Subunit Rotation Research Articles

The DARC site: a database of aligned ribosomal complexes

The ribosome is a highly dynamic machine responsible for protein synthesis within the cell. Cryo-electron microscopy (cryo-EM) and X-ray crystallography structures of ribosomal particles, alone and in complex with diverse ligands (protein factors, RNAs and small molecules), have revealed the dynamic nature of the ribosome and provided much needed insight into translation and its regulation. In the past years, there has been exponential growth in the deposition of cryo-EM maps into the Electron Microscopy Data Bank (EMDB) as well as atomic structures into the Protein Data Bank (PDB). Unfortunately, the deposited ribosomal particles usually have distinct orientations with respect to one another, which complicate the comparison of the available structures. To simplify this, we have developed a Database of Aligned Ribosomal Complexes, the DARC site (http://darcsite.genzentrum.lmu.de/darc/), which houses the available cryo-EM maps and atomic coordinates of ribosomal particles from the EMDB and PDB aligned within a common coordinate system. An easy-to-use, searchable interface allows users to access and download >130 cryo-EM maps and >300 atomic models in the format of brix and pdb files, respectively. The aligned coordinate system substantially simplifies direct visualization of conformational changes in the ribosome, such as subunit rotation and head-swiveling, as well as direct comparison of bound ligands, such as antibiotics or translation factors.

How to Change the Oligomeric State of a Circular Protein Assembly: Switch from 11-Subunit to 12-Subunit TRAP Suggests a General Mechanism

BackgroundMany critical cellular functions are performed by multisubunit circular protein oligomers whose internal geometry has evolved to meet functional requirements. The subunit number is arguably the most critical parameter of a circular protein assembly, affecting the internal and external diameters of the assembly and often impacting on the protein's function. Although accurate structural information has been obtained for several circular proteins, a lack of accurate information on alternative oligomeric states has prevented engineering such transitions. In this study we used the bacterial transcription regulator TRAP as a model system to investigate the features that define the oligomeric state of a circular protein and to question how the subunit number could be manipulated.Methodology/Principal FindingsWe find that while Bacillus subtilis and Bacillus stearothermophilus TRAP form 11-subunit oligomers, the Bacillus halodurans TRAP exclusively forms 12-subunit assemblies. Significantly, the two states of TRAP are related by a simple rigid body rotation of individual subunits around inter-subunit axes. We tested if such a rotation could be induced by insertion or deletion mutations at the subunit interface. Using wild type 11-subunit TRAP, we demonstrate that removal of five C-terminal residues at the outer side of the inter-subunit axis or extension of an amino acid side chain at the opposite, inner side, increased the subunit number from 11 to 12. Our findings are supported by crystal structures of TRAP oligomers and by native mass spectrometry data.Conclusions/SignificanceThe subunit number of the TRAP oligomer can be manipulated by introducing deletion or addition mutations at the subunit interface. An analysis of available and emerging structural data on alternative oligomeric states indicates that the same principles may also apply to the subunit number of other circular assemblies suggesting that the deletion/addition approach could be used generally to engineer transitions between different oligomeric states.

Structure and Dynamics of the Mammalian Ribosomal Pretranslocation Complex

Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pretranslocation complex reveal spontaneous, large-scale conformational changes, including an intersubunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favored within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.

Growth Hormone Receptor: Structure Function Relationships

Background: The growth hormone receptor (GHR) was the first class 1 cytokine receptor to be cloned, and it has been studied intensively. The crystal structures of the bound and unbound forms have been solved and the energetic contributions of residues involved in the binding interaction have been quantified. Two receptor subunits bind to opposite sides of the hormone through site 1 and site 2, and a third interaction occurs between receptors in the lower β-sandwich module at site 3. All three interactions are required for receptor activation, which was thought to be a consequence of hormone-induced receptor dimerization. However, substantial data support the existence of a constitutive receptor dimer that interacts via the transmembrane domain (TMD), with receptor activation triggered by a hormone-induced conformational change. Mutagenesis studies and crystal structure data indicate that receptor activation involves a relative rotation and scissor movement of subunits to activate the associated tyrosine kinase, Janus kinase 2 (JAK2). We have recently reported that a second tyrosine kinase, an Src-family kinase, also associates constitutively with the receptor and activates the Ras–extracellular-signal-regulated kinase pathway. Activation of this kinase requires a conformational change in a loop of the lower sandwich module, and a different orientation of the TMDs than needed for JAK2 activation. Conclusions: This structural difference could allow differential activation of these two kinases by GH analogues such as I179M human GH.

Structural Basis for Catalytic Activation of a Serine Recombinase

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 Å crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.

Recombining DNA by Protein Swivels

Two new reports on serine recombinases, one of a crystal snapshot in an alternate rotational conformer poised for DNA cleavage (Keenholtz et al., 2011), and a second employing single-DNA molecule approaches (Bai et al., 2011), provide strong support for the subunit rotation model for exchanging DNA strands.

The rotational spectra of the fluorobenzene⋯water and p-difluorobenzene⋯water dimers: Structure and internal dynamics

The rotational spectra of the weakly hydrogen-bonded complexes of fluorobenzene and p-difluorobenzene with water were measured in the frequency range from 3 to 15 GHz using pulsed molecular jet Fourier transform microwave spectrometers in Kiel and in Edmonton. Spectroscopic constants were derived from the spectra of several isotopologues of fluorobenzene⋯water, i.e. those containing H 2 16O, H 2 18O, HDO, and D 2O, and of p-difluorobenzene⋯water with H 2 16O, H 2 17O, H 2 18O, and D 2O. The structural parameters derived from the determined rotational constants correspond to planar (or nearly planar) structures of the complexes. The internal dynamics of the complexes are discussed with regard to the observed tunneling splittings of the rotational lines, which are a result of the hindered internal rotation of the water subunit.

Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding

The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140ΔV2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120-gp41 interactions, whereas a "flat open" concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N- and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes.

Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation.

We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small-molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross-linking, alanine-scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full-length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer-dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.

Unravelling the Symmetry Mismatch of the Two Coupled Rotary Motors of a Single FOF1-ATP Synthase by Three Color Fret

FoF1-ATP synthase is the enzyme that provides the ‘chemical energy currency’ adenosine triphosphate, ATP, for living cells. The enzyme consists of the membrane-embedded Fo part and the F1 part with three catalytic nucleotide binding sites. The formation of ATP is accomplished by a stepwise internal rotation of subunits within both parts. During ATP synthesis, proton translocation through the Fo part drives a 10-step rotary motion of the c10 ring with respect to the non-rotating subunits a and b2 [1]. This rotation is transmitted to the γ and ε subunits of the F1 part which perform three 120° steps per full rotation [2]. To localize transient elastic energy storage within the enzyme due to the symmetry mismatch, we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme. Cy5 is attached to one c subunit and EGFP to the a subunit of the Fo part [3]. The F1 part has a fluorophore bound at the ε subunit. To reduce photophysical artifacts due to spectral fluctuations of the fluorophores, a duty cycle-optimized alternating three laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors in a single enzyme revealed elastic deformations in the rotor parts of Fo and F1 during catalysis. [1] M. G. Düser, N. Zarrabi, D. J. Cipriano, S. Ernst, G. D.Glick, S.D. Dunn, M. Börsch (2009) EMBO Journal 28, 2689-2696. [2] B. Zimmermann, M. Diez, N. Zarrabi, P. Gräber, M. Börsch (2005) EMBO Journal 24, 2053-2063. [3] M. G. Düser, Y. Bi, N. Zarrabi, S.D. Dunn, M. Börsch (2008) J. Biol. Chem. 283, 33602-33610.

Kinesin Generates a Nucleotide-Dependent Distortion of the Microtubule Lattice

The kinesin molecular motors largely divide into two distinct functional forms: in the motile form, the energy of ATP hydrolysis powers travel along microtubules; in the depolymerizing form, ATP hydrolysis drives disassembly of microtubules. While significant advances have been made in the understanding structural basis of kinesin motility, the structural basis for ATP-driven depolymerization remains largely mysterious. We used high-resolution cryo-EM to visualize an unexpected, nucleotide-driven distortion of the microtubule structure when decorated by motile kinesins. This distortion is generated when ATP analogs bind in kinesin's active site and “pinch” two of kinesin's microtubule-binding domains together, changing the molecule's grip on the microtubule surface and leading to a relative rotation of alpha- and beta- tubulin subunits within the tubulin dimer. The tubulin subunit rotation is accompanied by a ∼2% longitudinal contraction of the microtubule lattice. We observed this effect in the presence of two highly-divergent motile kinesins: “conventional” plus-end directed kinesin, as well as minus-end directed ncd. We infer that this functional behavior is likely a general feature of the kinesin family proteins, and may inform the mechanism of the depolymerizing kinesins.

Time Resolved Crystallographic Analysis of Cooperative Ligand Binding and Ligand Migration in a Dimeric Hemoglobin

Invertebrate hemoglobins, which range in size from dimers to assemblies of hundreds of subunits, offer excellent models for the investigation of allosteric protein function. A recurring theme among cooperative invertebrate hemoglobins is a subunit pairing involving the heme-embedding E and F helices, despite a lack of sequence conservation in these helices. This is quite different from the assembly of vertebrate hemoglobins and suggests that such a pairing is amenable for regulating oxygen delivery. We have carried out extensive time-resolved crystallographic analysis on the simplest of these hemoglobins, the cooperative homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis. Limited ligand-linked subunit rotation permits the full allosteric transition to be followed in crystals of HbI. These studies have been combined with mutant and functional studies along with conventional crystallographic analysis to provide a comprehensive understanding of the communication between subunits and the movement of ligands through the protein. Investigation of ligand migration through this protein suggests, surprisingly, that ligands primarily escape through a distal histidine gate, despite its involvement in the subunit interface. As a result, ligand escape may require transient subunit movement, which is inhibited in the protein crystal lattice. These findings emphasize the central role of dynamics in protein function. Our studies are also directed towards understanding cooperative protein function in larger invertebrate hemoglobin assemblies. Preliminary time-resolved crystallogaphic analysis on the tetrameric Scapharca HbII suggests similar structural transitions as HbI and offers the possibility of investigating how changes in one subunit directly impact a neighboring subunit in the heterodimeric halves of HbII. In addition, we will discuss new insights that may provide unifying themes for the basis of cooperativity among diverse invertebrate hemoglobins that form EF dimeric pairing.

Genome wide cloning of maize meiotic recombinase Dmc1 and its functional structure through molecular phylogeny

The development of meiotic division and associated genetic recombination paved the way for evolutionary changes. However, the secondary and tertiary structure and functional domains of many of the proteins involved in genetic recombination have not been studied in detail. We used the human Dmc1 gene product along with secondary and tertiary domain structures of Escherichia coli RecA protein to help determine the molecular structure and function of maize Dmc1, which is required for synaptonemal complex formation and cell cycle progression. The maize recombinase Dmc1 gene was cloned and characterized, using rice Dmc1 cDNA as an orthologue. The deduced amino acid sequence was used for elaborating its 3-D structure, and functional analysis was made with the CDD software, showing significant identity of the Dmc1 gene product in Zea mays with that of Homo sapiens. Based on these results, the domains and motives of WalkerA and WalkerB as ATP binding sites, a multimer site (BRC) interface, the putative ssDNA binding L1 and L2 loops, the putative dsDNA binding helix-hairpin-helix, a polymerization motif, the subunit rotation motif, and a small N-terminal domain were proposed for maize recombinase Dmc1.

Archazolid A Binds to the Equatorial Region of the c-Ring of the Vacuolar H+-ATPase

The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC(50) value in the low nanomolar range. The binding site of archazolid is presumed to overlap with the binding site of the established plecomacrolide V-ATPase inhibitors bafilomycin and concanamycin in subunit c of the membrane-integral V(O) complex. Using a semi-synthetic derivative of archazolid for photoaffinity labeling of the V(1)V(O) holoenzyme we confirmed binding of archazolid to the V(O) subunit c. For the plecomacrolide binding site a model has been published based on mutagenesis studies of the c subunit of Neurospora crassa, revealing 11 amino acids that are part of the binding pocket at the interface of two adjacent c subunits (Bowman, B. J., McCall, M. E., Baertsch, R., and Bowman, E. J. (2006) J. Biol. Chem. 281, 31885-31893). To investigate the contribution of these amino acids to the binding of archazolid, we established in Saccharomyces cerevisiae mutations that in N. crassa had changed the IC(50) value for bafilomycin 10-fold or more and showed that out of the amino acids forming the plecomacrolide binding pocket only one amino acid (tyrosine 142) contributes to the binding of archazolid. Using a fluorescent derivative of N,N'-dicyclohexylcarbodiimide, we found that the binding site for archazolid comprises the essential glutamate within helix 4 of subunit c. In conclusion the archazolid binding site resides within the equatorial region of the V(O) rotor subunit c. This hypothesis was supported by an additional subset of mutations within helix 4 that revealed that leucine 144 plays a role in archazolid binding.

The Conformational Landscape of Nicotinoids: Solving the Conformational Disparity of Anabasine

The conformational landscape of the alkaloid anabasine (neonicotine) has been investigated by using rotational spectroscopy and ab initio calculations. The results allow a detailed comparison of the structural properties of the prototype piperidinic and pyrrolidinic nicotinoids (anabasine vs. nicotine). Anabasine adopts two most stable conformations in isolation conditions, for which we determined accurate rotational and nuclear quadrupole coupling parameters. The preferred conformations are characterized by an equatorial pyridine moiety and additional N-H equatorial stereochemistry at the piperidine ring (eq-eq; eq=equatorial). The two rings of anabasine are close to a bisecting arrangement, with the observed conformations differing by an approximately 180 degrees rotation of the pyridine subunit, denoted either syn or anti. The preference of anabasine for the eq-eq-syn conformation has been established by relative intensity measurements (syn/anti approximately 5(2)). The conformational preferences of free anabasine are directed by a weak N...H-C hydrogen bond interaction between the nitrogen lone pair at piperidine and the closest C-H bond in pyridine, with N...H distances ranging from 2.686 (syn) to 2.667 A (anti). Supporting ab initio calculations by using MP2 and the recent M05-2X density functional are provided, evaluating the predictive performance of both methods.

Critical Salt Bridges Guide Capsid Assembly, Stability, and Maturation Behavior in Bacteriophage HK97

HK97 is a double-stranded DNA bacteriophage that undergoes dramatic conformational changes during viral capsid maturation and for which x-ray structures, at near atomic resolution, of multiple intermediate and mature capsid states are available. Both amide H/(2)H exchange and crystallographic comparisons between the pre-expanded Prohead II particles and the expanded Head II of bacteriophage HK97 revealed quaternary interactions that remain fixed throughout maturation and appear to maintain intercapsomer integrity at all quasi- and icosahedral 3-fold axes. These 3-fold staples are formed from Arg and Glu residues and a metal binding site. Mutations of either Arg-347 or Arg-194 or a double mutation of E344Q and E363A resulted in purification of the phage in capsomer form (hexamers and pentamers). Mutants that did assemble had both decreased thermal stability and decreased in vitro expansion rates. Amide H/(2)H exchange mass spectrometry showed that in the wild type capsid some subunits had a bent "spine" helix (highly exchanging), whereas others were straight (less exchanging). Similar analysis of the never assembled mutant capsomers showed uniform amide exchange in all of these that was higher than that of the straight spine helices (characterized in more mature intermediates), suggesting that the spine helix is somewhat bent prior to capsid assembly. The result further supports a previously proposed mechanism for capsid expansion in which the delta domains of each subunit induce a high energy intermediate conformation, which now appears to include a bent helix during capsomer assembly.

R‐ to T‐ Transition in Fructose‐1,6‐bisphosphatase: Loop Displacement is the Penultimate Step to Inactivation

AMP transforms fructose‐1, 6‐bisphosphatase (FBPase) from its active R‐state to its inactive T‐state. During this quaternary transition, a catalytically essential loop (residues 52–72) leaves the active site (engaged conformation) and moves to its inactive (disengaged) conformation. Structures of FBPase and molecular dynamic simulations reveal the mechanism of loop displacement. The AMP complex of Asp10 FBPase provides the first example of an engaged dynamic loop in a near‐T quaternary state. Subunit rotation forces loop 190 (from subunit C1) into contact with the hinge (residues 52–57) of the dynamic loop of subunit C2. Additional conformational changes in residues 264–274 of subunit C2 reduce space available to the engaged conformation of loop 52–72. Target Molecular Dynamics indicates the absence of steric barriers in subunit rotations but requires the displacement of the loop 50–72 from its engaged conformation in order to achieve the T‐state. Principal Component Analysis reveals anti‐correlated motions, which cause steric clashes consistent with the displacement of loop 52–72 from its engaged conformation. This research is supported by NIH grant NS 10546.

Roles of the β subunit hinge domain in ATP synthase F 1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698–20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

Crystal Structures Of Yeast Mitochondrial ATPase with Uncoupling Mutations

Yeast mitochondrial ATP synthase is a transmembrane protein responsible for synthesis of more than 90% of ATP under aerobic conditions. The water soluble portion of ATP synthase, F1, is composed of five subunits with stoichiometry α3β3γδe and has a combined molecular weight of 360 kDa. The three active sites of ATPase are formed at the interfaces between alternating α- and β-subunits. ATPase is capable of efficient ATP hydrolysis, accompanied by rotation of central stalk subunits, γδe, within the α3β3 core and is capable of ATP synthesis if central subunits are forced to rotate in the opposite direction. A number of mutations in ATP synthase have been identified that result in the uncoupling of catalytic function and proton flow across the mitochondrial membrane. These uncoupling mutations cluster at the interface between γ-subunit and α3β3 catalytic core of ATPase. In this work, four X-ray crystal structures of ATPase with single amino acid substitutions αN67I, αF405S, βV279F, and γI270T were solved at resolutions ranging from 3.2 A to 2.74 A. This study will present a structural comparison of the mutant structures with the wild type structures to understand the mechanism of coupling. However, the crystal structures likely represent the ground state of catalytic reaction cycle while the mutations may result in notable distortions of enzyme structure during other stages of catalytic cycle. Additionally, the uncoupling of the ATPase may be caused by changes in the energy of interaction between the portions that are rotating in the molecular machine thereby altering transition to the higher energy states. Structural based hypotheses are presented to explain the role of these residues in the coupling of the enzyme. Supported by NIH R01GM0662223

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

Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) 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 gamma-subunit relative to the alpha-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 alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-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.