scholarly journals Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA

2021 ◽  
Vol 22 (2) ◽  
pp. 490
Author(s):  
Ana Marcela Giudici ◽  
Clara Díaz-García ◽  
Maria Lourdes Renart ◽  
Ana Coutinho ◽  
Manuel Prieto ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Selectivity Filter ◽  
Channel State ◽  
X Ray Crystallography ◽  
Combined Use ◽  
Alkylammonium Salts ◽  
And Function ◽  
Shed Light

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+–KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67–W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+–bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.

scholarly journals Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Katie D. Hickey ◽  
Mary M. Buhr
Keyword(s):  
Lipid Bilayer ◽  
Lipid Composition ◽  
Protein Function ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Outer Leaflet ◽  
Lipid Environment ◽  
Protein Incorporation ◽  
And Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+K+-ATPase or -amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+K+-ATPase within the membranes.

scholarly journals Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

2018 ◽  
Vol 115 (40) ◽  
pp. 9923-9928 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Manu Ben Johny ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Single Type ◽  
Regulatory Subunits ◽  
And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

scholarly journals Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 8 ◽  
Author(s):  
Laura Keller ◽  
Nicolas Bery ◽  
Claudine Tardy ◽  
Laetitia Ligat ◽  
Gilles Favre ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Phage Display Library ◽  
Small Gtpases ◽  
Intracellular Expression ◽  
Co Precipitation ◽  
Cytoskeleton Dynamics

RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.

scholarly journals Methods used to study the oligomeric structure of G-protein-coupled receptors

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Hui Guo ◽  
Su An ◽  
Richard Ward ◽  
Yang Yang ◽  
Ying Liu ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Experimental Techniques ◽  
Distribution Analysis ◽  
Wide Range ◽  
And Function ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.

scholarly journals A global multi-technique approach to study low-resolution solution structures

2005 ◽  
Vol 38 (6) ◽  
pp. 874-887 ◽  
Author(s):  
Marcelo Nöllmann ◽  
W. Marshall Stark ◽  
Olwyn Byron
Keyword(s):  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Optimization Method ◽  
Solution Structures ◽  
X Ray Crystallography ◽  
Angle Scattering ◽  
Monte Carlo Simulated Annealing

Finding the conformation of large macromolecular complexes has become an important problem in structural biology, which is not always soluble by high-resolution techniques such as X-ray crystallography and NMR spectroscopy. Solution biophysical properties can provide direct or indirect structural information on these large complexes. A general systematic approach to the construction of a structural model of the macromolecule consistent with all the experimental solution properties is currently lacking. In this paper, such an approach is presented, where generalized rigid-body modelling is combined with a Monte Carlo/simulated-annealing optimization method, to search over a large range of possible conformations for the structure that best fits solution experimental properties derived from small-angle scattering, fluorescence resonance energy transfer and analytical ultracentrifugation.

scholarly journals Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Yiwei Meng ◽  
Zhou Yang ◽  
Bin Cheng ◽  
Xinyu Nie ◽  
Shannan Li ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Ph Regulation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Limited Information ◽  
High Sequence Identity ◽  
Ph Profile ◽  
Content Type ◽  
Ion Translocation ◽  
And Function

ABSTRACT Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na+ and Li+ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na+ and Li+ expulsion, as well as complementary growth following exposure to 300 mM Na+ and 100 mM Li+ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile. IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu38, in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.

scholarly journals Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids

2019 ◽  
Author(s):  
Bijoy J. Desai ◽  
Ruben L. Gonzalez
Keyword(s):  
Amino Acids ◽  
Structural Biology ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Studies ◽  
Bioorthogonal Labeling ◽  
Biomolecular Complexes ◽  
And Function

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

scholarly journals Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Stijn van Dorp ◽  
Ruoyi Qiu ◽  
Ucheor B Choi ◽  
Minnie M Wu ◽  
Michelle Yen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Structural Basis ◽  
Resting Cells ◽  
Dual Roles ◽  
Er Calcium

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

scholarly journals Actin-binding compounds, discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

2020 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Lien A. Phung ◽  
Ewa Prochniewicz ◽  
Sarah Lichtenberger ◽  
Anna Wilson ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Striated Muscle ◽  
Muscle Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structure And Function ◽  
Actin Binding ◽  
Myosin Isoforms ◽  
And Function

AbstractWe have used spectroscopic and functional assays to evaluate the effects of a group of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. A previous high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay from our group identified a class of compounds that bind to actin and affect actomyosin structure and function. In the current study, we tested their effects on the two isoforms of striated muscle α-actins, skeletal and cardiac. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that these effects were different for the two actin isoforms, suggesting a different mode of action. To determine the effects of these compounds on sarcomeric function, we further tested their activity on skeletal and cardiac myofibrils. We found that several compounds affected ATPase activity of skeletal and cardiac myofibrils differently, suggesting different mechanisms of action of these compounds for the two muscle types. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

scholarly journals Probing the molecular mechanism of action of Deinococcus radiodurans RecD2 at single-molecule resolution

2021 ◽  
Author(s):  
Deb Purkait ◽  
Farhana Islam ◽  
Padmaja P. Mishra
Keyword(s):  
Single Molecule ◽  
Deinococcus Radiodurans ◽  
Excision Repair ◽  
Structural Characteristics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Unwinding ◽  
X Ray Crystallography ◽  
Domains Of Life ◽  
Molecular Mechanism Of Action

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. Helicases are responsible for resolving double-stranded DNA (dsDNA) into separate single-strands and this activity is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions towards its unusually high tolerance to gamma radiation and hydrogen peroxide. Although previous X-ray Crystallography studies have revealed the structural characteristics of the protein, the direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5 prime terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.

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