Differences in Conformational Behavior of ATA and TAT Sequences in Single Strand DNA Trimer

Abstract

The conformational behavior of single strand (ss) TAT and ATA trimers of DNA have been studied by computational chemistry tools including CICADA software interfaced with AMBER molecular mechanics and dynamics. The Single-Coordinate-Driving (SCD) method has been used in conjunction with molecular dynamics simulated annealing. It has been revealed that the conformational flexibility of each sequence differs substantially from the other one. Four common conformational families have been found for both trimers. These are: helical, reverse-stacked (base 3), half-stacked (base 3), reverse-stacked (base 1). However, the energies of conformers representing the families are different for both the studied systems. An additional conformational family, bulged, has been found for ss(ATA), while ss(TAT) has been found also in half-stacked (base 1) conformation. In general, ss(TAT) exhibits a higher number of low energy conformations while ss(ATA) shows one interesting low energy conformational interconversion between reverse-stacked (A3) family and half-stacked (A3) family. The high conformational variability of the trimers has been confirmed by flexibility analysis and by molecular dynamics simulations, which have also shown the conformational stability of single conformational families. It has been concluded that the methodology used is able to provide a very detailed picture of the conformational space of these molecules.     

Thermodynamic and Structural Properties of r(ACC) as Revealed by Ultraviolet Electronic Absorption,Circular Dichroism, 1H-NMR Spectroscopy and Monte Carlo Simulations

Abstract

UV absorption, circular dichroïsm (CD) and ¹H NMR, associated with Monte Carlo (MC) molecular structure simulations have been applied to the study of the trinucleoside diphosphate: r(ACC).

The MC study which has been conducted as a function of temperature, is based on random variations of the nucleotide conformational angles, i.e. phosphodiester chain torsional angles and sugar pucker pseudorotational angles. All of the chemical bond lengths and valence angles remained fixed during the structural simulation, except those of the sugar pucker. Six different initial structures have been selected in order to explore the molecular conformational space as completely as possible. This simulation procedure led to distinct families of equilibrium conformations at 283,298 and 318 K.

The thermodynamical parameters such as variations in entropy, enthalpy and also melting temperature (ΔS⁰ _x, ΔH⁰ _x and T_m) of the stacking (X) equilibrium were obtained from UV absorption and circular dichroïsm (CD) spectra recorded over a 80K temperature range. Chemical shifts (δ), vicinal coupling constants(³J _k) and cross-relaxation rates (σ_k,l) of trimers were measured at 400.13 MHz over a range of concentrations (2–13mM) and temperatures (283–333K). Least-squares fitting of the experimental chemical shifts to simple models of association (A) and stacking equilibria allowed separation of the variations in the δ values (Δδ_x and Δδ_A) due to either phenomenon. The three NMR data sets (Δδ_x, ³J^k,l and σ_k.l) were then evaluated for the minima conformers obtained with the MC simulations. Theoretical values of Δδ_x were estimated using the results of an ab initio study while the coupling constant data were simulated with Karplus-type equations. Finally, the relaxation data were simulated from the distance matrices using treatment for cases of both slow conformational exchange accompanied by rapid small-amplitude fluctuations about the minima structures.

A consistent picture of the large amplitude deformations (torsional angle variation) of these trimers has emerged from the present study. Optimized conformational blends at 283, 296 and 318K were obtained by least-squares fitting of the experimental data to the theoretical ones, while considering the populations as adjustable parameters. As it would be expected, the right-handed helical conformation (A-RNA type) is found to be the major stacked species, in the temperature range of 283 to 318K. Limited evidence for bulged structures has been obtained, whereas novel reverse-stacked and half-stacked conformers also presented theoretical data compatible with the NMR observables of aqueous r(ACC).     

NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

Conformational flexibility of two RNA trimers explored by computational tools and database search

Two RNA sequences, AAA and AUG, were studied by the conformational search program CICADA and by molecular dynamics (MD) in the framework of the AMBER force field, and also via thorough PDB database search. CICADA was used to provide detailed information about conformers and conformational interconversions on the energy surfaces of the above molecules. Several conformational families were found for both sequences. Analysis of the results shows differences, especially between the energy of the single families, and also in flexibility and concerted conformational movement. Therefore, several MD trajectories (altogether 16 ns) were run to obtain more details about both the stability of conformers belonging to different conformational families and about the dynamics of the two systems. Results show that the trajectories strongly depend on the starting structure. When the MD start from the global minimum found by CICADA, they provide a stable run, while MD starting from another conformational family generates a trajectory where several different conformational families are visited. The results obtained by theoretical methods are compared with the thorough database search data. It is concluded that all except for the highest energy conformational families found in theoretical result also appear in experimental data. Registry numbers: adenylyl-(3' --> 5')-adenylyl-(3' --> 5')-adenosine [917-44-2] adenylyl-(3' --> 5')-uridylyl-(3' --> 5')-guanosine [3494-35-7].     

Conformational Flexibility of Two RNA Trimers Explored by Computational Tools and Database Search

Abstract

Two RNA sequences, AAA and AUG, were studied by the conformational search program CICADA and by molecular dynamics (MD) in the framework of the AMBER force field, and also via thorough PDB database search. CICADA was used to provide detailed information about conformers and conformational interconversions on the energy surfaces of the above molecules. Several conformational families were found for both sequences. Analysis of the results shows differences, especially between the energy of the single families, and also in flexibility and concerted conformational movement. Therefore, several MD trajectories (altogether 16 ns) were run to obtain more details about both the stability of conformers belonging to different conformational families and about the dynamics of the two systems. Results show that the trajectories strongly depend on the starting structure. When the MD start from the global minimum found by CICADA, they provide a stable run, while MD starting from another conformational family generates a trajectory where several different conformational families are visited. The results obtained by theoretical methods are compared with the thorough database search data. It is concluded that all except for the highest energy conformational families found in theoretical result also appear in experimental data.

Registry numbers:

adenylyl-(3′ →5′)-adenylyl-(3′ →5′)-adenosine [917-44-2]

adenylyl-(3′ →5′)-uridylyl-(3′ →5′)-guanosine [3494-35-7]     

Characterization of the dynamic behavior of r(ACC) and r(AAC) with NMR relaxation data and both metropolis monte carlo and molecular dynamics simulations

The solution-state behavior of two triribonucleotides, adenylyl(3′-5′) adenylyl (3′-5′) cytidine [r(AAC)] and adenylyl (3′-5′) cytidylyl (3′-5′) cytidine [r(ACC)], was studied with spectroscopic and molecular modeling methods. Melting temperatures of 299 and 294 K for r(AAC) and r(ACC), respectively, were obtained from ultraviolet absorption (UV) and circular dichroism (CD) temperature profiles of the order-disorder transition. The behavior of the Raman marker modes is consistent with greater stability of r(AAC) compared to that of r(ACC). Nuclear magnetic resonance (nmr) relaxation data (homonuclear cross-relaxation rates, proton selective and nonselective longitudinal relaxation times, and carbon longitudinal relaxation times) were measured at 283, 296, and 318 K for both trimers. In parallel, the major types of conformations were explored with Metropolis Monte Carlo (MMC) and molecular dynamics (MD) simulations to obtain representations of both slow and fast events. Fitting of experimental data showed that although the MMC conformations do not represent an exhaustive list of conformers in solution, the canonical helical form (A-RNA type) should coexist at low temperature with significant populations of other less classical conformers such as half-stacked (HS), bulged (BU), and reverse-stacked (RS). Fitting of the experimental relaxation data ensemble at 283 K led to very different representations for the two trimers. r(AAC) was shown to have a fairly compact, rigid structure (angular order parameter, S²_ang ∼ 0.9, correlation time for internal motion, τ_e ∼ 0.1 ns), which undergoes fairly rapid overall tumbling characterized by the correlation time τ_c ∼ 0.6 ns, whereas r(ACC) exhibits much more flexibility (S²_ang ∼ 0.7, τ_e ∼ 0.1 ns) and slower molecular reorientation (τ_c ∼ 1.0 ns). The values of S²_ang tended to confirm that large amplitude fluctuations did not occur on the relaxation timescale (ns). In the course of this paper, a widely accepted concept was shown to be questionable. As regards the nmr relaxation data, simulations show that for fairly small nucleic acids (τ_c < 2.0 ns) the second term of the model-free spectral densities is not negligible for representative motional models (S²_ang values < 0.9 and τ_e values in the 0.05–0.2 ns range). The difference in the dynamic behavior of r(AAC) and r(ACC) can be explained by the greater propensity of the A-A sequence to stack as compared to that of A-C. © 1996 John Wiley & Sons, Inc.     

A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein

The crystallographic dimer of the C-terminal fragment (CTF) of the L7/L12 ribosomal protein has been subjected to molecular dynamics (MD) simulations. A 90 picosecond (ps) trajectory for the protein dimer, 19 water molecules and two counter ions has been calculated at constant temperature. Effects of intermolecular interactions on the structure and dynamics have been studied. The exact crystallographic symmetry is lost and the atomic fluctuations differ from one monomer to the other. The average MD structure is more stable than the X-ray one, as judged by accessible surface area and energy calculations. Crystal (non-dimeric) interactions have been simulated in another 40 ps trajectory by using harmonic restraints to represent intermolecular hydrogen bonds. The conformational changes with respect ot the X-ray structure are then virtually suppressed.The unrestrained dimer trajectory has been scanned for cooperative motions involving secondary structure elements. The intrinsic collective motions of the monomer are transmitted via intermolecular contacts to the dimer structure.The existence of a stable dimeric form of CTF, resembling the crystallographic one, has been documented. At the cost of fairly small energy expenditure the dimer has considerable conformational flexibility. This flexibility may endow the dimer with some functional potential as an energy transducer.     

Molecular dynamics simulations of a K+ channel blocker: Tc1 toxin from Tityus cambridgei

Toxins that block voltage-gated potassium (Kv) channels provide a possible template for improved homology models of the Kv pore. In assessing the interactions of Kv channels and their toxins it is important to determine the dynamic flexibility of the toxins. Multiple 10 ns duration molecular dynamics simulations combined with essential dynamics analysis have been used to explore the flexibility of four different Kv channel-blocking toxins. Three toxins (Tc1, AgTx and ChTx) share a common fold. They also share a common pattern of conformational dynamics, as revealed by essential dynamics analysis of the simulation results. This suggests that some aspects of dynamic behaviour are conserved across a single protein fold class. In each of these three toxins, the residue exhibiting minimum flexibility corresponds to a conserved lysine residue that is suggested to interact with the filter domain of the channel. Thus, comparative simulations reveal functionally important conservation of molecular dynamics as well as protein fold across a family of related toxins.     

Structure and Dynamics of Double Helical DNA in Torsion Angle Hyperspace: A Molecular Mechanics Approach

Abstract

Analysis of the conformational space populated by the torsion angles and the correlation between the conformational energy and the sequence of DNA are important for fully understanding DNA structure and function. Presence of seven variable torsion angles about single covalent bonds in DNA main chain puts a big challenge for such analysis. We have carried out restrained energy minimization studies for four representative dinucleosides, namely d(ApA):d(TpT), d(CpG):d(CpG), d(GpC):d(GpC) and d(CpA):d(TpG) to determine the energy hyperspace of DNA in context to the values of the torsion angles and the structural properties of the DNA conformations populating the favorable regions of this energy hyperspace. The torsion angles were manipulated by constraining their values at the reference points and then performing energy minimization. The energy minima obtained on the potential energy contour plots mostly correspond to the conformations populated in crystal structures of DNA. Some novel favorable conformations that are not present in crystal structure data are also found. The plots also suggest few low energy routes for conformational transitions or the associated energy barrier heights. Analyses of base pairing and stacking possibility reveal structural changes accompanying these transitions as well as the flexibility of different base steps towards variations in different torsion angles.     

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and Immunoglobulin M

Glycoprotein-glycans have recently been implicated to play a variety of functional roles. The same glycan chain have been found complexed with proteins of diverse functions. In this article two such glycan chains found attached to Fc regions of immunoglobulin G and immunoglobulin M have been studied. An extensive simulated annealing procedure have been adopted to arrive at a low-energy minimum of the two oligosaccharides. Molecular dynamics simulations have been performed to study the flexibility of the glycosidic linkages. It was found that both glycan chains can undergo conformational transitions and adopt folded and extended conformations. The two β(1–2) linkages of complex-type glycan had been found to prefer different conformational regime and the terminal fucose linked to the GlcNAc residue drastically modifies the GlcNAc β(1–4)GlcNAc linkage conformation. In the high-mannose type glycan chain α(1–3) linkages can induce flexibility in addition to the α(1–6) linkages. The results have been compared with recent experimental nmr and fluorescence energy transfer data. © 1998 John Wiley & Sons, Inc. Biopoly 45: 177–190, 1998     

Structural properties of plant and mammalian lipoxygenases. Temperature-dependent conformational alterations and membrane binding ability

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).     

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution

A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational 'leaps' to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques.     

Single-strand conformation polymorphism (SSCP) of oligodeoxyribonucleotides: an insight into solution structural dynamics of DNAs provided by gel electrophoresis and molecular dynamics simulations

Studies on the solution structure dynamics of RNA/DNA are becoming crucially important. The phenomena of SSCP (single-strand conformation polymorphism), small RNA dynamics in a cell, and others can be related to the conformational changes of single-stranded (ss) RNAs/DNAs in solution. However, little is known about those dynamics. Only the intra-structural transition of ssDNAs in solution has been reported based on Watson-Crick (W-C) base-pairing. Here, we found a general feature of the SSCP phenomenon by studying the simpler molecules of ss-oligodeoxyribonucleotides. A single base substitution or a positional exchange of nucleotide in a highly homologous series of ss-dodecanucleotides led to a change in the mobility-in-gel. This was unexpected, since most of these nucleotides [such as d(A(11)G) or d(A(11)C)] have no possibility of forming W-C base-pairing. MD (molecular dynamics) experiments revealed differences in shape and size between the dynamic structures of these molecules which could affect their mobility-in-gel. In addition, a high correlation was observed between the electrophoretic mobility and the size-related parameters such as end-to-end distance obtained from MD simulations. Because the simulation was considerably shorter (nanosecond) than the experimental time-scale (second), the result must be considered conservatively; but it is nevertheless encouraging for utilizing MD simulation for structural analysis of oligonucleotides.     

Criticality in the conformational phase transition among self-similar groups in intrinsically disordered proteins: Probed by salt-bridge dynamics

Intrinsically disordered proteins (IDP) serve as one of the key components in the global proteome. In contrast to globular proteins, they harbor an enormous amount of physical flexibility enforcing them to be retained in conformational ensembles rather than stable folds. Previous studies in an aligned direction have revealed the importance of transient dynamical phenomena like that of salt-bridge formation in IDPs to support their physical flexibility and have further highlighted their functional relevance. For this characteristic flexibility, IDPs remain amenable and accessible to different ordered binding partners, supporting their potential multi-functionality. The current study further addresses this complex structure-functional interplay in IDPs using phase transition dynamics to conceptualize the underlying (avalanche type) mechanism of their being distributed across and hopping around degenerate structural states (conformational ensembles). For this purpose, extensive molecular dynamics simulations have been done and the data analyzed from a statistical physics perspective. Investigation of the plausible scope of 'self-organized criticality' (SOC) to fit into the complex dynamics of IDPs was found to be assertive, relating the conformational degeneracy of these proteins to their functional multiplicity. In accordance with the transient nature of 'salt-bridge dynamics', the study further uses it as a probe to explain the structural basis of the proposed criticality in the conformational phase transition among self-similar groups in IDPs. The analysis reveal scale-invariant self-similar fractal geometries in the structural conformations of different IDPs. The insights from the study has the potential to be extended further to benefit structural tinkering of IDPs in their functional characterization and drugging.     

Thermodynamic contributions for the incorporation of GTA triplets within canonical TAT/TAT and C+GC/C+GC base-triplet stacks of DNA triplexes

Nucleic acid triple helices may be used in the control of gene expression. One limitation of using triplex-forming oligonucleotides as therapeutic agents is that their target sequences are limited to homopurine tracts. To increase the repertoire of sequences that can be targeted, it has been postulated that a guanine can target a thymidine forming a stable GTA mismatch triplet. In this work, we have used a combination of optical and calorimetric techniques to determine thermodynamic unfolding profiles of two triplexes containing a single GTA triplet, d(A(3)TA(3)C(5)T(3)AT(3)C(5)T(3)GT(3)) (ATA) and d(AGTGAC(5)TCACTC(5)TCGCT) (GTG), and their control triplexes, d(A(7)C(5)T(7)C(5)T(7)) (TAT7) and d(AGAGAC(5)TCTCTC(5)TCTCT) (AG5T). In general, the presence of a GTA mismatch in DNA triplexes is destabilizing; however, this destabilization is greater when placed in a C(+)GC/C(+)GC base-triplet stack than between a TAT/TAT stack. These destabilizations are accompanied by a reduced unfolding enthalpy of approximately 10 kcal/mol, suggesting a decrease in the base stacking contributions surrounding the mismatch. Relative to their corresponding control triplexes, the folding of ATA is accompanied by a lower counterion uptake and a similar proton uptake, while GTG folding is accompanied by an increase in the counterion and proton uptakes. These effects are consistent with the observed decrease in stacking interactions. The overall results indicate that the main difficulty of targeting pyrimidine interruptions is that the decrease in stacking contributions, due to the incorporation of a GTA mismatch, affects the stability of the neighboring base triplets. This suggests that nucleotide analogues that increase the strength of these base-triplet stacks will result in a more effective targeting of pyrimidine interruptions.     

Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.     

Evolution of structurally disordered proteins promotes neostructuralization

Protein structure is generally more conserved than sequence, but for regions that can adopt different structures in different environments, does this hold true? Understanding how structurally disordered regions evolve altered secondary structure element propensities as well as conformational flexibility among paralogs are fundamental questions for our understanding of protein structural evolution. We have investigated the evolutionary dynamics of structural disorder in protein families containing both orthologs and paralogs using phylogenetic tree reconstruction, protein structure disorder prediction, and secondary structure prediction in order to shed light upon these questions. Our results indicate that the extent and location of structurally disordered regions are not universally conserved. As structurally disordered regions often have high conformational flexibility, this is likely to have an effect on how protein structure evolves as spatially altered conformational flexibility can also change the secondary structure propensities for homologous regions in a protein family.     

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin

Determining the three-dimensional structure of myoglobin, the first solved structure of a protein, fundamentally changed the way protein function was understood. Even more revolutionary was the information that came afterward: protein dynamics play a critical role in biological functions. Therefore, understanding conformational dynamics is crucial to obtaining a more complete picture of protein evolution. We recently analyzed the evolution of different protein families including green fluorescent proteins (GFPs), β-lactamase inhibitors, and nuclear receptors, and we observed that the alteration of conformational dynamics through allosteric regulation leads to functional changes. Moreover, proteome-wide conformational dynamics analysis of more than 100 human proteins showed that mutations occurring at rigid residue positions are more susceptible to disease than flexible residue positions. These studies suggest that disease-associated mutations may impair dynamic allosteric regulations, leading to loss of function. Thus, in this study, we analyzed the conformational dynamics of the wild-type light chain subunit of human ferritin protein along with the neutral and disease forms. We first performed replica exchange molecular dynamics simulations of wild-type and mutants to obtain equilibrated dynamics and then used perturbation response scanning (PRS), where we introduced a random Brownian kick to a position and computed the fluctuation response of the chain using linear response theory. Using this approach, we computed the dynamic flexibility index (DFI) for each position in the chain for the wild-type and the mutants. DFI quantifies the resilience of a position to a perturbation and provides a flexibility/rigidity measurement for a given position in the chain. The DFI analysis reveals that neutral variants and the wild-type exhibit similar flexibility profiles in which experimentally determined functionally critical sites act as hinges in controlling the overall motion. However, disease mutations alter the conformational dynamic profile, making hinges more loose (i.e., softening the hinges), thus impairing the allosterically regulated dynamics.     

Conformational dynamics of the mitochondrial ADP/ATP carrier: a simulation study

The mitochondrial ADP/ATP carrier is a six helix bundle membrane transport protein, which couples the exit of ATP from the mitochondrial matrix to the entry of ADP. Extended (4×20 ns) molecular dynamics simulations of the carrier, in the presence and absence of bound inhibitor (carboxyatractyloside), have been used to explore the conformational dynamics of the protein in a lipid bilayer environment, in the presence and absence of the carboxyatractyloside inhibitor. The dynamic flexibility (measured as conformational drift and fluctuations) of the protein is reduced in the presence of bound inhibitor. Proline residues in transmembrane helices H1, H3 and H5 appear to form dynamic hinges. Fluctuations in inter-helix salt bridges are also observed over the time course of the simulations. Inhibitor-protein and lipid-protein interactions have been characterised in some detail. Overall, the simulations support a transport mechanism in which flexibility about the proline hinges enables a transition between a ‘closed’ and an ‘open’ pore-like state of the carrier protein.