Notchl is a member of a conserved family of large modular type I transmembrane receptors that control differentiation in multicellular animals. Notch function is mediated through a novel signal transduction pathway involving successive ligand-induced proteolytic cleavages that serve to release the intracellular domain of Notch, which then translocates to the nucleus and activates downstream transcription factors. The extracellular domain of all Notch receptors have three iterated LIN 12 modules that appear to act as negative regulatory domains, possibly by limiting proteolysis. Each LIN 12 module contains three disulfide bonds and three conserved aspartate (D) or asparagine (N) residues. To begin to understand the structural basis for LIN12 function, the first LIN12 module of human Notchl (rLIN12.1) has been expressed recombinantly in E. coli and purified in reduced form. In redox buffers, rLIN12.1 forms only one disulfide isomer in the presence of millimolar Ca++, whereas multiple disulfide isomers are observed in the presence of Mg ,-, and EDTA. One-dimensional proton nuclear magnetic resonance shows that Ca + induces a dramatic increase in chemical shift dispersion of the native rLIN12.1 amide protons, as seen for Ca ++ -binding LDL-A modules. We have identified conditions suitable for determining the solution structure of rLIN 12. 1 by NMR. Using recombinant '5N-labelled protein, we have completed backbone assignments of rLIN12.1 and side-chain assignments are in progress. In addition, we have successfully refolded a recombinant fragment of Notchl that spans all three LIN12 modules and intend to pursue solution structural studies of the entire LIN12 domain.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000995-27
Application #
6578198
Study Section
Project Start
2002-05-01
Project End
2003-04-30
Budget Start
Budget End
Support Year
27
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Marintchev, Assen; Edmonds, Katherine A; Marintcheva, Boriana et al. (2009) Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation. Cell 136:447-60
Frueh, Dominique P; Arthanari, Haribabu; Koglin, Alexander et al. (2009) A double TROSY hNCAnH experiment for efficient assignment of large and challenging proteins. J Am Chem Soc 131:12880-1
Frueh, Dominique P; Leed, Alison; Arthanari, Haribabu et al. (2009) Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in (15)N-(13)C-ILV methyl labeled proteins. J Biomol NMR 45:311-8
Lentz, Margaret R; Westmoreland, Susan V; Lee, Vallent et al. (2008) Metabolic markers of neuronal injury correlate with SIV CNS disease severity and inoculum in the macaque model of neuroAIDS. Magn Reson Med 59:475-84
Chen, Jingyang; Dupradeau, Francois-Yves; Case, David A et al. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites. Biochemistry 46:3096-107
Hyberts, Sven G; Heffron, Gregory J; Tarragona, Nestor G et al. (2007) Ultrahigh-resolution (1)H-(13)C HSQC spectra of metabolite mixtures using nonlinear sampling and forward maximum entropy reconstruction. J Am Chem Soc 129:5108-16
Lentz, Margaret R; Kim, John P; Westmoreland, Susan V et al. (2005) Quantitative neuropathologic correlates of changes in ratio of N-acetylaspartate to creatine in macaque brain. Radiology 235:461-8
Kim, John P; Lentz, Margaret R; Westmoreland, Susan V et al. (2005) Relationships between astrogliosis and 1H MR spectroscopic measures of brain choline/creatine and myo-inositol/creatine in a primate model. AJNR Am J Neuroradiol 26:752-9
Peled, S; Cory, D G; Raymond, S A et al. (1999) Water diffusion, T(2), and compartmentation in frog sciatic nerve. Magn Reson Med 42:911-8
Mo, H; Dai, Y; Pochapsky, S S et al. (1999) 1H, 13C and 15N NMR assignments for a carbon monoxide generating metalloenzyme from Klebsiella pneumoniae. J Biomol NMR 14:287-8

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