Core Capabilities &Approach: The Protein Core has accumulated and demonstrated the required expertise in recombinant protein expression and purification utilizing bacterial and insect cell systems. The protein expression platform will be centered on 6X-His and/or Strep tagged proteins, with or without other fusion partners, such as GB1, thioredoxin, NusA, MBP, and GST. Fusion constructs will contain TEV or thrombin cleavage sites for down-stream removal of the fusion protein. For each expression vector variant, multiple conditions will be tested to achieve optimal expression and yield: host, temperature, induction time, etc. For production in E. coli, the optimization approaches will include: 1) incremental truncation/extension of the coding sequence to define minimal folding domains, 2) mutagenesis or in vitro evolution of the coding sequence in the context of preserved biological function, and 3) a combination of) and 2). This approach will result in large numbers of constructs, requiring high-throughput cloning, expression, and solubility screening. These tasks have been successfully handled by the Core and yielded difficult proteins such as Vpr and Vpx in forms amenable to structure determination. Despite these efforts, production of soluble virus-derived or cellular proteins in E. coli may fail for some targets. In these cases, targets will be expressed in insect cells. The high-throughput methodologies for cloning, expression screening, and protein production in eukaryotic systems have been established and utilized for expression and purification of Trimsa, DCAFi, and DDBi. Purified proteins will be characterized by static multi-angle light scattering to assess their quaternary state, LC-ESI-TOF mass spectrometry to confirm purity, NMR isotope labeling efficiency and selenomethionine (Se-Met) labeling, before submitting samples for NMR and X-ray crystallography. Labeling protocolsf^39-242) are well-established and routinely used. For proteins purified from eukaryotic expression systems, post-translational modification will be analyzed using ESI-TOF and other mass spectrometry methods. To determine protein stability, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry will be performed as a function of temperature and chaotropic agents. iii. Project Component: The Protein Core has been the main driving force in the PCHPI's studies of Vpr and DCAFif48,86) and will continue to play a primary role in these studies (Ps).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Specialized Center (P50)
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Special Emphasis Panel (ZRG1-AARR-K)
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University of Pittsburgh
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Gupta, Rupal; Lu, Manman; Hou, Guangjin et al. (2016) Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies. J Phys Chem B 120:329-39
Van Oss, S Branden; Shirra, Margaret K; Bataille, Alain R et al. (2016) The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6. Mol Cell 64:815-825
Perilla, Juan R; Gronenborn, Angela M (2016) Molecular Architecture of the Retroviral Capsid. Trends Biochem Sci 41:410-20
Sharaf, Naima G; Ishima, Rieko; Gronenborn, Angela M (2016) Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study. Biochemistry 55:3864-73
Byeon, In-Ja L; Byeon, Chang-Hyeock; Wu, Tiyun et al. (2016) Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity. Biochemistry 55:2944-59
Saito, Akatsuki; Ferhadian, Damien; Sowd, Gregory A et al. (2016) Roles of Capsid-Interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74. J Virol 90:5808-23
Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L et al. (2016) In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway. Nat Commun 7:13689
Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik et al. (2016) The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes. J Biol Chem 291:11809-19
Ramalho, Ruben; Rankovic, Sanela; Zhou, Jing et al. (2016) Analysis of the mechanical properties of wild type and hyperstable mutants of the HIV-1 capsid. Retrovirology 13:17
Oum, Yoon Hyeun; Desai, Tanay M; Marin, Mariana et al. (2016) Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion. J Virol Methods 233:62-71

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