scholarly journals Ca2+ regulation of conformational states in the transport cycle of spin-labeled sarcoplasmic reticulum ATPase.

1979 ◽  
Vol 254 (8) ◽  
pp. 2968-2974
Author(s):  
C. Coan ◽  
S. Verjovski-Almeida ◽  
G. Inesi
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Conformational States ◽  
Transport Cycle

Cyclopiazonic Acid Effect on Ca2+-Dependent Conformational States of the Sarcoplasmic Reticulum ATPase. Implication for the Enzyme Turnover†

Biochemistry ◽  
1998 ◽  
Vol 37 (12) ◽  
pp. 4266-4274 ◽  
Author(s):  
Fernando Soler ◽  
Fernando Plenge-Tellechea ◽  
Isabel Fortea ◽  
Francisco Fernandez-Belda
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Cyclopiazonic Acid ◽  
Conformational States ◽  
Acid Effect ◽  
Enzyme Turnover

Watching conformational dynamics of ABC transporters with single-molecule tools

2015 ◽  
Vol 43 (5) ◽  
pp. 1041-1047 ◽  
Author(s):  
Florence Husada ◽  
Giorgos Gouridis ◽  
Ruslan Vietrov ◽  
Gea K. Schuurman-Wolters ◽  
Evelyn Ploetz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Abc Transporters ◽  
Fluorescent Dyes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Cell Volume Regulation ◽  
Size Exclusion ◽  
Conformational States ◽  
Functional Interpretation ◽  
Transport Cycle

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

scholarly journals Transient conformers of LacY are trapped by nanobodies

2015 ◽  
Vol 112 (45) ◽  
pp. 13839-13844 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Xiaoxu Jiang ◽  
Els Pardon ◽  
Jan Steyaert ◽  
...  
Keyword(s):  
Lactose Permease ◽  
Conformational States ◽  
Dynamic Membrane ◽  
Conformational Selection ◽  
Sugar Binding ◽  
Open Conformation ◽  
Transport Cycle ◽  
Fluorescence Change ◽  
Alternating Access ◽  
Selection Of

The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H+ by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.

scholarly journals Structural capture of an intermediate transport state of a CLC CI-/H+ antiporter

2018 ◽  
Author(s):  
Kunwoong Park ◽  
Hyun-Ho Lim
Keyword(s):  
Crystal Structures ◽  
Conformational Heterogeneity ◽  
Conformational States ◽  
Cellular Processes ◽  
Chloride Homeostasis ◽  
Transport Cycle ◽  
Multiple Conformations ◽  
Structural Levels

AbstractThe CLC family proteins are involved in a variety of cellular processes, where chloride homeostasis needs to be controlled. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Recent studies have revealed that the conformational heterogeneity of the critical glutamate residue, Gluex could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations of the Gluex has been suggested from combined structural snapshots of two different CLC antiporters. Thus, we aimed to investigate the presence of these three intermediate conformations in CLC-ec1, the most deeply studied CLC at both functional and structural levels. By comparing crystal structures of E148D, E148A mutant and wildtype CLC-ec1 with varying anion concentrations, we suggest that the Gluex indeed take at least three distinct conformational states in a single CLC antiporter, CLC-ec1.

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