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      Nanobodies and recombinant binders in cell biology

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          Abstract

          Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes.

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          A general method for the covalent labeling of fusion proteins with small molecules in vivo.

          Antje Keppler, Susanne Gendreizig, Thomas Gronemeyer … (2003)
          Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.
          Article 0 comments Cited 462 times – based on 0 reviews     Review now
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            Activation and allosteric modulation of a muscarinic acetylcholine receptor

            Andrew C. Kruse, Aaron Ring, Aashish Manglik … (2013)
            Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.
            Article 0 comments Cited 279 times – based on 0 reviews     Review now
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              Scaffold proteins: hubs for controlling the flow of cellular information.

              Matthew C Good, Jesse G. Zalatan, Wendell A Lim (2011)
              The spatial and temporal organization of molecules within a cell is critical for coordinating the many distinct activities carried out by the cell. In an increasing number of biological signaling processes, scaffold proteins have been found to play a central role in physically assembling the relevant molecular components. Although most scaffolds use a simple tethering mechanism to increase the efficiency of interaction between individual partner molecules, these proteins can also exert complex allosteric control over their partners and are themselves the target of regulation. Scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. As a result, scaffold proteins have been exploited by evolution, pathogens, and cellular engineers to reshape cellular behavior.
              Article 0 comments Cited 266 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): J Cell Biol
                Journal ID (iso-abbrev): J. Cell Biol
                Journal ID (publisher-id): jcb
                Journal ID (hwp): jcb
                Title: The Journal of Cell Biology
                Publisher: The Rockefeller University Press
                ISSN (Print): 0021-9525
                ISSN (Electronic): 1540-8140
                Publication date (Print): 8 June 2015
                Volume: 209
                Issue: 5
                Pages: 633-644
                Affiliations
                [1 ]Department of Biology II, Ludwig Maximilians University Munich and Center for Integrated Protein Science Munich, 82152 Planegg-Martinsried, Germany
                [2 ]Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
                [3 ]Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1050 Brussels, Belgium
                Author notes
                Correspondence to Heinrich Leonhardt: h.leonhardt@ 123456lmu.de
                Article
                Publisher ID: 201409074
                DOI: 10.1083/jcb.201409074
                PMC ID: 4460151
                PubMed ID: 26056137
                SO-VID: f42319fe-7c62-4509-bfb3-5e3568d88b61
                Copyright © © 2015 Helma et al.
                License:

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

                History
                Date received : 15 September 2014
                Date accepted : 19 May 2015
                Categories
                Subject: Reviews
                Subject: Review
                Subject: 3

                ScienceOpen disciplines: Cell biology
                Data availability:
                ScienceOpen disciplines: Cell biology

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