Project Details


The broad long term objectives are twofold. One is to develop
methods that will extend the power of solution NMR beyond the study of
native globular proteins to the study of nonnative and nonglobular
proteins. The second objective is to use NMR to understand protein
folding mechanisms by studying structures of partially folded proteins,
and by assessing the effect of amino acid substitutions on structure,
stability and folding kinetics. These studies will form the basis for
using NMR to understand how interruptions in the Gly-X-Y pattern, found
in collagen diseases like Osteogenesis Imperfecta and Ehlers Danlos
Syndrome, can result in serious disease.
The first aim is to characterize the partially folded state of
guinea pig alpha-lactalbumin to elucidate the nature of protein folding
intermediates. More specifically, we wish to learn which regions of the
molten globule state contain regions of secondary structure and whether
tertiary interactions are important in stabilizing these regions of
secondary structure. We will characterize the partially folded state by
1H NMR methods, and by isotope labelling and heteronuclear 2D and 3D NMR
experiments. Key mutants will be made to assess the effects of sequence
change on secondary structure and tertiary interactions of the partially
folded state.
The second aim is to obtain, for the first time, individual residue
assignments and the NMR solution structure of triple helical peptides, to
determine the role of individual amino acids in stabilizing the triple
helix and to understand how key residues direct protein folding. We will
examine the effects, by 1D NMR, of (Gly-X-Y) sequence changes on the
amount of triple helix formed, and on the kinetics and thermodynamics of
folding. To obtain the solution structure we will first design synthetic
triple helical peptides to facilitate the spin system identification
process. Then we propose 1H NMR experiments as well as heteronuclear NMR
experiments that should allow us to perform sequential resonance
assignments, and distinguish inter from intra strand NOE's.
Effective start/end date8/1/927/31/96


  • Structural Biology


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