scholarly journals Interaction of the Motor Protein SecA and the Bacterial Protein Translocation Channel SecYEG in the Absence of ATP

2019 ◽  
Author(s):  
Klemens Winkler ◽  
Andreas Karner ◽  
Andreas Horner ◽  
Christof Hannesschlaeger ◽  
Denis Knyazev ◽  
...  
Keyword(s):  
High Speed ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Resonance Energy Transfer ◽  
Motor Protein ◽  
Resonance Energy ◽  
Power Stroke ◽  
Secretory Proteins ◽  
Force Microscopy ◽  
Stroke Mechanism

ABSTRACTTranslocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecA’s two-helix finger into SecYEG’s aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

scholarly journals Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

2018 ◽  
Author(s):  
Tomas Fessl ◽  
Daniel Watkins ◽  
Peter Oatley ◽  
William J. Allen ◽  
Robin A. Corey ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Insertion ◽  
Initiation Phase ◽  
Dependent Transport

AbstractProtein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon – SecYMKEG – resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Förster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allenet al.doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

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.

scholarly journals Movements of Mycoplasma mobile gliding machinery detected by high-speed atomic force microscopy

2021 ◽  
Author(s):  
Kohei Kobayashi ◽  
Noriyuki Kodera ◽  
Taishi Kasai ◽  
Yuhei O Tahara ◽  
Takuma Toyonaga ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Internal Structure ◽  
Sodium Azide ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Solid Surfaces ◽  
Force Microscopy ◽  
Special Mechanism ◽  
Atomic Force ◽  
Internal Structures

ABSTRACTMycoplasma mobile, a parasitic bacterium, glides on solid surfaces, such as animal cells and glass by a special mechanism. This process is driven by the force generated through ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells are unclear. In this study, we detected the movements of the internal structure by scanning cells immobilized on a glass substrate using high-speed atomic force microscopy (HS-AFM). By scanning the surface of a cell, we succeeded in visualizing particles, 2 nm in hight and aligned mostly along the cell axis with a pitch of 31.5 nm, consistent with previously reported features based on electron microscopy. Movements of individual particles were then analyzed by HS-AFM. In the presence of sodium azide, the average speed of particle movements was reduced, suggesting that movement is linked to ATP hydrolysis. Partial inhibition of the reaction by sodium azide enabled us to analyze particle behavior in detail, showing that the particles move 9 nm right, relative to the gliding direction, and 2 nm into the cell interior in 330 ms, then return to their original position, based on ATP hydrolysis.IMPORTANCEThe Mycoplasma genus contains bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide by a special mechanism linked to their infection and survival. The special machinery for gliding can be divided into surface and internal structures that have evolved from rotary motors represented by ATP synthases. This study succeeded in visualizing the real-time movements of the internal structure by scanning from the outside of the cell using an innovative high-speed atomic force microscope, and then analyzing their behaviors.

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.

Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer

2009 ◽  
Vol 297 (2) ◽  
pp. C290-C298 ◽  
Author(s):  
Kristin Rule Gleitsman ◽  
Michihiro Tateyama ◽  
Yoshihiro Kubo
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Motor Protein ◽  
Resonance Energy ◽  
Outer Hair Cells ◽  
Nonlinear Capacitance ◽  
Fluorescence Resonance

Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The nonlinear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, of which its precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a fluorescence resonance energy transfer (FRET)-based technique. We heterologously expressed prestin labeled with fluorophores at the COOH- or NH2-terminus in human embryonic kidney-293T cells, and monitored FRET changes on depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the COOH-termini and between the COOH- and NH2-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish nonlinear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the COOH-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements.

scholarly journals Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis

2013 ◽  
Vol 288 (29) ◽  
pp. 20785-20796 ◽  
Author(s):  
Rebecca S. Cooper ◽  
Guillermo A. Altenberg
Keyword(s):  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Binding Domains ◽  
Partial Opening ◽  
Contact Models ◽  
Bacterial Lipid ◽  
Atp Binding Cassette

In ATP-binding cassette proteins, the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The intermonomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mm MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant contact models, for the mode of operation of ATP-binding cassette proteins.

scholarly journals Signal processing for molecular and cellular biological physics: an emerging field

2013 ◽  
Vol 371 (1984) ◽  
pp. 20110546 ◽  
Author(s):  
Max A. Little ◽  
Nick S. Jones
Keyword(s):  
Signal Processing ◽  
Optical Tweezers ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Digital Signal ◽  
Force Microscopy ◽  
Cellular Processes ◽  
Atomic Force ◽  
Non Gaussian ◽  
Autocorrelated Noise

Recent advances in our ability to watch the molecular and cellular processes of life in action—such as atomic force microscopy, optical tweezers and Forster fluorescence resonance energy transfer—raise challenges for digital signal processing (DSP) of the resulting experimental data. This article explores the unique properties of such biophysical time series that set them apart from other signals, such as the prevalence of abrupt jumps and steps, multi-modal distributions and autocorrelated noise. It exposes the problems with classical linear DSP algorithms applied to this kind of data, and describes new nonlinear and non-Gaussian algorithms that are able to extract information that is of direct relevance to biological physicists. It is argued that these new methods applied in this context typify the nascent field of biophysical DSP. Practical experimental examples are supplied.

High-Speed Fluorescence Microscopy: Lifetime Imaging in the Biomedical Sciences

1995 ◽  
Vol 1 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Ammasi Periasamy ◽  
Xue F. Wang ◽  
Pawel Wodnick ◽  
Gerald W. Gordon ◽  
Seongwook Kwon ◽  
...  
Keyword(s):  
High Speed ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hpv Infection ◽  
Free Calcium ◽  
Fluorescence Lifetime Imaging ◽  
Biomedical Sciences ◽  
Fluorescence Excitation ◽  
Lifetime Imaging ◽  
Microscopic Techniques

The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past 30 to 40 years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena that occur over very short time periods (nanosecond to millisecond) or over short distances (10 to 1000 Å). However, the recent combination of (1) very rapidly gated and sensitive image intensifiers and (2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses the implementation of a fluorescence lifetime imaging microscope (FLIM) and provides a review of some of the applications of such an instrument. These include measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca2+)i and the detection of human papillomavirus (HPV) infection in clinical cervicovaginal smears.

scholarly journals Chained structure of dimeric F1-like ATPase in Mycoplasma mobile gliding machinery

2021 ◽  
Author(s):  
Takuma Toyonaga ◽  
Takayuki Kato ◽  
Akihiko Kawamoto ◽  
Noriyuki Kodera ◽  
Tasuku Hamaguchi ◽  
...  
Keyword(s):  
High Speed ◽  
Atp Hydrolysis ◽  
3D Structure ◽  
Gliding Motility ◽  
Force Transmission ◽  
Animal Cells ◽  
Α Subunit ◽  
Force Microscopy ◽  
Atomic Force ◽  
Internal Structures

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. Interestingly, the internal structure of the probable gliding motor has 28 protein chains, each of which has 17 particles composed of homologs of the catalytic α- and β-subunits of F1-ATPase. In this study, we isolated chain particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated chain particles were composed of five proteins, MMOBs 1660 (α-subunit homolog), 1670 (β-subunit homolog), 1630, 1620, and 4530, and showed ATP hydrolyzing activity. The 2D structure, with dimensions of 35 and 26 nm, showed a hexameric ring dimer about 12 nm in diameter, resembling F1-ATPase catalytic (αβ)3. We isolated the F1-like ATPase unit, which is composed of MMOBs 1660, 1670, and 1630. Furthermore, we isolated the complex in chain form and analyzed the 3D structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31 nm intervals. An atomic model of F1-ATPase catalytic (αβ)3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism.

scholarly journals A Coumarin-Benzothiazole Derivative as a FRET-Based Chemosensor of Adenosine 5′-Triphosphate

Chemosensors ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 34 ◽  
Author(s):  
Gabr ◽  
Ibrahim ◽  
Tripathi ◽  
Prabhakar
Keyword(s):  
Aqueous Solution ◽  
Energy Transfer ◽  
Atp Hydrolysis ◽  
Limit Of Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Interaction ◽  
Plot Analysis ◽  
Sensing Platform ◽  
Ratiometric Probe

A coumarin-benzothiazole ratiometric probe of ATP was designed and synthesized. The probe is based on incorporation of benzothiazole scaffold as a donor and coumarin nucleus as an acceptor in a single Förster resonance energy transfer/fluorescence resonance energy transfer (FRET) sensing platform. The sensor can detect ATP in aqueous solution with high selectivity over other nucleotide polyphosphate (NPP) anions. Binding of ATP to the sensor results in modulation of FRET efficiency between the donor and the acceptor which afforded a linear relationship between FRET signal and ATP (0.1–10 μM). A limit of detection (LOD) of 94.5 nM was quantified for FRET sensing of ATP by the probe. In addition, Job plot analysis revealed 1:1 binding interaction between the probe and ATP. The FRET probe was successfully utilized in monitoring ATP hydrolysis by apyrase in aqueous solution.

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