scholarly journals Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes

2017 ◽  
Vol 139 (33) ◽  
pp. 11559-11567 ◽  
Author(s):  
Ting Zeng ◽  
Silke Leimkühler ◽  
Ulla Wollenberger ◽  
Vincent Fourmond
Keyword(s):  
Conformational Changes ◽  
Sulfite Oxidase ◽  
Rate Limiting

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

scholarly journals A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip

2016 ◽  
Vol 149 (1) ◽  
pp. 85-103 ◽  
Author(s):  
Shaweta Gupta ◽  
Srirupa Chakraborty ◽  
Ridhima Vij ◽  
Anthony Auerbach
Keyword(s):  
Conformational Changes ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Bubble Formation ◽  
Transmembrane Helix ◽  
Energy Landscapes ◽  
Rate Limiting Step ◽  
Sequence Domain ◽  
Rate Limiting

Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing (“gating”) between resting and active conformations under the influence of neurotransmitters. Here, to explore the mechanisms that link the transmitter-binding sites (TBSs) with the distant gate, we use mutant cycle analyses to measure coupling between residue pairs, phi value analyses to sequence domain rearrangements, and current simulations to reproduce a microsecond shut component (“flip”) apparent in single-channel recordings. Significant interactions between amino acids separated by >15 Å are rare; an exception is between the αM2–M3 linkers and the TBSs that are ∼30 Å apart. Linker residues also make significant, local interactions within and between subunits. Phi value analyses indicate that without agonists, the linker is the first region in the protein to reach the gating transition state. Together, the phi pattern and flip component suggest that a complete, resting↔active allosteric transition involves passage through four brief intermediate states, with brief shut events arising from sojourns in all or a subset. We derive energy landscapes for gating with and without agonists, and propose a structure-based model in which resting→active starts with spontaneous rearrangements of the M2–M3 linkers and TBSs. These conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a “bubble” that collapses to initiate ion conduction. The energy landscapes suggest that twisting is the most energetically unfavorable step in the resting→active conformational change and that the rate-limiting step in the reverse process is bubble formation.

CDC20 assists its catalytic incorporation in the mitotic checkpoint complex

Science ◽  
2020 ◽  
Vol 371 (6524) ◽  
pp. 67-71 ◽  
Author(s):  
Valentina Piano ◽  
Amal Alex ◽  
Patricia Stege ◽  
Stefano Maffini ◽  
Gerardo A. Stoppiello ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mitotic Checkpoint ◽  
Catalytic Complex ◽  
Controlled Systems ◽  
Cellular Pathways ◽  
Mitotic Checkpoint Complex ◽  
Rate Limiting

Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.

scholarly journals Maximum Entropy Production Theorem for Transitions between Enzyme Functional States and Its Applications

Entropy ◽  
2019 ◽  
Vol 21 (8) ◽  
pp. 743 ◽  
Author(s):  
Davor Juretić ◽  
Juraj Simunić ◽  
Željana Bonačić Lošić
Keyword(s):  
Free Energy ◽  
Entropy Production ◽  
Conformational Changes ◽  
Triosephosphate Isomerase ◽  
Biological Evolution ◽  
Maximal Entropy ◽  
Total Entropy ◽  
Functional States ◽  
Rate Limiting ◽  
Total Entropy Production

Transitions between enzyme functional states are often connected to conformational changes involving electron or proton transport and directional movements of a group of atoms. These microscopic fluxes, resulting in entropy production, are driven by non-equilibrium concentrations of substrates and products. Maximal entropy production exists for any chosen transition, but such a maximal transitional entropy production (MTEP) requirement does not ensure an increase of total entropy production, nor an increase in catalytic performance. We examine when total entropy production increases, together with an increase in the performance of an enzyme or bioenergetic system. The applications of the MTEP theorem for transitions between functional states are described for the triosephosphate isomerase, ATP synthase, for β-lactamases, and for the photochemical cycle of bacteriorhodopsin. The rate-limiting steps can be easily identified as those which are the most efficient in dissipating free-energy gradients and in performing catalysis. The last step in the catalytic cycle is usually associated with the highest free-energy dissipation involving proton nanocurents. This recovery rate-limiting step can be optimized for higher efficiency by using corresponding MTEP requirements. We conclude that biological evolution, leading to increased optimal catalytic efficiency, also accelerated the thermodynamic evolution, the synergistic relationship we named the evolution-coupling hypothesis.

scholarly journals Conformational Changes Represent the Rate-Limiting Step in the Transport Cycle of Maize SUCROSE TRANSPORTER1

2013 ◽  
Vol 25 (8) ◽  
pp. 3010-3021 ◽  
Author(s):  
Carmen Derrer ◽  
Anke Wittek ◽  
Ernst Bamberg ◽  
Armando Carpaneto ◽  
Ingo Dreyer ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Transport Cycle ◽  
Rate Limiting

scholarly journals Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qin Yu ◽  
Dongchun Ni ◽  
Julia Kowal ◽  
Ioannis Manolaridis ◽  
Scott M. Jackson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Reaction Cycle ◽  
Rate Limiting Step ◽  
Exogenous Substrate ◽  
Endogenous Substrate ◽  
Inhibitory Antibodies ◽  
Rate Limiting ◽  
Drug Pharmacokinetics ◽  
Cytoplasmic Side

AbstractABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.

Structure-function relationships in an anion-translocating ATPase

2000 ◽  
Vol 28 (4) ◽  
pp. 520-526
Author(s):  
H. Bhattacharjee ◽  
T. Zhou ◽  
J. Li ◽  
D. L. Gatti ◽  
A. R. Walmsley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Abc Transporters ◽  
Efflux Pump ◽  
Allosteric Activation ◽  
Ancestral Gene ◽  
Binding Domains ◽  
Fluorescence Measurements ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette (‘ABC’) transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.

scholarly journals Weakening the IF2-fMet-tRNA Interaction Suppresses the Lethal Phenotype Caused by GTPase Inactivation

2021 ◽  
Vol 22 (24) ◽  
pp. 13238
Author(s):  
Jerneja Tomsic ◽  
Enrico Caserta ◽  
Cynthia L. Pon ◽  
Claudio O. Gualerzi
Keyword(s):  
Conformational Changes ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Gtp Hydrolysis ◽  
Lethal Phenotype ◽  
Dominant Lethal ◽  
Thermally Driven ◽  
Rate Limiting ◽  
Late Stages

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.

scholarly journals Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1

2020 ◽  
Author(s):  
Yanxin Liu ◽  
Ming Sun ◽  
Alexander G. Myasnikov ◽  
Daniel Elnatan ◽  
Nicolas Delaeter ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Activation Mechanism ◽  
Client Protein ◽  
Protein Maturation ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Client Proteins ◽  
Rate Limiting

AbstractHsp90 is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of cellular “client” proteins. The ATP-driven client maturation process is regulated by a large number of co-chaperones. Among them, Aha1 is the most potent activator of Hsp90 ATPase activity and thus dramatically affects Hsp90’s client proteins. To understand the Aha1 activation mechanism, we determined full-length Hsp90:Aha1 structures in six different states by cryo-electron microscopy, including nucleotide-free semi-closed, nucleotide-bound pre-hydrolysis, and semi-hydrolyzed states. Our structures demonstrate that the two Aha1 domains can each interact with Hsp90 in two different modes, uncovering a complex multistep activation mechanism. The results show that Aha1 accelerates the chemical step of ATP hydrolysis like a conventional enzyme, but most unusually, catalyzes the rate-limiting large-scale conformational changes of Hsp90 fundamentally required for ATP hydrolysis. Our work provides a structural framework to guide small molecule development targeting this critical modulator of client protein maturation.

scholarly journals Kinetic Relationship between the Voltage Sensor and the Activation Gate in spHCN Channels

2007 ◽  
Vol 130 (1) ◽  
pp. 71-81 ◽  
Author(s):  
Andrew Bruening-Wright ◽  
Fredrik Elinder ◽  
H. Peter Larsson
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Ionic Current ◽  
Current Data ◽  
Terminal Region ◽  
Hcn Channels ◽  
Mode Shift ◽  
Channel Opening ◽  
Rate Limiting ◽  
Voltage Activation

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarizations that cause an inward movement of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. The mechanism of how the motion of S4 charges triggers channel opening is unknown. Here, we used voltage clamp fluorometry (VCF) to detect S4 conformational changes and to correlate these to the different activation steps in spHCN channels. We show that S4 undergoes two distinct conformational changes during voltage activation. Analysis of the fluorescence signals suggests that the N-terminal region of S4 undergoes conformational changes during a previously characterized mode shift in HCN channel voltage dependence, while a more C-terminal region undergoes an additional conformational change during gating charge movements. We fit our fluorescence and ionic current data to a previously proposed 10-state allosteric model for HCN channels. Our results are not compatible with a fast S4 motion and rate-limiting channel opening. Instead, our data and modeling suggest that spHCN channels open after only two S4s have moved and that S4 motion is rate limiting during voltage activation of spHCN channels.

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