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How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine Vilius Kurauskas, Audrey Hessel, Peixiang Ma, Paola Lunetti, Katharina Weinhäupl, Lionel Imbert, Bernhard Brutscher, Martin S. King, Remy Sounier, Vincenza Dolce, Edmund Richard Stephan Kunji, Loredana Capobianco, Christophe Chipot, Francois Dehez, Beate Bersch, and Paul Schanda J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b00269 • Publication Date (Web): 03 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018
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The Journal of Physical Chemistry Letters
How
Detergent
Impacts
Membrane
Proteins:
Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine Vilius Kurauskas Weinhäupl
[a]
[a]
, Audrey Hessel
, Lionel Imbert [e]
Vincenza Dolce François Dehez
[a]
[a]
, Bernhard Brutscher [c]
, Edmund R. S. Kunji
[f]*
, Peixiang Ma
, Beate Bersch
[a]
[a]
[a]
[c]
, Martin S. King
, Loredana Capobianco
, Paul Schanda
[b]
, Paola Lunetti
[b]*
, Katharina
, Rémy Sounier
[d]
,
[f]
, Christophe Chipot ,
[a]*
[a] Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France [b] Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy; [c] MRC-MBU, Univ. Cambridge, Cambridge CB2 0XY, UK [d] CNRS, INSERM, Univ. Montpellier, 34094 Montpellier,France [e] Dept of Pharmacy, Univ. Calabria, 87036 Arcavacata di Rende, Italy [f] LPCT, UMR 7019 Univ. Lorraine, CNRS and Lab. Internat. Associé & Univ. Illinois at Urbana-Champaign, F-54500 Vandoeuvre-lès-Nancy.
* Corresponding Authors:
[email protected] ,
[email protected],
[email protected] ACS Paragon Plus Environment
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The Journal of Physical Chemistry Letters
ABSTRACT Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent–membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in PDC, they systematically correspond to non-functional states.
nonspeci c binding
DPC
disrupted 3ry structure
TOC Graphic
+A T +GP TP
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extensive millisecond dynamics
temperature-unfolding
KEYWORDS. CPMG NMR; protein stability; transport activity; detergent-protein interactions; MD simulation
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The Journal of Physical Chemistry Letters
Mitochondrial carriers (MCs) constitute a large family of transport proteins of ca. 30-35 kDa molecular weight that play important roles in the intracellular translocation of metabolites, 1
nucleotides, and coenzymes . The transport mechanism involves the alternating exposure of a 2
central cavity to the cytosolic side (c-state) or the mitochondrial matrix (m-state) . Crystallographic structures of the most prominent member, the ADP/ATP carrier (AAC), 3,4
extracted from the the native mitochondrial membrane
could only be obtained in the presence
of the strong inhibitor carboxyatractyloside (CATR) which locks the carrier in an aborted c-state. These structures revealed a central cavity, in which CATR is bound, surrounded by six transmembrane helices. No other atomic-resolution structures of MCs are available, underscoring the difficulty of obtaining crystals of these dynamic proteins. Solution-NMR may overcome the 5–
difficulties that crystallography faces for dynamic MPs. Substrate, lipid and inhibitor binding 10
as well as dynamics
8,11
, detected by NMR have been reported and related to functional
mechanisms of this class of membrane proteins. Other biophysical techniques, e.g. functional/thermostability assays and molecular-dynamics simulations, have challenged some of these interpretations
12–14
. Solving this apparent controversy has been hampered by the lack of
direct experimental atomic-resolution data and stringent control experiments. Here, we resort to an extensive set of NMR experiments to address the structure substrate interactions and dynamics of MCs in DPC alongside with functional/thermostability assays and moleculardynamics simulations and conclude on their physiological significance. To probe the structural integrity of the yeast ADP/ATP carrier 3 (AAC3) in different detergents, we first investigated its thermal stability, consistently with our previous
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investigations
13,14
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. We produced AAC3 in the yeast mitochondrial membrane, extracted it with
the mild detergent dodecylmaltoside (DDM), diluted it into solutions of either DDM, lauryl maltoside neopentyl glycol (LMNG) or DPC, and performed thermostability shift assay (TSA) experiments
14,15
. In DDM and LMNG, a typical protein melting curve reveals a cooperative
thermal denaturation event with an apparent melting temperature of ca. 49°C (Figure 1, blue/green lines). In the CATR-bound state, the melting temperature is dramatically increased to 80-90°C. In contrast, the same protein preparation diluted into DPC already shows a high fluorescence signal at low temperature, and no transition in the course of the temperature scan, irrespective of the presence of CATR. These observations indicate that AAC3 in DPC does not 13
form a stable tertiary structure, nor binds CATR, in line with previous findings . To understand this behavior at the atomic level, we turned to solution-NMR of three members of the MC family, namely AAC3, GDP/GTP carrier (GGC1) and ornithine carrier (ORC1). Given their low sequence identity (
