scholarly journals Single-molecule observations of neck linker conformational changes in the kinesin motor protein

2006 ◽  
Vol 13 (10) ◽  
pp. 887-894 ◽  
Author(s):  
Michio Tomishige ◽  
Nico Stuurman ◽  
Ronald D Vale
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Motor Protein ◽  
Kinesin Motor

scholarly journals Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

2020 ◽  
Author(s):  
Stefan Niekamp ◽  
Nico Stuurman ◽  
Nan Zhang ◽  
Ronald D. Vale
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
In Silico ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Motor Protein ◽  
Single Molecule Imaging ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule binding domains (MTBD) that step along the track. Here, we simultaneously tracked two MTBDs and one AAA ring of a single dynein, as it undergoes hundreds of steps with nanometer precision using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take different sized steps. This variability in the movement between AAA ring and MTBD results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

scholarly journals Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

2021 ◽  
Vol 118 (31) ◽  
pp. e2101391118
Author(s):  
Stefan Niekamp ◽  
Nico Stuurman ◽  
Nan Zhang ◽  
Ronald D. Vale
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
In Silico ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Motor Protein ◽  
Single Molecule Imaging ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

scholarly journals A Single-Molecule View on Kinesin Motor-Protein Cooperation in Intraflagellar Transport in Living C. elegans

2015 ◽  
Vol 108 (2) ◽  
pp. 599a
Author(s):  
Bram Prevo ◽  
Pierre J.J. Mangeol ◽  
Felix Oswald ◽  
Jonathan M. Scholey ◽  
Erwin J.G. Peterman
Keyword(s):  
Single Molecule ◽  
Motor Protein ◽  
Intraflagellar Transport ◽  
Kinesin Motor ◽  
C Elegans

Pneumatically-driven Microfluidic Device for Evaluating Active Transport by Kinesin Motor Protein

2016 ◽  
Vol 136 (9) ◽  
pp. 384-389
Author(s):  
Kazuya Fujimoto ◽  
Hirofumi Shintaku ◽  
Hidetoshi Kotera ◽  
Ryuji Yokokawa
Keyword(s):  
Active Transport ◽  
Microfluidic Device ◽  
Motor Protein ◽  
Kinesin Motor

scholarly journals Real-time analysis of RAG complex activity in V(D)J recombination

2016 ◽  
Vol 113 (42) ◽  
pp. 11853-11858 ◽  
Author(s):  
Jennifer Zagelbaum ◽  
Noriko Shimazaki ◽  
Zitadel Anne Esguerra ◽  
Go Watanabe ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Signal Sequence ◽  
High Mobility ◽  
Time Analysis ◽  
Complex Activity ◽  
Synaptic Complex ◽  
Single Molecule Fret ◽  
Real Time Analysis

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

scholarly journals Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

2014 ◽  
Vol 143 (4) ◽  
pp. 449-464 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outward Current ◽  
Side Chain ◽  
Ion Binding ◽  
Inward Current ◽  
Na Release ◽  
K Transport ◽  
External K

A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

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