Core Capabilities &Approach: NMR screening will be carried out by 2-D ^SN-^H HSQC spectroscopy. Initial NMR spectra will be recorded at 20?C on uniformly [15]N-labeled proteins. The proteins will be categorized into one of four groups based on the HSQC spectral dispersion and HetNOE data: """"""""good"""""""", """"""""promising"""""""", """"""""poor"""""""", or """"""""unfolded"""""""". This information will be fed back to the Protein Core for iterative improvement of constructs, etc. HSQC spectra will be used to screen putative interactions with cellular host proteins. If necessary, screening will be carried out on dilute samples (~10(uM) and using 3 mm NMR tubes (150uml). Any spectral changes that occur upon addition of possible binding proteins (unlabeled) will be monitored. Three scenarios are possible: 1) resonances may shift gradually in position upon addition of a binding partner, indicating that binding is weak (Kd>10uM). In this case, the structure of the complex can be determined with an excess of binding partner, after shifting the equilibrium to saturation(243-245);2) HSQC resonances may broaden or disappear due to exchange on the microsecond-millisecond timescale or due to the large size of the complex. In this case, experimental conditions, such as temperature, salts, and buffers will be changed, or 2H,i5N-labeled samples prepared. Samples of this kind will enter the crystallization pipleline;3) new resonances are observed in the HSQC spectrum. In this case, addition of successive aliquots of the binding protein will result in an increase in peak intensity for the new resonances with a concomitant decrease and disappearance of the free original resonances (slow exchange regime), indicative of tight binding (Kd<~1uM). For these cases, Kas will be determined and structure determination will be pursued. NMR structure determination: We will prepare mixed NMR samples containing one component labeled with 3C/15N and the other component unlabeled(13). Total [1]H/[13]C/[15]N NMR assignments will be obtained using 3-D HNCACB, HNCA, HN(CO)CACB, HN(CO)CA, H(CCO)NH-TOCSY, C(CO)HN-TOCSY and HCCH-TOCSY experiments and standard protocols(246-251), the final structures will be calculated using CYANA and XPLOR-NIH(252,253) incorporating all NOE distance, dihedral angle and RDC constraints. iii. Project Component: In addition to screening activities, the NMR Core will be involved in P1 (structures of CA), P4 (conformational dynamics of RT), P5 (DCAF1 domains), and P6 (APOBEC family proteins).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center (P50)
Project #
2P50GM082251-06
Application #
8528176
Study Section
Special Emphasis Panel (ZRG1-AARR-K (50))
Project Start
Project End
Budget Start
2012-09-30
Budget End
2013-07-31
Support Year
6
Fiscal Year
2012
Total Cost
$456,958
Indirect Cost
$110,718
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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