What can be learned about the enzyme ATPase from single-molecule studies of its subunit F<sub>1</sub>?

Volkan-Kacso, Sandor; Marcus, R. A.

doi:10.1017/s0033583517000129

Cited by 6 publications

(3 citation statements)

References 49 publications

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“…It is however thanks to single molecule experiments with soluble and liposome embedded F1F0 ATP synthase that we have such a detailed picture of the F1 binding change mechanism. 165 Apart from the ATP synthase, the groups of Jeuken and Stamou have performed single molecule proton pumping measurements with the quinol oxidase of E. coli 162 and the plasmamembrane P-type ATPase 166 , in which the pH change within the lumen of small unilamellar vesicles (SUVs) was followed by fluorescent pH sensors. Surprisingly, in both systems they have observed long phases of enzymatic inactivity or even passive proton leakage through the protein and have attributed their findings to a sensitive enzyme regulation by the local environment (but see Berg et al for a different finding).…”

Section: Rational Design Of Fluorescent Dyes To Follow Enzyme Functionmentioning

confidence: 99%

Current problems and future avenues in proteoliposome research

Amati

Graf

Deutschmann

et al. 2020

Biochemical Society Transactions

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Membrane proteins (MPs) are the gatekeepers between different biological compartments separated by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing number of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extraction and purification of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the experimental system. In this review, we focus on proteoliposomes that have become an indispensable experimental system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resolution. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymatic cascades, a very promising experimental tool considering our increasing knowledge of the interplay of different cellular components.

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Section: Rational Design Of Fluorescent Dyes To Follow Enzyme Functionmentioning

confidence: 99%

Current problems and future avenues in proteoliposome research

Amati

Graf

Deutschmann

et al. 2020

Biochemical Society Transactions

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“…It produces a rotation of the γ shaft relative to its barrel-like α3β3 stator structure, and is powered by the hydrolysis of ATP (3). The chemistry and rotation in F1-ATPase are the reverse of that in ATP synthase, and the study of rotation in the former can reveal the rotary mechanism in the latter (4).…”

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confidence: 99%

Method to extract multiple states in F ₁ -ATPase rotation experiments from jump distributions

Volkan-Kacso

Zhu

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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A method is proposed for analyzing fast (10 µs) single-molecule rotation trajectories in F 1 adenosinetriphosphatase (F 1 -ATPase). This method is based on the distribution of jumps in the rotation angle that occur in the transitions during the steps between subsequent catalytic dwells. The method is complementary to the "stalling" technique devised by H. Noji et al. [Biophys. Rev. 9, 103-118, 2017], and can reveal multiple states not directly detectable as steps. A bimodal distribution of jumps is observed at certain angles, due to the system being in either of 2 states at the same rotation angle. In this method, a multistate theory is used that takes into account a viscoelastic fluctuation of the imaging probe. Using an established sequence of 3 specific states, a theoretical profile of angular jumps is predicted, without adjustable parameters, that agrees with experiment for most of the angular range. Agreement can be achieved at all angles by assuming a fourth state with an ∼10 µs lifetime and a dwell angle about 40 • after the adenosine 5 -triphosphate (ATP) binding dwell. The latter result suggests that the ATP binding in one β subunit and the adenosine 5 -diphosphate (ADP) release from another β subunit occur via a transient whose lifetime is ∼10 µs and is about 6 orders of magnitude smaller than the lifetime for ADP release from a singly occupied F 1 -ATPase. An internal consistency test is given by comparing 2 independent ways of obtaining the relaxation time of the probe. They agree and are ∼15 µs.F-ATPase | single-molecule imaging | concerted dynamics | 4-state model | ADP release Author contributions: S† References and equations used to estimate the quantities in columns 3-8 are grouped in 6 groups in order according to the columns to which they refer.Groups are separated by semicolons, e.g., items before the first semicolon refer to column 3.Volkán-Kacsó et al.

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“…In the present system, the F1-ATPase, the Brønsted slope for ATP addition and ejection are close to 0.5, found both from interpreting stalling experiments (Adachi et al, 2012;Volkán-Kacsó and Marcus, 2015) and from a theoretical prediction using quantities from other independent ensemble and single-molecule experiments. Meanwhile, for the hydrolysis step it is close to unity (Adachi et al, 2012;Volkán-Kacsó and Marcus, 2017b), and correspondingly for the synthesis step it is close to zero, all of which provides mechanistic information.…”

Section: T T T T Andmentioning

confidence: 86%

F1-ATPase Rotary Mechanism: Interpreting Results of Diverse Experimental Modes With an Elastic Coupling Theory

Volkan-Kacso

Marcus

2022

Front. Microbiol.

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In this chapter, we review single-molecule observations of rotary motors, focusing on the general theme that their mechanical motion proceeds in substeps with each substep described by an angle-dependent rate constant. In the molecular machine F1-ATPase, the stepping rotation is described for individual steps by forward and back reaction rate constants, some of which depend strongly on the rotation angle. The rotation of a central shaft is typically monitored by an optical probe. We review our recent work on the theory for the angle-dependent rate constants built to treat a variety of single-molecule and ensemble experiments on the F1-ATPase, and relating the free energy of activation of a step to the standard free energy of reaction for that step. This theory, an elastic molecular transfer theory, provides a framework for a multistate model and includes the probe used in single-molecule imaging and magnetic manipulation experiments. Several examples of its application are the following: (a) treatment of the angle-dependent rate constants in stalling experiments, (b) use of the model to enhance the time resolution of the single-molecule imaging apparatus and to detect short-lived states with a microsecond lifetime, states hidden by the fluctuations of the imaging probe, (c) treatment of out-of-equilibrium “controlled rotation” experiments, (d) use of the model to predict, without adjustable parameters, the angle-dependent rate constants of nucleotide binding and release, using data from other experiments, and (e) insights obtained from correlation of kinetic and cryo-EM structural data. It is also noted that in the case where the release of ADP would be a bottleneck process, the binding of ATP to another site acts to accelerate the release by 5–6 orders of magnitude. The relation of the present set of studies to previous and current theoretical work in the field is described. An overall goal is to gain mechanistic insight into the biological function in relation to structure.

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What can be learned about the enzyme ATPase from single-molecule studies of its subunit F₁?

Cited by 6 publications

References 49 publications

Current problems and future avenues in proteoliposome research

Current problems and future avenues in proteoliposome research

Method to extract multiple states in F ₁ -ATPase rotation experiments from jump distributions

F1-ATPase Rotary Mechanism: Interpreting Results of Diverse Experimental Modes With an Elastic Coupling Theory

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What can be learned about the enzyme ATPase from single-molecule studies of its subunit F1?

Cited by 6 publications

References 49 publications

Current problems and future avenues in proteoliposome research

Current problems and future avenues in proteoliposome research

Method to extract multiple states in F 1 -ATPase rotation experiments from jump distributions

F1-ATPase Rotary Mechanism: Interpreting Results of Diverse Experimental Modes With an Elastic Coupling Theory

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What can be learned about the enzyme ATPase from single-molecule studies of its subunit F₁?

Method to extract multiple states in F ₁ -ATPase rotation experiments from jump distributions