Project: Research project

Project Details


DESCRIPTION: The comprehensive goal of this proposal is to determine the
mechanisms by which proteins fold into their native three dimensional
structures. Many fully unfolded proteins spontaneously refold when placed
in the proper environment indicating that the information needed to form the
three- dimensional native structure is contained in their amino acid
sequences. However, the rules that determine how sequence directs the
folding are unknown. To further address the protein folding problem it is
necessary to determine the structures and kinetics of the intermediates in
the folding pathways. Previous work has indicated that "misfolded" states
can significantly influence a protein's progress toward formation of the
native structure. When prevented from misfolding formation of the native
structure can become very fast, on the order of 1-20 ms. Further, many
proteins are known to exhibit "burst phases" of refolding that occur in less
than a few milliseconds. Consequently it would be of value if techniques
were developed that could follow ultrafast events in protein folding and
could be capable of providing structural information on these early
intermediates. Typical mixing methods (eg stopped flow) currently in use
are too slow to allow these measurements and are limited by instrument
response times. Although selected faster measurements have been carried out
there are few of these studies published and the applicability of these
methods is very limited. What is proposed here is a more generally
applicable expansion of modern optical methods to include submillisecond
kinetics measurements of protein folding. The measurements make use of
rapid mixing technology developed in the investigator's laboratory to allow
for spectroscopic studies as early as 100 m sec after initiation of the
folding process. The specific measurements to be done include: resonance
Raman spectroscopy to follow changes in the axial coordination of the heme;
UV Resonance Raman to utilize tyrosine and tryptophan residues as reporters
of the local environment about their side chains; fluorescence lifetime
studies of tryptophan side chains to gain a measure of distance from trp to
the heme as well as a measure of the size of the protein; UV-circular
dichroism to measure the extent of alpha helix formation; and IR and raman
spectroscopies to study secondary structure by deconvolution of amide
stretching bands.
Effective start/end date5/1/974/30/01


  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences


  • Spectroscopy


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