UMass Chan AffiliationsThompson Lab
Department of Biochemistry and Molecular Pharmacology
Amino Acids, Peptides, and Proteins
Chemical and Pharmacologic Phenomena
Chemicals and Drugs
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AbstractPotent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.
Elife. 2018 Apr 20;7. pii: 34311. doi: 10.7554/eLife.34311. Link to article on publisher's site
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/40639
Full author list omitted for brevity. For the full list of authors, see article.
RightsCopyright 2018 Muller et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Except where otherwise noted, this item's license is described as Copyright 2018 Muller et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Role of Disulfide Bond Rearrangement in Newcastle Disease Virus Entry: A DissertationJain, Surbhi (2008-06-26)Newcastle disease virus (NDV), an avian paramyxovirus, enters the host cell by fusion of viral and host cell membranes. The fusion of two membranes is mediated by the viral fusion (F) protein. The F protein, like other class I fusion proteins, is thought to undergo major conformational changes during the fusion process. The exact mechanism that leads to major refolding of F protein is not clear. Recently, it has been proposed that disulfide bond reduction in the fusion protein of some viruses may be involved in the conformational changes in fusion proteins. In some viruses, the reduction of disulfide bonds in the fusion protein is mediated by host cell disulfide isomerases belonging to the protein disulfide isomerase (PDI) family. In this study, the role of disulfide bond isomerization in the entry of NDV was analyzed. Using inhibitors of thiol-disulfide isomerases, we found that blocking the reduction of disulfide bonds in the fusion protein inhibited cell-cell fusion as well as virus entry into the host cell. Also, over-expression of isomerases belonging to the PDI family significantly enhanced cell-cell fusion. Taken together, these results suggest that free thiols play an important role in fusion mediated by NDV glycoproteins. Using a thiol specific, membrane impermeable biotin, MPB, we found that free thiols are produced in cell surface-expressed NDV F protein. The production of free thiols was inhibited by inhibitors of thiol-disulfide isomerases. Over-expression of isomerases belonging to the PDI family enhanced detection of free thiols in F protein. In F protein, present in virions or in virus-like particles, free thiols were detected only after the particles were attached to target cells. Taken together, these results suggest that free thiols are produced in F protein and the production of free thiols is mediated by host cell thiol-disulfide isomerases. Using conformation sensitive antibodies, we also studied the conformation of cell surface-expressed F protein in the presence ofthiol-disulfide isomerase inhibitors or in cells over-expressing thiol-disulfide isomerases. In the presence of thiol-disulfide isomerase inhibitors, the cell surface-expressed F protein was in a prefusion conformation while in cells over-expressing thiol-disulfide isomerases the F protein was in a post-fusion conformation. We also correlated the production of free thiols to the conformational changes in F protein. Using temperature-arrested intermediates or F protein with mutations in heptad repeat domains, which are defective in attaining intermediate conformations, we found that free thiols are produced before any of the proposed conformational changes in F protein. Also, the production of free thiols in F protein was found to be independent of its activation by hemagglutinin-neuraminidase (HN) protein. These results suggest that free thiols are probably required for the activation of F protein during membrane fusion.
Contribution of Ordered Water Molecules and a Crucial Phenylalanine to Cooperative Pathway(s) in Scapharca Dimeric Hemoglobin: a DissertationPardanani, Animesh Dev (1997-06-01)The homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis binds oxygen cooperatively and thus offers a simple model system for studying communication between two chemically identical sites. Although the individual subunits of HbI have the same myoglobin-fold as mammalian hemoglobins, the quaternary assemblage is radically different. Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins. Analysis of structures of this hemoglobin in the liganded (02or CO) and unliganded states has provided a framework for understanding the role of individual amino acid side-chains in mediating cooperativity. The work presented in this dissertation has directly tested the central tenets of the proposed structural mechanism for cooperativity in HbI, illuminating the key roles played by residue Phe 97 and interface water molecules in intersubunit communication. Heterologous expression of Scapharca dimeric hemoglobin: A synthetic gene has been utilized to express recombinant RbI in Escherichia coli. The HbI apoprotein constitutes 5-10% of the total bacterial protein in this system. Addition of the heme precursor δ-aminolevulinic acid to the expression culture results in a ~3-fold increase in the production of soluble hemoglobin. Recombinant HbI has been successfully purified to homogeneity, resulting in a final yield of 80-100 mg of pure holoprotein from a 12 L expression culture. Analysis of recombinant HbI reveals its oxygen binding properties to be indistinguishable from native HbI. It was necessary to correct a protein sequence error by mutating residue Asn 56 to aspartate in order to obtain diffraction quality crystals, that are isomorphous to native HbI crystals. These recombinant HbI crystals diffract to high resolution, permitting the functional effects of mutant HbI proteins to be correlated with detailed structural analysis.