The 3-dimensional structure and interaction energetics of a peptide which binds and inhibits the glycolytic enzymes aldolase and glyceraldehyde-3- phosphate dehydrogenase will be investigated by NMR and constrained molecular dynamics. The peptide is from erythrocyte band 3 protein. A number of studies on the association of band 3 with glycolytic enzymes have shown that binding leads to full but reversible inactivation of the enzymes. Moreover, phosphorylation of the N-terminal tyrosine of band 3 by an endogenous protein tyrosine kinase prevents binding. These observations, together with others, suggest that band 3 contributes to the control of glycolysis by an inhibitory interaction between band 3 and the glycolytic enzymes which is modulated through tyrosine phosphorylation. Effects of band 3 association are well reproduced by a pentadecameric peptide from the N-terminus of the cytoplasmic domain of band 3. Thus we have undertaken structural studies of this peptide, and variants of it, when bound to the glycolytic enzymes in order to understand the binding interactions and possible mechanism of inactivation. The structure of the bound peptide will be determined by using exchange-transferred nuclear Overhauser effect (NOE) experiments. Since the evaluation of interproton distances is the basis of structure determination by NMR, we will investigate by NMR simulation studies a multiple spin-pair analysis procedure for equating transferred NOE intensities to distances. The accuracy of the distances evaluated from a partial rate-matrix approach corresponding to exchange systems relative to that estimated from a 2-spin approximation will be examined. The procedures found most reliable by the simulation studies will be applied in the NMR structural studies of the band 3 peptide complexes. Modeling of the complex structure will be done with the bound peptide structure determined by NMR and the known crystallographic structures of the glycolytic enzymes. Structure refinement will consider the agreement with NMR distances and simulated transferred NOE spectra, as well as interaction energies and structural characteristics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29GM039478-01A3
Application #
3466771
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1991-08-01
Project End
1996-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
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