scholarly journals Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ling Wang ◽  
Zachary Lee Johnson ◽  
Michael R Wasserman ◽  
Jesper Levring ◽  
Jue Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Multidrug Resistance Protein 1 ◽  
Rate Limiting Step ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Resistance Protein ◽  
Rate Limiting

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

scholarly journals Nanoscale Dynamics of Cellulase TrCel7A Digesting Cellulose

2021 ◽  
Author(s):  
Zachary K. Haviland ◽  
Daguan Nong ◽  
Kate L. Vasquez Kuntz ◽  
Thomas J. Starr ◽  
Dengbo Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cellulose Degradation ◽  
Average Distance ◽  
Static State ◽  
Single Molecule Tracking ◽  
Rate Limiting Step ◽  
Three State Model ◽  
Enzyme Molecules ◽  
20 Nm ◽  
Rate Limiting

AbstractUnderstanding how cellulases catalyze the digestion of lignocellulose is a major goal of bioenergy research. Cel7A fromTrichoderma reeseiis a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. We used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeledTrCel7A binding to and moving processively along immobilizedGluconoacetobactercellulose. Enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzymes bound into a static state and dissociated without detectable movement. Processive enzymes moved an average distance of 39 nm at an average speed of 3.2 nm/s. Static binding episodes preceding and following processive runs were of similar duration to static binding events that lacked any processive movement. Transient jumps of >20 nm were observed, but no diffusive behavior indicative of a diffusive search of the enzyme for a free cellulose strand end was observed. These data were integrated into a three-state model in whichTrCel7A molecules can bind from solution into either a static or a processive state, and can reversibly switch between static and processive states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state either by dissociation from the cellulose surface or initiation of a processive run.

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.

scholarly journals Identification of human multidrug resistance protein 1 (MRP1) mutations and characterization of a G671V substitution

2001 ◽  
Vol 46 (11) ◽  
pp. 656-663 ◽  
Author(s):  
S. Conrad ◽  
H.-M. Kauffmann ◽  
K. Ito ◽  
R. G. Deeley ◽  
S. P. C. Cole ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Multidrug Resistance Protein ◽  
Multidrug Resistance Protein 1 ◽  
Resistance Protein

Myosin learns to walk

2001 ◽  
Vol 114 (11) ◽  
pp. 1981-1998
Author(s):  
Amit Mehta
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Kinetic Scheme ◽  
Myosin V ◽  
Step Size ◽  
Class V ◽  
Adp Release ◽  
Solution Kinetic ◽  
Conventional Kinesin ◽  
Rate Limiting

Recent experiments, drawing upon single-molecule, solution kinetic and structural techniques, have clarified our mechanistic understanding of class V myosins. The findings of the past two years can be summarized as follows: (1) Myosin V is a highly efficient processive motor, surpassing even conventional kinesin in the distance that individual molecules can traverse. (2) The kinetic scheme underlying ATP turnover resembles those of myosins I and II but with rate constants tuned to favor strong binding to actin. ADP release precedes dissociation from actin and is rate-limiting in the cycle. (3) Myosin V walks in strides averaging ∼36 nm, the long pitch pseudo-repeat of the actin helix, each step coupled to a single ATP hydrolysis. Such a unitary displacement, the largest molecular step size measured to date, is required for a processive myosin motor to follow a linear trajectory along a helical actin track.

Wavelet Based Structural Analysis of Electroless Deposits in the Diffusion Limited Regime

1994 ◽  
Vol 367 ◽  
Author(s):  
A. Arneodo ◽  
F. Argoul ◽  
A. Kuhn ◽  
J.F. Muzy
Keyword(s):  
Electroless Deposition ◽  
Side Reactions ◽  
Statistical Features ◽  
Diffusion Limited ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Fractal Patterns ◽  
Rate Limiting ◽  
Comparative Structural Characterization

AbstractWe discuss the actual relevance of thin gap geometry electrodeposition to generate fractal patterns that mimic the morphology of Witten and Sander's diffusion-limited aggregates (DLA). Eliminating migration and convection, as well as electrochemical side reactions, we show that electroless deposition is a good candidate to meet the requirements for diffusion to be the rate limiting step of the growth process. We use the wavelet transform microscope to achieve a comparative structural characterization of both experimental electroless deposits and numerical DLA clusters. The fact that five-fold symmetry and Fibonacci hierarchical ordering are found as common predominant statistical features is, to our knowledge, the first demonstration, relying on an appropriate structural fractal analysis, of the existence of DLA morphologies in an experimental context.

scholarly journals Single-molecule observation of nucleotide induced conformational changes in basal SecA-ATP hydrolysis

2018 ◽  
Vol 4 (10) ◽  
pp. eaat8797 ◽  
Author(s):  
Nagaraju Chada ◽  
Kanokporn Chattrakun ◽  
Brendan P. Marsh ◽  
Chunfeng Mao ◽  
Priya Bariya ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Conformational Dynamics ◽  
Real Space ◽  
Biochemical Activity ◽  
Force Microscopy ◽  
Atomic Force ◽  
Reversible Transitions ◽  
Space Measurement

SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.

Vapor Transport Deposition and Characterization of Polycrystalline CdTe Solar Absorbers

2003 ◽  
Vol 763 ◽  
Author(s):  
James M. Kestner ◽  
Sarah McElvain ◽  
Colin A. Wolden ◽  
Stephen Kelly ◽  
Tim R. Ohno ◽  
...  
Keyword(s):  
Vapor Transport ◽  
Buffer Layers ◽  
High Rate ◽  
X Ray Diffraction ◽  
Solar Absorbers ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Substrate Temperatures ◽  
Vapor Transport Deposition

AbstractVapor transport deposition is being developed for high-rate synthesis of CdTe thin films. Films have been deposited at rates in excess of 20 μm/min. Thegrowth ratedependenceon source temperature yielded an apparent activation energy of 42 kcal/mol, in good agreement with the theoretical value for CdTe sublimation (45.7 kcal/mol). For substrate temperatures greater than 400°C the rate limiting step was resublimation. This phenomenon had a dramatic influence on morphology, although x-ray diffraction of all films indicated a strong (111) orientation. A preliminary device optimization investigating the effect of CdTe deposition temperature, post-deposition CdCl2 anneal parameters, alternative back contacts, and high-resistance buffer layers yielded a best cell with efficiency of 9.8% (704 mV Voc, 21.0mA/cm2 Jsc, 66% FF).

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