Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics

During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated.

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Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

Biochemical Society Transactions ◽

10.1042/bst20150138 ◽

2015 ◽

Vol 43 (5) ◽

pp. 1023-1032 ◽

Cited By ~ 10

Author(s):

Thomas Stockner ◽

Anna Mullen ◽

Fraser MacMillan

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Abc Transporters ◽

Epr Spectroscopy ◽

Structural Information ◽

Dynamic Properties ◽

Molecular System ◽

Synergistic Effects ◽

Md Simulations ◽

Substrate Spectrum

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented.

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Nucleosome dynamics

Biochemical Society Symposium ◽

10.1042/bss0730109 ◽

2006 ◽

Vol 73 ◽

pp. 109-119 ◽

Cited By ~ 2

Author(s):

Chris Stockdale ◽

Michael Bruno ◽

Helder Ferreira ◽

Elisa Garcia-Wilson ◽

Nicola Wiechens ◽

...

Keyword(s):

High Resolution ◽

Chromatin Structure ◽

Current Knowledge ◽

Dynamic Properties ◽

Structure And Dynamics ◽

Nucleosome Dynamics ◽

Sharp Focus ◽

Recent Advances ◽

Insight Into ◽

Chromatin Structure And Dynamics

In the 30 years since the discovery of the nucleosome, our picture of it has come into sharp focus. The recent high-resolution structures have provided a wealth of insight into the function of the nucleosome, but they are inherently static. Our current knowledge of how nucleosomes can be reconfigured dynamically is at a much earlier stage. Here, recent advances in the understanding of chromatin structure and dynamics are highlighted. The ways in which different modes of nucleosome reconfiguration are likely to influence each other are discussed, and some of the factors likely to regulate the dynamic properties of nucleosomes are considered.

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Correlation of membrane protein conformational and functional dynamics

Nature Communications ◽

10.1038/s41467-021-24660-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Raghavendar Reddy Sanganna Gari ◽

Joel José Montalvo‐Acosta ◽

George R. Heath ◽

Yining Jiang ◽

Xiaolong Gao ◽

...

Keyword(s):

Membrane Protein ◽

Conformational Changes ◽

Single Channel ◽

Conformational Dynamics ◽

Md Simulations ◽

High Temporal Resolution ◽

Dependent Manner ◽

Functional Dynamics ◽

Ph Dependent ◽

Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

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Multi-Frequency EPR Spectroscopy Studies of the Structure and Conformational Changes of Site-Directed Spin Labelled Membrane Proteins

Supramolecular Structure and Function 8 ◽

10.1007/0-306-48662-8_10 ◽

2005 ◽

pp. 157-177 ◽

Cited By ~ 1

Author(s):

Heinz-Jürgen Steinhoff

Keyword(s):

Membrane Proteins ◽

Conformational Changes ◽

Epr Spectroscopy ◽

Spectroscopy Studies

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Characterization of Bifunctional Spin Labels for Investigating the Structural and Dynamic Properties of Membrane Proteins Using EPR Spectroscopy

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.7b07631 ◽

2017 ◽

Vol 121 (39) ◽

pp. 9185-9195 ◽

Cited By ~ 8

Author(s):

Indra D. Sahu ◽

Andrew F. Craig ◽

Megan M. Dunagum ◽

Robert M. McCarrick ◽

Gary A. Lorigan

Keyword(s):

Membrane Proteins ◽

Epr Spectroscopy ◽

Dynamic Properties ◽

Spin Labels

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FcαRI binding at the IgA1 CH2–CH3 interface induces long-range conformational changes that are transmitted to the hinge region

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807478115 ◽

2018 ◽

Vol 115 (38) ◽

pp. E8882-E8891 ◽

Cited By ~ 7

Author(s):

Monica T. Posgai ◽

Sam Tonddast-Navaei ◽

Manori Jayasinghe ◽

George M. Ibrahim ◽

George Stan ◽

...

Keyword(s):

Binding Site ◽

Long Range ◽

Conformational Changes ◽

Lectin Binding ◽

Coarse Grained ◽

Alanine Scanning Mutagenesis ◽

Long Distance ◽

Receptor Binding Site ◽

Binding Interface ◽

Functional Dynamics

IgA effector functions include proinflammatory immune responses triggered upon clustering of the IgA-specific receptor, FcαRI, by IgA immune complexes. FcαRI binds to the IgA1–Fc domain (Fcα) at the CH2–CH3 junction and, except for CH2 L257 and L258, all side-chain contacts are contributed by the CH3 domain. In this study, we used experimental and computational approaches to elucidate energetic and conformational aspects of FcαRI binding to IgA. The energetic contribution of each IgA residue in the binding interface was assessed by alanine-scanning mutagenesis and equilibrium surface plasmon resonance (SPR). As expected, hydrophobic residues central to the binding site have strong energetic contributions to the FcαRI:Fcα interaction. Surprisingly, individual mutation of CH2 residues L257 and L258, found at the periphery of the FcαRI binding site, dramatically reduced binding affinity. Comparison of antibody:receptor complexes involving IgA or its precursor IgY revealed a conserved receptor binding site at the CH2–CH3 junction (or its equivalent). Given the importance of residues near the CH2–CH3 junction, we used coarse-grained Langevin dynamics simulations to understand the functional dynamics in Fcα. Our simulations indicate that FcαRI binding, either in an asymmetric (1:1) or symmetric (2:1) complex with Fcα, propagated long-range conformational changes across the Fc domains, potentially impacting the hinge and Fab regions. Subsequent SPR experiments confirmed that FcαRI binding to the Fcα CH2–CH3 junction altered the kinetics of HAA lectin binding at the IgA1 hinge. Receptor-induced long-distance conformational transitions have important implications for the interaction of aberrantly glycosylated IgA1 with anti-glycan autoantibodies in IgA nephropathy.

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Coarse-Grained Models Reveal Functional Dynamics - I. Elastic Network Models – Theories, Comparisons and Perspectives

Bioinformatics and Biology Insights ◽

10.4137/bbi.s460 ◽

2008 ◽

Vol 2 ◽

pp. BBI.S460 ◽

Cited By ~ 21

Author(s):

Lee-Wei Yang ◽

Choon-Peng Chng

Keyword(s):

Conformational Changes ◽

Conformational Dynamics ◽

Computational Cost ◽

Network Models ◽

Coarse Graining ◽

Coarse Grained ◽

Elastic Network ◽

Elastic Network Models ◽

Time Space ◽

Functional Dynamics

In this review, we summarize the progress on coarse-grained elastic network models (CG-ENMs) in the past decade. Theories were formulated to allow study of conformational dynamics in time/space frames of biological interest. Several highlighted models and their underlined hypotheses are introduced in physical depth. Important ENM offshoots, motivated to reproduce experimental data as well as to address the slow-mode-encoded configurational transitions, are also introduced. With the theoretical developments, computational cost is significantly reduced due to simplified potentials and coarse-grained schemes. Accumulating wealth of data suggest that ENMs agree equally well with experiment in describing equilibrium dynamics despite their distinct potentials and levels of coarse-graining. They however do differ in the slowest motional components that are essential to address large conformational changes of functional significance. The difference stems from the dissimilar curvatures of the harmonic energy wells described for each model. We also provide our views on the predictability of ‘open to close’ (open→close) transitions of biomolecules on the basis of conformational selection theory. Lastly, we address the limitations of the ENM formalism which are partially alleviated by the complementary CG-MD approach, to be introduced in the second paper of this two-part series.

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Structure and dynamics of catalytically competent but labile paramagnetic metal-hydrides: the Ti(iii)-H in homogeneous olefin polymerization

Chemical Science ◽

10.1039/d0sc04967k ◽

2020 ◽

Vol 11 (46) ◽

pp. 12436-12445

Author(s):

Enrico Salvadori ◽

Mario Chiesa ◽

Antonio Buonerba ◽

Alfonso Grassi

Keyword(s):

Epr Spectroscopy ◽

Olefin Polymerization ◽

Metal Hydrides ◽

Open Shell ◽

Geometrical Structures ◽

Structure And Dynamics ◽

Paramagnetic Metal

Catalytically competent titanium-hydrides are reactive and difficult to isolate. We use EPR spectroscopy to define the electronic and geometrical structures as well as dynamics of an open-shell Ti-H active in syndiospecific olefin polymerization.

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