Visualizing conformational dynamics of proteins in solution and at the cell membrane

Gordon, Sharona E.; Munari, Mika; Zagotta, William N.

doi:10.7554/elife.37248

Cited by 49 publications

(110 citation statements)

References 46 publications

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“…3d). The FRET efficiencies decreased with distance, but as previously observed for tmFRET 20 , the distance dependence was shallower than predicted by the Förster equation. This shallower distance dependence has been attributed, in part, to heterogeneity of the interatomic distances in proteins.…”

supporting

confidence: 77%

“…The FRET efficiencies produced by E1 and E2 were similar because of the low degree of background quenching 20,36 (Extended Data Fig. 6).…”

Section: Ementioning

confidence: 82%

“…2a and Fig. 3a) 20 . TETAC is a cysteine-reactive compound with a short linker to a cyclen ring that binds transition metal ions with subnanomolar affinity 21 .…”

mentioning

confidence: 88%

“…For ACCuRET experiments, stabilization buffer (SBT) (130 mM KCl; 30 mM Trizma Base; 0.2 mM EDTA, pH 7.4) was used for the bath solution. The Cu 2+ -TETAC was prepared as previously described 20 . TETAC (Toronto Research Chemicals, Toronto, Canada) was made as a 100 mM stock in DMSO and stored at −20°C.…”

Section: Oocyte Expression and Electrophysiologymentioning

confidence: 99%

“…2a). These advantages recently enabled the accurate measurement of absolute distances and distance changes in protein backbones that are associated with structural rearrangements in soluble and membrane proteins 20 .…”

mentioning

confidence: 99%

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The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

Dai

Aman

DiMaio

et al. 2019

Preprint

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Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body 1 . They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells 1 . In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration 2 . VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore 3-6 . These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD) 3 and within the membrane's electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization 7 . HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization 8-10 . The mechanism underlying this "reverse gating" is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels 11 , no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.

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supporting

confidence: 77%

“…The FRET efficiencies produced by E1 and E2 were similar because of the low degree of background quenching 20,36 (Extended Data Fig. 6).…”

Section: Ementioning

confidence: 82%

“…2a and Fig. 3a) 20 . TETAC is a cysteine-reactive compound with a short linker to a cyclen ring that binds transition metal ions with subnanomolar affinity 21 .…”

mentioning

confidence: 88%

Section: Oocyte Expression and Electrophysiologymentioning

confidence: 99%

mentioning

confidence: 99%

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The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

Dai

Aman

DiMaio

et al. 2019

Preprint

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Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

Kyrychenko,

Ladokhin

2023

The Chemical Record

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Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence‐based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information. Here, we review the fundamentals and summarize the latest progress in applications of different classes of fluorescent probes and their specific quenchers, aimed at studies of protein folding and protein‐membrane interactions. Specifically, we discuss various environment‐sensitive dyes, FRET probes, probes for short‐distance measurements, and several probe‐quencher pairs for studies of membrane penetration of proteins and peptides. The goals of this review are: (a) to familiarize the readership with the general concept that complex biological systems often require both a probe and a quencher to decipher mechanistic details of functioning and (b) to provide example of the immediate applications of the described methods.

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Dynamics and Hydration of Proteins Viewed by Fluorescence Methods: Investigations for Protein Engineering and Synthetic Biology

Sýkora

Prokop

Damborský

et al. 2022

Springer Series on Fluorescence

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Protein engineering and synthetic biology are currently very active areas of research and development. In the pursuit of engineering proteins with specific capabilities, it has become evident that the scrutiny of structural and geometrical properties does not suffice to achieve the proposed goals. The dynamics and hydration of specific protein areas seem to be of higher influence than it has been once thought. This chapter introduces three different fluorescence spectroscopy techniques (time-dependent fluorescent shift, HMC hydration assay based on unnatural amino acid fluorescence, and photoinduced electron transfer–fluorescence correlation spectroscopy) that allow for assessing the dynamics and hydration of proteins in a site-specific fashion and showcase their usefulness in advancing the design of more efficient enzymes. Systematic application of these techniques to various biomolecular systems will allow a thorough description of these important protein properties, which are rarely taken into account during protein engineering and synthetic biology projects.

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Visualizing conformational dynamics of proteins in solution and at the cell membrane

Cited by 49 publications

References 46 publications

The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

Dynamics and Hydration of Proteins Viewed by Fluorescence Methods: Investigations for Protein Engineering and Synthetic Biology

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