| 1. |
- Lemak, Alexander, et al.
(författare)
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A novel strategy for NMR resonance assignment and protein structure determination
- 2011
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Ingår i: JOURNAL OF BIOMOLECULAR NMR. - : Springer Science Business Media. - 0925-2738 .- 1573-5001. ; 49:1, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- The quality of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy is contingent on the number and quality of experimentally-derived resonance assignments, distance and angular restraints. Two key features of protein NMR data have posed challenges for the routine and automated structure determination of small to medium sized proteins; (1) spectral resolution - especially of crowded nuclear Overhauser effect spectroscopy (NOESY) spectra, and (2) the reliance on a continuous network of weak scalar couplings as part of most common assignment protocols. In order to facilitate NMR structure determination, we developed a semi-automated strategy that utilizes non-uniform sampling (NUS) and multidimensional decomposition (MDD) for optimal data collection and processing of selected, high resolution multidimensional NMR experiments, combined it with an ABACUS protocol for sequential and side chain resonance assignments, and streamlined this procedure to execute structure and refinement calculations in CYANA and CNS, respectively. Two graphical user interfaces (GUIs) were developed to facilitate efficient analysis and compilation of the data and to guide automated structure determination. This integrated method was implemented and refined on over 30 high quality structures of proteins ranging from 5.5 to 16.5 kDa in size.
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| 2. |
- Sheng, Yi, et al.
(författare)
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Molecular basis of Pirh2-mediated p53 ubiquitylation
- 2008
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Ingår i: NATURE STRUCTURAL and MOLECULAR BIOLOGY. - : Springer Science and Business Media LLC. - 1545-9985 .- 1545-9993. ; 15:12, s. 1334-1342
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Tidskriftsartikel (refereegranskat)abstract
- Pirh2 (p53-induced RING-H2 domain protein; also known as Rchy1) is an E3 ubiquitin ligase involved in a negative-feedback loop with p53. Using NMR spectroscopy, we show that Pirh2 is a unique cysteine-rich protein comprising three modular domains. The protein binds nine zinc ions using a variety of zinc coordination schemes, including a RING domain and a left-handed beta-spiral in which three zinc ions align three consecutive small beta-sheets in an interleaved fashion. We show that Pirh2-p53 interaction is dependent on the C-terminal zinc binding module of Pirh2, which binds to the tetramerization domain of p53. As a result, Pirh2 preferentially ubiquitylates the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 regulates protein turnover of the transcriptionally active form of p53.
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