. These studies provide unique
information about how the energetics of one interaction
in a protein are influenced by changes in flexibility
caused by altering other interactions. Cooperativity of
this type is likely to play a key role in both determining
the preferred folding pathway of a proteins and its
functional properties. In the case of BPTI, we are
examining how changes in flexibility determine whether
the folded protein acts as a protease inhibitor or as a
substrate that is hydrolyzed.
Selected Publications
Beeser, S. A., Oas, T. G. & Goldenberg, D. P. (1998).
Determinants of backbone dynamics in native BPTI: Cooperative
influence of the 14-38 disulfide and the Tyr 35 side chain.
J. Mol. Biol. , 284, 1581-1596.Medline
Price-Carter, M., Hull, M. S. & Goldenberg, D. P. (1998).
Roles of individual disulfide bonds in the stability and folding of
an w-conotoxin.
Biochemistry , 37, 9851-9861. Medline
Bulaj, G. & Goldenberg, D. P. (1999).
Early events in the disulfide-coupled folding of BPTI.
Protein Sci. , 8, 1825-1842. Medline
Goldenberg, D. P. (1999).
Finding the right fold.
Nature Struct. Biol. , 6, 987-990. Medline
Bulaj, G. & Goldenberg, D. P. (2001).
f-Values for BPTI folding intermediates and implications for
transition states.
Nature Struct. Biol. , 8, 326-330. Medline
Goldenberg, D. P., Koehn, R. E., Gilbert, D. E. & Wagner, G. (2001).
Solution structure and backbone dynamics of an w-conotoxin
precursor. Medline
Protein Sci. , 10, 538-550.
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