Single-molecule structural dynamics of EF-G--ribosome interaction during translocation

EF-G catalyzes translocation of mRNA and tRNAs within the ribosome during protein synthesis. Detection of structural states in the reaction sequence that are not highly populated can be facilitated by studying the process one molecule at a time. Here we present single-molecule studies of translocation showing that, for ribosomes engaged in poly(Phe) synthesis, fluorescence resonance energy transfer (FRET) between the G' domain of EF-G and the N-terminal domain of ribosomal protein L11 occurs within two rapidly interconverting states, having FRET efficiencies of 0.3 and 0.6. The antibiotic fusidic acid increases the population of the 0.6 state, indicating that it traps the ribosome.EF-G complex in a preexisting conformation formed during translation. Only the 0.3 state is observed when poly(Phe) synthesis is prevented by omission of EF-Tu, or in studies on vacant ribosomes. These results suggest that the 0.6 state results from the conformational lability of unlocked ribosomes formed during translocation. An idling state, possibly pertinent to regulation of protein synthesis, is detected in some ribosomes in the poly(Phe) system.     

Soluble apyrases release adp during ATP hydrolysis

A soluble form of CD39 was expressed and purified from High-Five insect cells. The soluble CD39 is a monomer with a molecular weight of 54,000. The k(cat) and K(m) of the purified soluble CD39 were 4.6 s(-1) and 12 microM for ATP and 1.3 s(-1) and 7 microM for ADP as substrates, respectively. One nucleotide binding site was detected on the monomer only in the presence of Ca(2+). In contrast to the membrane bound CD39, soluble CD39 released ADP as an intermediate during ATP hydrolysis, as did the soluble potato apyrase.     

Extensive conformational transitions are required to turn on ATP hydrolysis in myosin

Conventional myosin is representative of biomolecular motors in which the hydrolysis of adenosine triphosphate (ATP) is coupled to large-scale structural transitions both in and remote from the active site. The mechanism that underlies such “mechanochemical coupling,” especially the causal relationship between hydrolysis and allosteric structural changes, has remained elusive despite extensive experimental and computational analyses. In this study, using combined quantum mechanical and molecular mechanical simulations and different conformations of the myosin motor domain, we provide evidence to support that regulation of ATP hydrolysis activity is not limited to residues in the immediate environment of the phosphate. Specifically, we illustrate that efficient hydrolysis of ATP depends not only on the proper orientation of the lytic water but also on the structural stability of several nearby residues, especially the Arg238-Glu459 salt bridge (the numbering of residues follows myosin II in Dictyostelium discoideum) and the water molecule that spans this salt bridge and the lytic water. More importantly, by comparing the hydrolysis activities in two motor conformations with very similar active-site (i.e., Switches I and II) configurations, which distinguished this work from our previous study, the results clearly indicate that the ability of these residues to perform crucial electrostatic stabilization relies on the configuration of residues in the nearby N-terminus of the relay helix and the “wedge loop.” Without the structural support from those motifs, residues in a closed active site in the post-rigor motor domain undergo subtle structural variations that lead to consistently higher calculated ATP hydrolysis barriers than in the pre-powerstroke state. In other words, starting from the post-rigor state, turning on the ATPase activity requires not only displacement of Switch II to close the active site but also structural transitions in the N-terminus of the relay helix and the “wedge loop,” which have been proposed previously to be ultimately coupled to the rotation of the converter subdomain 40 Å away.     

Intermediate complex of ATP hydrolysis and synthesis by muscle proteins

Myosin catalyzed exchange between ³²P_i and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of ³²P_i in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange ³²P_i and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of ³²P_i incorporation is proposed and the importance of the M*-ADP complex for force generation is suggested.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

Influence of phalloidin on ATP hydrolysis during actin polymerization

The influence of phalloidin on the ATP hydrolysis associated with actin polymerization was investigated. Whereas in the absence of phalloidin actin-bound ATP was totally hydrolyzed during polymerization, ATP hydrolysis was not complete after actin polymerization in the presence of phalloidin: 5-10% of ATP remained unhydrolyzed and disappeared only after 2 days.     

Single-molecule studies of complex systems: the replisome

A complete, system-level understanding of biological processes requires comprehensive information on the kinetics and thermodynamics of the underlying biochemical reactions. A wide variety of structural, biochemical, and molecular biological techniques have led to a quantitative understanding of the molecular properties and mechanisms essential to the processes of life. Yet, the ensemble averaging inherent to these techniques limits us in understanding the dynamic behavior of the molecular participants. Recent advances in imaging and molecular manipulation techniques have made it possible to observe the activity of individual enzymes and record "molecular movies" that provide insight into their dynamics and reaction mechanisms. An important future goal is extending the applicability of single-molecule techniques to the study of larger, more complex multi-protein systems. In this review, the DNA replication machinery will be used as an example to illustrate recent progress in the development of various single-molecule techniques and its contribution to our understanding of the orchestration of multiple enzymatic processes in large biomolecular systems.     

ATP hydrolysis during SOS induction in Escherichia coli.

Changes in cellular ATP concentration during SOS induction in strains of Escherichia coli with different levels of RecA and LexA proteins were studied. UV irradiation of RecA+ strains induced a twofold increase in the ATP concentration around the first 20 min, followed by a decrease to the values of nonirradiated cells. On the other hand, mutants defective in RecA protein or with either deficient RecA protease activity or cleavage-resistant LexA repressor did not show any decrease, suggesting that ATP consumption is related to LexA repressor hydrolysis. Furthermore, strains presenting a constitutive synthesis of RecA protein showed the same changes in ATP concentration as the wild-type strain. Likewise, the presence in a RecA+ strain of a LexA(Def) protein, which is defective in its capacity for binding specifically to SOS operators, did not disturb the changes in ATP when compared with the LexA+ RecA+ strain. Moreover, after UV irradiation, a LexA(Def) RecA- double mutant showed an important increase in ATP concentration, which remained elevated for at least 120 min after UV treatment.     

Molecular determinants of the ATP hydrolysis asymmetry of the CCT chaperonin complex

The eukaryotic cytosolic chaperonin CCT is a molecular machine involved in assisting the folding of proteins involved in important cellular processes. Like other chaperonins, CCT is formed by a double‐ring structure but, unlike all of them, each ring is composed of eight different, albeit homologous subunits. This complexity has probably to do with the specificity in substrate interaction and with the mechanism of protein folding that takes place during the chaperonin functional cycle, but its detailed molecular basis remains unknown. We have analyzed the known proteomes in search of residues that are differentially conserved in the eight subunits, as predictors of functional specificity (specificity‐determining positions; SDPs). We have found that most of these SDPs are located near the ATP binding site, and that they define four CCT clusters, corresponding to subunits CCT3, CCT6, CCT8 and CCT1/2/4/5/7. Our results point to a spatial organisation of the CCT subunits in two opposite areas of the ring and provide a molecular explanation for the previously described asymmetry in the hydrolysis of ATP. Proteins 2014; 82:703–707. © 2014 Wiley Periodicals, Inc.     

Thermodynamic analysis of structural transitions during GNNQQNY aggregation

Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutzfeldt‐Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full‐length amyloid proteins is not necessary for understanding amyloid formation. In this study, we simulate GNNQQNY, the N‐terminal prion‐determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. Utilizing a coarse‐grained model permits equilibration on relevant time scales. Replica‐exchange molecular dynamics is used to gather simulation statistics at multiple temperatures and clear energy traps that would aversely impact results. Investigating the association of 3‐, 6‐, and 12‐chain GNNQQNY systems by calculating thermodynamic quantities and orientational order parameters, we determine the aggregation pathway by studying aggregation states of GNNQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H‐bond formation, leading to the formation of β‐sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak. Proteins 2013; 81:1141–1155. © 2013 Wiley Periodicals, Inc.     

Kinetics of tryptophan fluorescence enhancement in myofibrils during ATP hydrolysis

The mechanism of ATP hydrolysis in myofibrils can be studied by following the time course of tryptophan fluorescence. Stoichiometric quantities of ATP produce an enhancement of the tryptophan fluorescence in stirred suspensions of rabbit psoas myofibrils at pCa greater than 7. Approximately 1 mol of ATP/myosin head is required to obtain the maximum fluorescence enhancement of 4-6%. Upon the addition of quantities of ATP greater than 1 mol/mol of myosin head, the fluorescence rapidly increases to a steady state, which lasts for a period that is proportional to the amount of ATP added. The fluorescence then decays to the initial level with a half-time of approximately 40 s at 20 degrees C. Hydrolysis of [gamma-32P]ATP at pCa greater than 7 in myofibrils has an initial burst of approximately 0.7 mol/mol of myosin head that is followed by a constant rate of hydrolysis. The duration of the steady state hydrolysis is identical to the duration of the enhancement of tryptophan fluorescence. A lower limit of 5 X 10(5) M-1 S-1 was obtained for the second order rate constant of the fluorescence enhancement by ATP. At pCa of 4, the duration of the fluorescence enhancement is one-tenth to one-twentieth as long as at pCa greater than 7; this is consistent with the increased steady state rate of ATP hydrolysis at higher calcium concentrations. The time course of the fluorescence enhancement observed in myofibrils during ATP hydrolysis is qualitatively and quantitatively similar to that observed with actomyosin-S1 in solution. These results suggest that the kinetic mechanism of ATP hydrolysis that has been well established by studies of actomyosin-S1 in solution also occurs in myofibrils.     

Electron cryomicroscopy of acto-myosin-S1 during steady-state ATP hydrolysis.

The structure of the complex of actin and myosin subfragment-1 (S1) during steady-state ATP hydrolysis has been examined by electron microscopy. This complex is normally dissociated by ATP in vitro but was stabilized here by low ionic strength. Optimal conditions for attachment were established by light-scattering experiments that showed that approximately 70% of S1 could be bound in the presence of ATP. Micrographs of the unstained complex in vitreous water suggest that S1 attaches to actin in a variety of configurations in ATP; this contrasts with the single attached configuration seen in the presence of ADP. The data are therefore compatible with the idea that a change in attached configuration of the myosin cross-bridge is the origin of muscle force. In control experiments where ATP was allowed to hydrolyze completely the binding of the S1 seemed cooperative.     

Configuration of the two kinesin motor domains during ATP hydrolysis

To understand the mechanism of kinesin movement we have investigated the relative configuration of the two kinesin motor domains during ATP hydrolysis using fluorescence polarization microscopy of ensemble and single molecules. We found that: (i) in nucleotide states that induce strong microtubule binding, both motor domains are bound to the microtubule with similar orientations; (ii) this orientation is maintained during processive motion in the presence of ATP; (iii) the neck-linker region of the motor domain has distinct configurations for each nucleotide condition tested. Our results fit well with a hand-over-hand type movement mechanism and suggest how the ATPase cycle in the two motor domains is coordinated. We propose that the motor neck-linker domain configuration controls ADP release.     

Binding of myosin to actin in myofibrils during ATP hydrolysis

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.     

Vanadate-induced trapping of nucleotides by purified maltose transport complex requires ATP hydrolysis

The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK(2) transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl(2) or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.     

Possible role of structural changes in ATP hydrolysis by subfragment-1 and myosin

Dependence of the rate of ATP hydrolysis with subfragment-I and temperature of SF-I, denaturation on the concentration of heavy water in solution was studied. The value of kinetic isotope effect V/Vx linearly increases with the rise of the volume portion of heavy water in solution and at X-1 it equals 1.9. The temperature of protein denaturacticn increases with X rise, the pattern of this relationship looking as an arched curve. The results differ from those earlier obtained on myosin which points to the absence of essential contribution of structural dynamic changes to enzymic hydrolysis of ATP by subfragment-I.     

ATP hydrolysis in a marine bacterium.

The membrane-bound adenosine triphosphatase of marine pseudomonad B-16, when solubilized, is able to rebind to depleted membrane residues of the bacterium and to those of Escherichia coli.     

Kinetic mechanism for formation of the active,dimeric UvrD helicase-DNA complex

Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase.