Distinct Structural Transitions Research Articles

Identification of an Aggregation-Prone Structure of Tau

The aggregation and deposition of normally soluble proteins is the hallmark of several devastating neurodegenerative disorders. For proteins such as tau in Alzheimer's disease and α-synuclein in Parkinson's disease, aggregation involves a transition from an intrinsically disordered monomer to a highly structured fiber. While understanding the role of these proteins in neurodegeneration requires elucidation of the structural basis of self-association, the conformational heterogeneity of disordered proteins makes their structural characterization inherently challenging. Here we use single molecule Förster resonance energy transfer to measure the conformational ensemble of tau in the absence and presence of heparin to identify critical conformational changes relevant to the initiation of aggregation. We find that different domains of tau display distinct conformational properties that are strongly correlated with their degree of disorder and that may relate to their roles in aggregation. Moreover, we observe that heparin binding induces a distinct two-state structural transition in tau characterized by a loss of long-range contacts and a concomitant compaction of the microtubule binding domain. Our results describe a conformational intermediate of tau that precedes the formation of aggregates and could serve as a target for tau-focused therapeutics.

Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements

Double-stranded DNA is a dynamic molecule whose structure can change depending on conditions. While there is consensus in the literature about many structures DNA can have, the state of highly-stretched DNA is still not clear. Several groups have shown that DNA in the torsion-unconstrained B-form undergoes an "overstretching" transition at a stretching force of around 65pN, which leads to approximately 1.7-fold elongation of the DNA contour length. Recent experiments have revealed that two distinct structural transitions are involved in the overstretching process: (i) a hysteretic "peeling" off one strand from its complementary strand, and (ii) a nonhysteretic transition that leads to an undetermined DNA structure. We report the first simultaneous determination of the entropy (ΔS) and enthalpy changes (ΔH) pertaining to these respective transitions. For the hysteretic peeling transition, we determined ΔS∼20cal/(K.mol) and ΔH∼7kcal/mol. In the case of the nonhysteretic transition, ΔS∼-3cal/(K.mol) and ΔH∼1kcal/mol. Furthermore, the response of the transition force to salt concentration implies that the two DNA strands are spatially separated after the hysteretic peeling transition. In contrast, the corresponding response after the nonhysteretic transition indicated that the strands remained in close proximity. The selection between the two transitions depends on DNA base-pair stability, and it can be illustrated by a multidimensional phase diagram. Our results provide important insights into the thermodynamics of DNA overstretching and conformational structures of overstretched DNA that may play an important role in vivo.

ATP binding controls distinct structural transitions of Escherichia coli DNA gyrase in complex with DNA

DNA gyrase is a molecular motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. A critical step in this reaction is the formation of a chiral DNA wrap on a similar scale to the nucleosome. Here we observe gyrase structural dynamics using a single-molecule assay in which gyrase drives the processive, stepwise rotation of a nanosphere attached to the side of a stretched DNA molecule. Analysis of rotational pauses and measurements of DNA contraction reveal multiple ATP-modulated structural transitions. DNA wrapping is coordinated with the ATPase cycle and proceeds via an unanticipated structural intermediate that dominates the kinetics of supercoiling. Our findings reveal a conformational landscape of loosely coupled transitions funneling the motor toward productive energy transduction, a feature that may be common to the reaction cycles of other DNA and protein remodeling machines.

Molecular-Level Characterization of the Breathing Behavior of the Jungle-Gym-type DMOF-1 Metal–Organic Framework

Fundamental insights into the molecular mechanisms that determine the breathing behavior of the jungle-gym-type DMOF-1 metal-organic framework upon adsorption of benzene and isopropyl alcohol are gained from computer simulations. In all cases, good agreement is obtained between the calculated and experimental structural parameters. In the case of benzene adsorption, DMOF-1 is predicted to exist in a narrow pore configuration at high loadings and/or low temperature. A structural transition into a large pore configuration is then observed as the temperature increases and/or the loading decreases, which is directly related to the spatial distribution and molecular interactions of the benzene molecules within the pores. The isopropyl alcohol adsorption simulations indicate that DMOF-1 undergoes two distinct structural transitions (from large pore to narrow pore and then back to large pore) as the number of adsorbed molecules increases, which is explained in terms of the formation of hydrogen bonds between the isopropyl molecules and the framework.

Single-Molecule Thermodynamics of Three Distinct DNA Structural Transitions under Large Tension

Torsion unconstrained double-stranded DNA can undergo three structural transitions under large tension. At forces around 65 pN, a well known DNA overstretching transition occurs during which the DNA elongates by about 1.7-fold. Recent experiments have revealed two distinct structural transitions during DNA overstretching: a “strand-unpeeling” of one strand from the other, and a “B-to-S” transition to a mysterious double-stranded “S-DNA”. In addition to DNA overstretching transition, DNA without nicks or free ends can also undergo an internal melting transition to two parallel DNA strands under tension at > 100 pN in physiological solution conditions or under lower tension at sufficiently low salt concentration or high temperature. Here, we report thermodynamics measurements for the entropy and enthalpy changes during these transitions. In addition, the force-responses of these DNA structures are directly determined in Single-molecule experiments. With these experimentally determined thermodynamic and mechanical quantities, the selections of the transitions are summarized in phase diagrams.

High pressure structures of “111” type iron-based superconductors predicted from first-principles

The high-pressure crystal structures of the "111" type iron-based superconductors: NaFeAs, LiFeP and LiFeAs have been systematically explored by using particle-swarm structural searches. It was found that though these iron-based superconductors are chemically similar, they adopted distinct structural phase transitions: P4/nmm→Cmcm→P3m1 for NaFeAs, P4/nmm→Cmcm→I4mm for LiFeP, and P4/nmm→P3m1 →I4mm→P6(3)/mmc for LiFeAs under high pressure. The high pressure orthorhombic Cmcm phase preserved the structural features of FeX(4)(X = As, P) tetrahedral layers present in the ambient-pressure P4/nmm structure. However, the FeX(4) tetrahedrons in the Cmcm phase were clearly distorted, leading to changes in the electronic behavior around the Fermi level. Under higher pressures, the FeX(4) layered structural features were no longer persistent and three-dimensional crystal structures were stabilized in other P3m1, I4mm, and P6(3)/mmc phases, which featured FeAs(5)/FeAs(6) hexahedron and octahedrons, FeX(5) tetragonal pyramids, and FeAs(6) octahedrons, respectively. Analysis of the electronic density of states suggests that most of the high pressure phases are metallic except for the tetragonal I4mm phase, which possesses a narrow band gap. This semiconducting state might relate to the tetragonal pyramid structure formed by FeX(5) unit, which might be favorable for charge localization.

Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.

Characteristics of Prunus serotina seed oil

Oils from Prunus serotina raw and toasted seeds extracted with hexane and supercritical CO 2 were evaluated for their physicochemical characteristics. Supercritical CO 2 extracted the least oil (21.3%), with high absorbing carotenoid pigments. P. serotina oil had characteristically high refractive index and density with three typical absorbance peaks in the UVC (100–290 nm) range centred at 260, 270 and 280 nm. The oil was highly polyunsaturated and abundant in oleic (35%), α-elostearic (27%), linoleic (27%), palmitic (4%), stearic (4%) and β-elostearic (1%) acids. P. serotina seed oil exhibited at least three distinct thermal structural transitions between −35 and −13 °C with two reversing transitions between −19 and −12 °C. Thermal oxidation by differential scanning calorimetry (DSC) revealed a three step oxidation of P. serotina seed oil with the mean onset and oxidation temperatures at 121 and 130–273 °C, respectively, depending on processing. Supercritical CO 2 extraction and toasting significantly affected the thermal and oxidation characteristics, fluorescence, and fatty acids of oils.

MOLECULAR DYNAMIC SIMULATIONS ON THE FOLDING AND CONFORMATIONAL INSIGHTS OF THE TRUNCATED PEPTIDES

A total of 120 ns molecular dynamics simulations was used to study the folding and conformational aspects of six peptides with different lengths (Pep19–25, Pep15–25, Pep1–25, Pep15–39, Pep1–40, and Pep1–50) truncated from the αβ-tubulin dimer. These truncated peptides were found to undergo distinct structural transitions, with Pep1–25 and Pep1–50 folding into their respective stable conformations whereas on the contrary for the others. All the six truncated peptides are more or less compact than the corresponding segments in the αβ-tubulin dimer. The most striking contraction was observed in Pep1–25, which folds in a similar manner of β-hairpin. Pep1–50 has the least contraction and its folded conformation is the closest to that in the αβ-tubulin dimer. Moreover, the same conversions of β12–β23 from helices to hydrogen-bonded turns were witnessed in both Pep1–50 and the αβ-tubulin dimer. The structural instabilities of Pep19–25, Pep15–25, Pep15–39, and Pep1–40 were caused by the lack of long-distance interactions or/and the absence of key residues, with the details given in the discussions. The folding and conformational divergences of six truncated peptides were also observed in their active peptide segments ( Ap 15–25). Ap 15–25 in Pep1–50 achieves the best agreements with the αβ-tubulin dimer, implying that the local structure of Ap 15–25 in the αβ-tubulin dimer can be well reserved in Pep1–50 rather than in the other truncated peptides. The long-distance interactions, especially the key residues (e.g. β48-Arg), play a crucial role in the correct folding of Ap 15–25. The correct folding into the stable conformations is a prerequisite for the peptides to implement their catalytic actions, and therefore the present results are helpful to the future designs of active peptides.

Quantitative Image Analysis Reveals Distinct Structural Transitions during Aging in Caenorhabditis elegans Tissues

Aging is associated with functional and structural declines in many body systems, even in the absence of underlying disease. In particular, skeletal muscles experience severe declines during aging, a phenomenon termed sarcopenia. Despite the high incidence and severity of sarcopenia, little is known about contributing factors and development. Many studies focus on functional aspects of aging-related tissue decline, while structural details remain understudied. Traditional approaches for quantifying structural changes have assessed individual markers at discrete intervals. Such approaches are inadequate for the complex changes associated with aging. An alternative is to consider changes in overall morphology rather than in specific markers. We have used this approach to quantitatively track tissue architecture during adulthood and aging in the C. elegans pharynx, the neuromuscular feeding organ. Using pattern recognition to analyze aged-grouped pharynx images, we identified discrete step-wise transitions between distinct morphologies. The morphology state transitions were maintained in mutants with pharynx neurotransmission defects, although the pace of the transitions was altered. Longitudinal measurements of pharynx function identified a predictive relationship between mid-life pharynx morphology and function at later ages. These studies demonstrate for the first time that adult tissues undergo distinct structural transitions reflecting postdevelopmental events. The processes that underlie these architectural changes may contribute to increased disease risk during aging, and may be targets for factors that alter the aging rate. This work further demonstrates that pattern analysis of an image series offers a novel and generally accessible approach for quantifying morphological changes and identifying structural biomarkers.

Observation of Noncooperative Folding Thermodynamics in Simulations of 1BBL

One of the predictions of the energy landscape theory of protein folding is the possibility of barrierless, “downhill” folding under certain conditions. The protein 1BBL has been proposed to fold by such a downhill mechanism, though this is a matter of some dispute. We carried out extensive replica exchange molecular dynamics simulations on 1BBL in explicit solvent to address this controversy and provide a microscopic picture of its folding thermodynamics. Our simulations show two distinct structural transitions in the folding of 1BBL. A low-temperature transition involves a disordering of the protein's tertiary structure without loss of secondary structure. A distinct, higher temperature transition involves the complete loss of secondary structure and dissolution of the hydrophobic core. In contrast, control simulations of the 1BBL homolog E3BD show a single high temperature unfolding transition. Further simulations of 1BBL at high ionic strength show a significant destabilization of helix II but not helix I, suggesting that the apparent folding cooperativity of 1BBL may be highly dependent on experimental conditions. Although our simulations cannot provide definitive evidence of downhill folding in 1BBL, they clearly show evidence of a complex, non-two-state folding process.

Purification and Structural Characterization of Human ERp29

ERp29 is a major resident of the endoplasmic reticulum (ER) and is postulated to play an important molecular chaperone role in most animal cells. Human ERp29 was isolated to homogeneity in high yield by using a bacterial expression system. Its secondary structure was studied by circular dichroism (CD), Fourier transformed infrared spectroscopy (FTIR) and Raman spectroscopy and it was found that human ERp29 comprises significant alpha-helical structure. The details of its temperature-induced conformational changes was studied by CD and FTIR for the first time, revealing that the protein is stable below 50 degrees C and has two distinct structural transitions between 50 degrees C and 70 degrees C. This may shed light on ERp29's inability to protect substrate proteins against thermal aggregation.

Nanomechanical properties of desmin intermediate filaments

Desmin intermediate filaments play important role in the mechanical integrity and elasticity of muscle cells. The mechanisms of how desmin contributes to cellular mechanics are little understood. Here, we explored the nanomechanics of desmin by manipulating individual filaments with atomic force microscopy. In complex, hierarchical force responses we identified recurring features which likely correspond to distinct properties and structural transitions related to desmin’s extensibility and elasticity. The most frequently observed feature is an initial unbinding transition that corresponds to the removal of ∼45-nm-long coiled-coil dimers from the filament surface with 20–60 pN forces in usually two discrete steps. In tethers longer than 60 nm we most often observed force plateaus studded with bumps spaced ∼16 nm apart, which are likely caused by a combination of protofilament unzipping, dimer–dimer sliding and coiled-coil-domain unfolding events. At high stresses and strains non-linear, entropic elasticity was dominant, and sometimes repetitive sawtooth force transitions were seen which might arise because of slippage within the desmin protofilament. A model is proposed in which mechanical yielding is caused by coiled-coil domain unfolding and dimer–dimer sliding/slippage, and strain hardening by the entropic elasticity of partially unfolded protofilaments.

Thermal and Alkaline Denaturation of Bovine β-Casein

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.

Single-molecule transition-state analysis of RNA folding.

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.

Model-dielectric-function analysis of ion-implanted Si(100) wafers

Optical properties of Si+, P+, and Ar+ ion-implanted Si(100) wafers have been studied using spectroscopic ellipsometry. The Si+, P+, and Ar+ ions are implanted at 150 keV with fluences ranging from 1×1014 to 2×1015 cm−2 at room temperature. A model dielectric function (MDF), which was developed for modeling the optical constants of perfectly crystalline semiconductors, has been applied to investigate the optical response of the ion-implanted Si(100) wafers. The MDF analysis indicates a distinct structural transition from the crystalline to amorphous phase at some ion fluences around 1014–1015cm−2. Since the critical points do not have any validity in amorphous material, the band gaps used in the MDF are not a result of the Bragg gaps at the Brillouin-zone boundaries, but are considered to arise from the short-range order determined by the covalent bonding. Using these results, we obtain an expression, D=(1.6×108/M)2.2 cm−2 which enables us to estimate the crystalline-amorphous phase transition fluence D for silicon wafers implanted with optional ion species of mass number M. No clear change in the morphology of silicon surface after ion implantation has also been confirmed by atomic force microscopy.

Loss of superconductivity with the addition of Al to MgB2 and a structural transition in Mg1-x AlxB2.

The basic magnetic and electronic properties of most binary compounds have been well known for decades. The recent discovery of superconductivity at 39 K in the simple binary ceramic compound magnesium diboride, MgB2, was therefore surprising. Indeed, this material has been known and structurally characterized since the mid 1950s (ref. 2), and is readily available from chemical suppliers (it is commonly used as a starting material for chemical metathesis reactions). Here we show that the addition of electrons to MgB2, through partial substitution of Al for Mg, results in the loss of superconductivity. Associated with the Al substitution is a subtle but distinct structural transition, reflected in the partial collapse of the spacing between boron layers near an Al content of 10 per cent. This indicates that superconducting MgB2 is poised very near a structural instability at slightly higher electron concentrations.

24] Application of circular dichroism to study RNA folding transitions

Circular dichroism (CD) spectroscopy has been widely used to study secondary structure formation in both proteins and nucleic acids. In this chapter, the application of CD is used to study RNA folding transitions. Modern commercially available CD spectrometers are capable of simultaneous CD and absorbance measurements. The amplitude of the applied photomultiplier voltage (high tension) directly relates to absorbance of the sample and is recorded along with the CD signal. This enables to measure transitions by both probes without any additional measurements. The chapter also discusses the use of CD in combination with Mg2+ or urea titrations to study the secondary and tertiary structural transitions in RNAs. Two-domain ribozyme P RNA have been from Bacillus subtilis folds with two distinct structural transitions, an unfolded to intermediate, U-to-I, transition and an intermediate to native transition. The U-to-I transition occurs at micromolar Mg2+ concentrations and is readily monitored by changes in absorbance (A260) and CD at 260 nm. These signals are primarily sensitive to the formation of helical structure. Tertiary transition occurs in millimolar Mg2+ range with a Hill coefficient of about 4. The formation of the tertiary structure of RNA is the result of structural changes involving the ribosephosphate backbone and non-Watson–Crick base pairing of nucleotides.

Identification of Two Distinct Structural Transitions in the Dissociation of Peptides from Class II Proteins of the Major Histocompatibility Complex

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTIdentification of Two Distinct Structural Transitions in the Dissociation of Peptides from Class II Proteins of the Major Histocompatibility ComplexBenjamin J. McFarland, Andrea J. Sant, and Craig BeesonView Author Information Department of Chemistry, University of Washington Seattle, Washington 98112 Department of Pathology, University of Chicago Chicago, Illinois 60637 Cite this: J. Am. Chem. Soc. 1999, 121, 48, 11225–11226Publication Date (Web):November 13, 1999Publication History Received28 May 1999Published online13 November 1999Published inissue 1 December 1999https://doi.org/10.1021/ja9917767Copyright © 1999 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views34Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (32 KB) Get e-AlertsSUBJECTS:Dissociation,Kinetic parameters,Kinetics,Noncovalent interactions,Peptides and proteins Get e-Alerts

A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL

Single-point mutants of GroEL were constructed with tryptophan replacing a tyrosine residue in order to examine nucleotide-induced structural transitions spectrofluorometrically. The tyrosine residues at positions 203, 360, 476 and 485 were mutated. Of these, the probe at residue 485 gave the clearest fluorescence signals upon nucleotide binding. The probe at 360 reported similar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated timescales, all of which precede hydrolysis of the nucleotide. All four of these rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is proposed to be a T-to-R transition. The next kinetic phase is a rearrangement to the open state identified by electron cryo-microscopy and this we designate an R to R∗ transition. Both of these rearrangements can occur when only a single ring of GroEL is loaded with ATP, and the results are consistent with the occupied ring behaving in a concerted, cooperative manner. At higher ATP concentrations both rings can be loaded with the nucleotide and the R to R∗ transition is accelerated. The resultant GroEL:ATP14 species can then undergo two final rearrangements, RR∗→[RR]+→[RR]#. These final slow steps are completely blocked when ADP occupies the second ring, i.e. it does not occur in the GroEL:ATP7:ADP7 or the GroEL:ATP7 species. All equilibrium and kinetic data conform to a minimal model in which the GroEL ring can exist in five distinct states which then give rise to seven types of oligomeric conformer: TT, TR, TR∗, RR, RR∗, [RR]+ and [RR]#, with concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by inter-ring contacts. This kinetic behaviour is consistent with the GroEL ring passing through distinct functional states in a binding-encapsulation-folding process, with the T-form having high substrate affinity (binding), the R-form being able to bind GroES but retaining substrate affinity (encapsulation), and the R∗-form retaining high GroES affinity but allowing the substrate to dissociate into the enclosed cavity (folding). ADP induces only one detectable rearrangement (designated T to T∗) which has no properties in common with those elicited by ATP. However, asymmetric ADP binding prevents ATP occupying both rings and, hence, restricts the system to the T∗T, T∗R and T∗R∗ complexes.