Studying Structural Dynamics of Potassium Channels by Single-Molecule FRET

Wang, Shizhen; Brettmann, Joshua; Nichols, Colin G.

doi:10.1007/978-1-4939-7362-0_13

Cited by 14 publications

(15 citation statements)

References 16 publications

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“…Leak and direct excitation corrections were not applied, since leakage was lower than 0.06 and direct excitation was essentially undetectable. Traces were inspected and selected manually following criteria described in previous publications 13 , 50 . The bin size of all time histograms was set at 0.02x recording time, ensuring equal contribution from each trace to avoid dominant effects of long traces 17 .…”

Section: Methodsmentioning

confidence: 99%

Potassium channel selectivity filter dynamics revealed by single-molecule FRET

et al. 2019

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Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+, but not Na+, ions are perfectly coordinated. Using single molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentrations. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels non-selective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild type KirBac1.1 channels are K+-selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, while K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

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Section: Methodsmentioning

confidence: 99%

Potassium channel selectivity filter dynamics revealed by single-molecule FRET

et al. 2019

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“…This is particularly true for membrane peptides and proteins for which there is no high-resolution structure available or when the structural information is limited. This is reflected in the extensive use of SDFL methods to study changes in conformational dynamics in different classes of membrane proteins like pore-forming peptides and proteins (Nagahama et al, 2002; Parker and Feil, 2005; Raghuraman and Chattopadhyay, 2007a; Haldar et al, 2008; Ho et al, 2013), GPCR (Yao et al, 2006; Daggett and Sakmar, 2011; Dekel et al, 2012; Alexiev and Farrens, 2014), potassium channels (Cha and Bezanilla, 1997, 1998; Cha et al, 1999; Raghuraman et al, 2014), inward-rectifying potassium channels (Wang et al, 2016, 2018, 2019), mechanosensitive ion channels (Wang et al, 2014; Martinac, 2017), ligand-gated ion channels (Sasmal and Lu, 2014), membrane transporters (Liu and Sharom, 1996; Verhalen et al, 2012; Terry et al, 2018), and intrinsically disordered proteins (Ferreon et al, 2009). Therefore, the wide applicability of SDFL approaches to study diverse systems makes fluorescence a sophisticated yet reliable technique for ensemble and single molecule measurements in both in vitro and in vivo .…”

Section: Site-directed Fluorescence (Sdfl) Approachesmentioning

confidence: 99%

Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins

Raghuraman

Chatterjee

Das

2019

Front. Mol. Biosci.

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Membrane proteins mediate a number of cellular functions and are associated with several diseases and also play a crucial role in pathogenicity. Due to their importance in cellular structure and function, they are important drug targets for ~60% of drugs available in the market. Despite the technological advancement and recent successful outcomes in determining the high-resolution structural snapshot of membrane proteins, the mechanistic details underlining the complex functionalities of membrane proteins is least understood. This is largely due to lack of structural dynamics information pertaining to different functional states of membrane proteins in a membrane environment. Fluorescence spectroscopy is a widely used technique in the analysis of functionally-relevant structure and dynamics of membrane protein. This review is focused on various site-directed fluorescence (SDFL) approaches and their applications to explore structural information, conformational changes, hydration dynamics, and lipid-protein interactions of important classes of membrane proteins that include the pore-forming peptides/proteins, ion channels/transporters and G-protein coupled receptors.

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“…Membrane proteins are also labeled simultaneously with two distinct biophysical reporters to investigate their conformational transitions through FRET (Taraska, 2012; Taraska and Zagotta, 2010) or to independently track structural rearrangements in two different regions of the protein (Kalstrup and Blunck, 2013; Kalstrup and Blunck, 2018). Thus far, two-color labeling of membrane proteins has been achieved using pairs of cysteine residues (Glauner et al, 1999; Koch, 2005; Posson and Selvin, 2008; Wang et al, 2018), where it is difficult to monitor the site-specific attachment of fluorophores and often suffers from complexities arising from mixed populations of proteins containing one or both fluorophores. Cysteine mutagenesis has also been combined with fluorescently-labeled ligands (Posson and Selvin, 2008), transition metal binding sites (Billesbølle et al, 2016; Taraska et al, 2009), lanthanide metal binding peptide tags (Vázquez-Ibar et al, 2002) or fluorescent non-canonical amino acids (Gordon et al, 2018; Kalstrup and Blunck, 2013) to achieve site-specific labeling of membrane proteins with two different biophysical reporters.…”

Section: Resultsmentioning

confidence: 99%

Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis

Gupta

Toombes

Swartz

2019

eLife

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The functional mechanisms of membrane proteins are extensively investigated with cysteine mutagenesis. To complement cysteine-based approaches, we engineered a membrane protein with thiol-independent crosslinkable groups using azidohomoalanine (AHA), a non-canonical methionine analogue containing an azide group that can selectively react with cycloalkynes through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We demonstrate that AHA can be readily incorporated into the Shaker Kv channel in place of methionine residues and modified with azide-reactive alkyne probes in Xenopus oocytes. Using voltage-clamp fluorometry, we show that AHA incorporation permits site-specific fluorescent labeling to track voltage-dependent conformational changes similar to cysteine-based methods. By combining AHA incorporation and cysteine mutagenesis in an orthogonal manner, we were able to site-specifically label the Shaker Kv channel with two different fluorophores simultaneously. Our results identify a facile and straightforward approach for chemical modification of membrane proteins with bioorthogonal chemistry to explore their structure-function relationships in live cells.

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Studying Structural Dynamics of Potassium Channels by Single-Molecule FRET

Cited by 14 publications

References 16 publications

Potassium channel selectivity filter dynamics revealed by single-molecule FRET

Potassium channel selectivity filter dynamics revealed by single-molecule FRET

Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins

Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis

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