scholarly journals Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

2015 ◽  
Vol 112 (15) ◽  
pp. 4660-4665 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Ewa Prochniewicz ◽  
David D. Thomas
Keyword(s):  
Light Chain ◽  
Contractile Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Efficiency ◽  
Power Stroke ◽  
Time Resolved ◽  
Muscle Disorders ◽  
Essential Light Chain ◽  
C Terminus

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.

scholarly journals Actin-Myosin Interaction: Structure, Function and Drug Discovery

2018 ◽  
Vol 19 (9) ◽  
pp. 2628 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Ewa Prochniewicz ◽  
David Thomas
Keyword(s):  
Fluorescent Probe ◽  
Light Chain ◽  
High Performance ◽  
Catalytic Domain ◽  
Contractile Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Muscle Disorders ◽  
Myosin Interaction

Actin-myosin interactions play crucial roles in the generation of cellular force and movement. The molecular mechanism involves structural transitions at the interface between actin and myosin’s catalytic domain, and within myosin’s light chain domain, which contains binding sites for essential (ELC) and regulatory light chains (RLC). High-resolution crystal structures of isolated actin and myosin, along with cryo-electron micrographs of actin-myosin complexes, have been used to construct detailed structural models for actin-myosin interactions. However, these methods are limited by disorder, particularly within the light chain domain, and they do not capture the dynamics within this complex under physiological conditions in solution. Here we highlight the contributions of site-directed fluorescent probes and time-resolved fluorescence resonance energy transfer (TR-FRET) in understanding the structural dynamics of the actin-myosin complex in solution. A donor fluorescent probe on actin and an acceptor fluorescent probe on myosin, together with high performance TR-FRET, directly resolves structural states in the bound actin-myosin complex during its interaction with adenosine triphosphate (ATP). Results from these studies have profound implications for understanding the contractile function of actomyosin and establish the feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Time-Resolved Fluorescence Resonance Energy Transfer Shows that the Bacterial Multidrug ABC Half-Transporter BmrA Functions as a Homodimer†

Biochemistry ◽  
2005 ◽  
Vol 44 (11) ◽  
pp. 4312-4321 ◽  
Author(s):  
Olivier Dalmas ◽  
Marie-Ange Do Cao ◽  
Miguel R. Lugo ◽  
Frances J. Sharom ◽  
Attilio Di Pietro ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance

scholarly journals Role of metAB in Methionine Metabolism and Optimal Chicken Colonization in Campylobacter jejuni

2020 ◽  
Vol 89 (1) ◽  
pp. e00542-20
Author(s):  
Brandon Ruddell ◽  
Alan Hassall ◽  
Orhan Sahin ◽  
Qijing Zhang ◽  
Paul J. Plummer ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Differential Distribution ◽  
Time Resolved ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Adenosyl Methionine ◽  
Strain Type

ABSTRACTCampylobacter jejuni is a zoonotic pathogen and is one of the leading causes of human gastroenteritis worldwide. C. jejuni IA3902 (representative of the sheep abortion clone) is genetically similar to C. jejuni W7 (representative of strain type NCTC 11168); however, there are significant differences in the ability of luxS mutants of these strains to colonize chickens. LuxS is essential for the activated methyl cycle and generates homocysteine for conversion to l-methionine. Comparative genomics identified differential distribution of the genes metA and metB, which function to convert homoserine for downstream production of l-methionine, between IA3902 and W7, which could enable a secondary pathway for l-methionine biosynthesis in a W7 ΔluxS but not in an IA3902 ΔluxS strain. To test the hypothesis that the genes metA and metB contribute to l-methionine production and chicken colonization by Campylobacter, we constructed two mutants for phenotypic comparison, the W7 ΔmetAB ΔluxS and IA3902 ΔluxS::metAB mutants. Quantitative reverse transcription-PCR and tandem mass spectrometry protein analysis were used to validate MetAB transcription and translation as present in the IA3902 ΔluxS::metAB mutant and absent in the W7 ΔmetAB ΔluxS mutant. Time-resolved fluorescence resonance energy transfer fluorescence assays demonstrated that l-methionine and S-adenosyl methionine concentrations decreased in the W7 ΔmetAB ΔluxS mutant and increased in the IA3902 ΔluxS::metAB mutant. Assessment of chicken colonization revealed that the IA3902 ΔluxS::metAB strain partially rescued the colonization defect of the IA3902 ΔluxS strain, while the W7 ΔmetAB ΔluxS strain showed significantly decreased colonization compared to that of the wild-type and the W7 ΔluxS strain. These results indicate that the ability to maintain l-methionine production in vivo, conferred by metA and metB in the absence of luxS, is critical for normal chicken colonization by C. jejuni.

scholarly journals The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

2020 ◽  
Author(s):  
Jiaxing Chen ◽  
Sofia Zaer ◽  
Paz Drori ◽  
Joanna Zamel ◽  
Khalil Joron ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Structural Heterogeneity ◽  
Conformational Space ◽  
Conformational Ensemble ◽  
Time Resolved ◽  
Intrinsically Disordered ◽  
Transient Nature

AbstractThe intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

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