Manuel Diez

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase.

Synthesis of ATP from ADP and phosphate, catalyzed by F0F1-ATP synthases, is the most abundant physiological reaction in almost any cell. F0F1-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F0F1-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The γ subunit rotates stepwise during proton transport–powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Stepwise rotation of the γ-subunit of EF0F1-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer1

The EF0F1‐ATP synthase mutants bQ64C and γT106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b‐subunit and with a cyanine (Cy5) at the γ‐subunit. After reconstitution into liposomes, these double‐labelled enzymes catalyzed ATP synthesis at a rate of 33 s−1. Fluorescence of TMR and Cy5 was measured with a confocal set‐up for single‐molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET‐states were identified, indicating stepwise rotation of the γ‐subunit of EF0F1.

Movements of the ε-subunit during catalysis and activation in single membrane-bound H+-ATP synthase

F0F1‐ATP synthases catalyze proton transport‐coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the ε‐subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence‐labeled F0F1 from Escherichia coli was incorporated into liposomes. Single‐molecule fluorescence resonance energy transfer (FRET) revealed that the ε‐subunit rotates stepwise showing three distinct distances to the b‐subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of ε during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of ε. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active–inactive transition was associated with a conformational change of ε within the central stalk.

In vitro assembly of mosaic hepatitis B virus capsid-like particles (CLPs): Rescue into CLPs of assembly-deficient core protein fusions and FRET-suited CLPs

Hepatitis B virus core protein self‐assembles into icosahedral, highly immunogenic capsid‐like particles (CLPs) that can serve as molecular platforms for heterologous proteins. Insertion into the centrally located c/e1 epitope leads to surface display, fusion to the C terminus to internal disposition of the foreign domains. However, symmetry‐defined space restrictions on the surface and particularly inside the CLPs limit the size of usable heterologous fusion partners. Further, CLPs carrying differing foreign domains are desirable for applications such as multivalent vaccines, and for structure probing by distance sensitive interactions like fluorescence resonance energy transfer (FRET). Here, we report an in vitro co‐assembly system for such mosaic‐CLPs allowing successful CLP formation with a per se assembly‐deficient fusion protein, and of CLPs from two different fluoroprotein‐carrying fusions that exert FRET in an assembly‐status dependent way.

Stepwise rotation of the γ-subunit of EF o F 1 -ATP synthase during ATP synthesis: a single-molecule FRET approach

FoF1-ATP synthases couple proton translocation with the synthesis of ATP using two rotary motors within the enzyme. To monitor inter-subunit movements during catalysis, we selectively attached two fluorophores to the F1 part, sulforhodamine B at one of three β-subunits and Cy5 at the γ-subunit. Reassembly with Fo parts embedded in liposomes yielded functional holoenzymes. Fluorescence resonance energy transfer (FRET) was investigated in photon bursts of freely diffusing liposomes with reconstituted ATP synthases using a confocal set-up for single-molecule detection. Incubation with AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies. Upon ATP addition, a repeating sequence of three distinct FRET efficiencies was observed, indicating the stepwise movement of the γ-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the γ-subunit under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition). The direction of rotation is opposite to the direction observed during ATP hydrolysis.

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Subunit movements within the H+-ATP synthase from chloroplasts (CF0F1) are investigated during ATP synthesis. The γ-subunit (γCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5′-(β,γ-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one α-subunit. The labeled CF0F1 is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the γ-subunit relative to the α-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each αβ-pair. Without catalysis the central stalk interacts with only one specific αβ-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

Conformational Changes of H+ ATP Synthase Upon ATP Hydrolysis Detected by Fluorescence Correlation Spectroscopy

H+ -ATPases catalyze the synthesis of ATP from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. The enzyme consists of two parts: the hydrophobic, membrane integrated F0-part is involved in proton transport and contains the subunits a, b and c with a likely stoichiometry ab2c12 for the Escherichia coli enzyme. The nucleotide binding sites are located in the hydrophilic F1-part with subunit composition α3β3γδe [1]. During ATP hydrolysis intersubunit rotation of the γ-subunit in the F1-part (which was dissociated from the holoenzyme) was observed with single fluorescence labeled enzymes in realtime using enhanced videomicroscopy [2 - 4]. ATP synthesis and hydrolysis occur at the catalytic binding sites of the β-subunits. In the crystal structure of the F1-ATPase three different conformations of the three β-subunits were detected [5]. From these structural data a detailed mechanistic model of the F1-ATPase as a ‘stepped rotatory motor protein’ was developed [6]. According to this model, large conformational changes in the lower part of the β-subunits are expected during catalysis. We investigated the diffusion of the F1-part before and after binding of ATP with fluorescence correlation spectroscopy (FCS). The β-subunit was specifically labeled with a newly synthesized sulfonyl fluoride derivative of Sulforhodamine G. The labeled amino acid is not known yet. Preliminary FCS measurements with EF1 are shown (Fig.1.). Upon ATP binding the translational diffusion time is decreased by about 15 percent due to changes in size and shape of the enzymes during the catalytic cycle. Similar results were obtained for EF1-ATPase with a fluorescence label on the γ-subunit [7]. These FCS measurements are supported by new electron microscopy data, which show a significant shrinking up of F1-part of the enzyme upon binding of the non-hydrolysable ATP derivative AMPPNP. The diameter of the F1-part decreased mainly in the upper half of the F1-part upon binding of AMPPNP [8].