Rate Of Interconversion Research Articles

Conformational Energy Landscape of the Ritonavir Molecule.

Conformational polymorphism of ritonavir, a well-known pharmaceutical drug, is intricately linked to its efficacy in the treatment of acquired immunodeficiency syndrome (AIDS). Polymorphic transition from the crystalline form I to form II leads to the loss of bioactivity. The constituent ritonavir molecules adopt a trans configuration about the carbamate torsion angle in the form I crystal, and a cis configuration in the form II crystal. Investigating the energetics and mechanistic features of conformational transitions at the single molecule level is a key step toward decoding the complex features of the solid state polymorphism. In this work, we employ the energy landscape framework to investigate the conformational transitions of an isolated ritonavir molecule. The landscape is explored using discrete path sampling (DPS) and visualized in terms of disconnectivity graphs. We identify two distinct funnels corresponding to the two molecular forms that are identified by crystallography. The two regions can be reliably distinguished using the carbamate torsion angle, and the corresponding interconversion rates are predicted to follow Arrhenius behavior. The results provide mechanistic insight into pathways for cis ↔ trans interconversion at the molecular level and may also help in elucidating the polymorphic transitions in the crystal state.

A novel transition pathway of ligand-induced topological conversion from hybrid forms to parallel forms of human telomeric G-quadruplexes.

The folding topology of DNA G-quadruplexes (G4s) depends not only on their nucleotide sequences but also on environmental factors and/or ligand binding. Here, a G4 ligand, 3,6-bis(1-methyl-4-vinylpyridium iodide)-9-(1-(1-methyl-piperidinium iodide)-3,6,9-trioxaundecane) carbazole (BMVC-8C3O), can induce topological conversion of non-parallel to parallel forms in human telomeric DNA G4s. Nuclear magnetic resonance (NMR) spectroscopy with hydrogen-deuterium exchange (HDX) reveals the presence of persistent imino proton signals corresponding to the central G-quartet during topological conversion of Tel23 and Tel25 G4s from hybrid to parallel forms, implying that the transition pathway mainly involves local rearrangements. In contrast, rapid HDX was observed during the transition of 22-CTA G4 from an anti-parallel form to a parallel form, resulting in complete disappearance of all the imino proton signals, suggesting the involvement of substantial unfolding events associated with the topological transition. Site-specific imino proton NMR assignments of Tel23 G4 enable determination of the interconversion rates of individual guanine bases and detection of the presence of intermediate states. Since the rate of ligand binding is much higher than the rate of ligand-induced topological conversion, a three-state kinetic model was evoked to establish the associated energy diagram for the topological conversion of Tel23 G4 induced by BMVC-8C3O.

Configuration Control in the Synthesis of Homo- and Heteroleptic Bis(oxazolinylphenolato/thiazolinylphenolato) Chelate Ligand Complexes of Oxorhenium(V): Isomer Effect on Ancillary Ligand Exchange Dynamics and Implications for Perchlorate Reduction Catalysis.

This study develops synthetic strategies for N,N-trans and N,N-cis Re(O)(LO-N)2Cl complexes and investigates the effects of the coordination spheres and ligand structures on ancillary ligand exchange dynamics and catalytic perchlorate reduction activities of the corresponding [Re(O)(LO-N)2](+) cations. The 2-(2'-hydroxyphenyl)-2-oxazoline (Hhoz) and 2-(2'-hydroxyphenyl)-2-thiazoline (Hhtz) ligands are used to prepare homoleptic N,N-trans and N,N-cis isomers of both Re(O)(hoz)2Cl and Re(O)(htz)2Cl and one heteroleptic N,N-trans Re(O)(hoz)(htz)Cl. Selection of hoz/htz ligands determines the preferred isomeric coordination sphere, and the use of substituted pyridine bases with varying degrees of steric hindrance during complex synthesis controls the rate of isomer interconversion. The five corresponding [Re(O)(LO-N)2](+) cations exhibit a wide range of solvent exchange rates (1.4 to 24,000 s(-1) at 25 °C) and different LO-N movement patterns, as influenced by the coordination sphere of Re (trans/cis), the noncoordinating heteroatom on LO-N ligands (O/S), and the combination of the two LO-N ligands (homoleptic/heteroleptic). Ligand exchange dynamics also correlate with the activity of catalytic reduction of aqueous ClO4(-) by H2 when the Re(O)(LO-N)2Cl complexes are immobilized onto Pd/C. Findings from this study provide novel synthetic strategies and mechanistic insights for innovations in catalytic, environmental, and biomedical research.

Conformational Analysis of P,N‐Containing Eight‐Membered Heterocycles and Their Pt/Ni Complexes in Solution

AbstractIn solution, 1,5‐diaza‐3,7‐diphosphacyclooctanes (PR2NR′2) are in equilibrium between the crown (CW; major) and chair–boat (CB; minor) forms. Aromatic substituent at the nitrogen atom shifts the equilibrium in favor of the CW form. [Pt(PR2NR′2)]Cl2 complexes exist in solution in the CB (CB*) conformation irrespective of the substituent at the heteroatoms. Aromatic substituents on N notably decrease the barrier of CB–CB* conformational exchange. Biligand complexes ([M(PR2NR′2)2]2+, M = Pt, Ni) are in CBCB1 conformation with distortion of the metal coordination plane. The CBCB1 form is in degenerate exchange with the CBCB1* form. In general, distortion of the metal coordination plane and the intramolecular exchange barriers are higher in Ni complexes than in Pt ones. An Ar group at the nitrogen atom increases the heterocycle interconversion rate. Bulky P‐substituents increase the distortion of the metal coordination plane and slow down its “twisting” rate. Counterions with stronger coordinating ability increase the barriers of intramolecular conversion.

Modulating Conformational States in the Glutamate Transporter Homologue Gltph using Protective Osmolytes

Gltph is a sodium dependent aspartate transporter from archaea that is structurally homologous to glutamate transporters and, therefore, provides a model for excitatory amino acid transporters (EAATs). Crystal structures suggest a model for transport, but do not provide structures for intermediate or transition states that must mediate transport. Moreover, conditions that are used for crystallography as well as the crystal lattice may drive the protein into conformations that are not highly populated in bilayers under conditions that support transport. In the present work, distance distributions were measured for Gltph in bilayers though site directed spin labeling (SDSL) and double electron-electron resonance (DEER) to determine if the crystal structures are representative of biologically relevant conformations. Distances were measured both across the subunits of the trimer and within an individual monomer. The protective osmolyte sucrose was used to modulate the populations of individual states and to determine if the conformational states observed by pulse EPR in bilayers were in equilibrium. Distance distributions for the single mutant S278R1 on hairpin loop (HP) 1 revealed three populations with average distances that were close to distances predicted from crystal structures. However, the width of the distance distributions suggests that there are biologically relevant states that are not seen in the crystal structures. The distributions were altered in the presence of sucrose, favoring shorter distances and narrower distributions. These sucrose mediated effects were not equivalent between substrate loaded states or the empty transporter, suggesting that either the conformational equilibrium or the rates of interconversion between states may be modulated by substrate binding.This work was supported by NIGMS, GM035215.

A Comprehensive Description of the Homo and Heterodimerization Mechanism of the Chemokine Receptors CCR5 and CXCR4

Signal transduction across cellular membranes is controlled by G protein-coupled receptors (GPCRs). In this family of transmembrane proteins the binding of extracellular physiological ligands stabilise the active conformation of receptors, leading to cellular response. It is widely accepted that members of the largest GPCR family self-assemble as dimers or higher-order but the functional consequences of the dimerization was described only for few receptors. The chemokines receptors are GPCRs implicated in many physiological processes, including the functioning and maintenance of the immune system. These receptors represent prime targets for therapeutic intervention in a wide spectrum of inflammatory and autoimmune diseases, heart diseases, and HIV. The CXCR4 and CCR5 receptors are two of the manly studied playing crucial roles in different pathologies. It was recently shown that inhibition of the CCR5-CXCR4 heterodimer formation reduces atherosclerosis in a hyperlipidemic mouse model. Furthermore the entry of HIV-1 virus into host cells requires CXCR4 and CCR5 in addition to its main receptor CD4. In this scenario the use of computational techniques able to describe complex biological processes such as protein dimerization acquires a great importance. Combining coarse-grained (CG) molecular dynamics and well-tempered metadynamics (MetaD) we are able to describe the mechanism of dimer formation, capturing multiple association and dissociation events allowing to compute a detailed free energy landscape of the process. CG-MetaD is an enhanced sampling method particularly suitable to describe processes with very slow rates of interconversion among the possible states of the system (i.e. protein dimerization). This approach provides an accurate and comprehensive description of the dimerization free energy landscape, thereby revealing critical motions and important structural-dynamical features (i.e. transient complex, role of lipids) involved in the homo and heterodimer formation of the CCR5 and CXCR4 receptors.

Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity

Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime. Adding to the sensitivity gains realized from rejecting non-modulatable background, the selective signal recovery of immobilized vs diffusing intracellular rsFastLime suggests that DM-SAFIRe can detect weak protein–protein interactions that are normally obscured by large fractions of unbound FPs.

Modulation of helix stability of indolocarbazole-pyridine hybrid foldamers.

Through variation of the central pyridine in an indolocarbazole-pyridine hybrid foldamer, the kinetic stabilities of helical conformations were determined and compared based on the inter-conversion rates of P and M helical isomers.

Light-Controlled Interconversion between a Self-Assembled Triangle and a Rhombicuboctahedral Sphere.

Stimuli-responsive structural reorganizations play an important role in biological processes, often in combination with kinetic control scenarios. In supramolecular mimics of such systems, light has been established as the perfect external trigger. Here, we report on the light-driven structural rearrangement of a small, self-assembled Pd3L6 ring based on photochromic dithienylethene (DTE) ligands into a rhombicuboctahedral Pd24L48 sphere measuring about 6.4 nm across. When the wavelength is changed, this interconversion can be fully reversed, as confirmed by NMR and UV/Vis spectroscopy as well as mass spectrometry. The sphere was visualized by AFM, TEM, and GISAXS measurements. Due to dissimilarities in the photoswitch conformations, the interconversion rates between the two assemblies are drastically different in the two directions.

Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity.

Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime. Adding to the sensitivity gains realized from rejecting non-modulatable background, the selective signal recovery of immobilized vs diffusing intracellular rsFastLime suggests that DM-SAFIRe can detect weak protein-protein interactions that are normally obscured by large fractions of unbound FPs.

Modeling of Cancer Stem Cell State Transitions Predicts Therapeutic Response

Cancer stem cells (CSCs) possess capacity to both self-renew and generate all cells within a tumor, and are thought to drive tumor recurrence. Targeting the stem cell niche to eradicate CSCs represents an important area of therapeutic development. The complex nature of many interacting elements of the stem cell niche, including both intracellular signals and microenvironmental growth factors and cytokines, creates a challenge in choosing which elements to target, alone or in combination. Stochastic stimulation techniques allow for the careful study of complex systems in biology and medicine and are ideal for the investigation of strategies aimed at CSC eradication. We present a mathematical model of the breast cancer stem cell (BCSC) niche to predict population dynamics during carcinogenesis and in response to treatment. Using data from cell line and mouse xenograft experiments, we estimate rates of interconversion between mesenchymal and epithelial states in BCSCs and find that EMT/MET transitions occur frequently. We examine bulk tumor growth dynamics in response to alterations in the rate of symmetric self-renewal of BCSCs and find that small changes in BCSC behavior can give rise to the Gompertzian growth pattern observed in breast tumors. Finally, we examine stochastic reaction kinetic simulations in which elements of the breast cancer stem cell niche are inhibited individually and in combination. We find that slowing self-renewal and disrupting the positive feedback loop between IL-6, Stat3 activation, and NF-κB signaling by simultaneous inhibition of IL-6 and HER2 is the most effective combination to eliminate both mesenchymal and epithelial populations of BCSCs. Predictions from our model and simulations show excellent agreement with experimental data showing the efficacy of combined HER2 and Il-6 blockade in reducing BCSC populations. Our findings will be directly examined in a planned clinical trial of combined HER2 and IL-6 targeted therapy in HER2-positive breast cancer.

Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems.

Cyclophilins catalyze cis ↔ trans isomerization of peptidyl-prolyl bonds, influencing protein folding along with a breadth of other biological functions such as signal transduction. Here, we have determined the microscopic rate constants defining the full enzymatic cycle for three human cyclophilins and a more distantly related thermophilic bacterial cyclophilin when catalyzing interconversion of a biologically representative peptide substrate. The cyclophilins studied here exhibit variability in on-enzyme interconversion as well as an up to 2-fold range in rates of substrate binding and release. However, among the human cyclophilins, the microscopic rate constants appear to have been tuned to maintain remarkably similar isomerization rates without a concurrent conservation of apparent binding affinities. While the structures and active site compositions of the human cyclophilins studied here are highly conserved, we find that the enzymes exhibit significant variability in microsecond to millisecond time scale mobility, suggesting a role for the inherent conformational fluctuations that exist within the cyclophilin family as being functionally relevant in regulating substrate interactions. We have additionally modeled the relaxation dispersion profile given by the commonly employed Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD) experiment when applied to a reversible enzymatic system such as cyclophilin isomerization and identified a significant limitation in the applicability of this approach for monitoring on-enzyme turnover. Specifically, we show both computationally and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed substrate binding and release in reversible systems even at saturating substrate concentrations unless the on-enzyme interconversion rate is much faster than the substrate release rate.

Real sequence effects on the search dynamics of transcription factors on DNA.

Recent experiments show that transcription factors (TFs) indeed use the facilitated diffusion mechanism to locate their target sequences on DNA in living bacteria cells: TFs alternate between sliding motion along DNA and relocation events through the cytoplasm. From simulations and theoretical analysis we study the TF-sliding motion for a large section of the DNA-sequence of a common E. coli strain, based on the two-state TF-model with a fast-sliding search state and a recognition state enabling target detection. For the probability to detect the target before dissociating from DNA the TF-search times self-consistently depend heavily on whether or not an auxiliary operator (an accessible sequence similar to the main operator) is present in the genome section. Importantly, within our model the extent to which the interconversion rates between search and recognition states depend on the underlying nucleotide sequence is varied. A moderate dependence maximises the capability to distinguish between the main operator and similar sequences. Moreover, these auxiliary operators serve as starting points for DNA looping with the main operator, yielding a spectrum of target detection times spanning several orders of magnitude. Auxiliary operators are shown to act as funnels facilitating target detection by TFs.

Harnessing selenocysteine reactivity for oxidative protein folding.

Although oxidative folding of disulfide-rich proteins is often sluggish, this process can be significantly enhanced by targeted replacement of cysteines with selenocysteines. In this study, we examined the effects of a selenosulfide and native versus nonnative diselenides on the folding rates and mechanism of bovine pancreatic trypsin inhibitor. Our results show that such sulfur-to-selenium substitutions alter the distribution of key folding intermediates and enhance their rates of interconversion in a context-dependent manner.

Nuts and Bolts of CF3 and CH 3 NMR Toward the Understanding of Conformational Exchange of GPCRs.

With the advent of efficient protein expression and functional purification protocols, it is now possible to reconstitute many G protein-coupled receptors (GPCRs) in detergent micelles at concentrations of 25 μM or more. Such concentrations are sufficient for studies of conformational states and dynamics relating to function and the mechanism of activation of GPCRs, using solution state NMR. In particular, methyl spectroscopy, in the form of one-dimensional (19)F NMR or two-dimensional ((1)H,(13)C) NMR, provides high fidelity spectra which reveal detailed features associated with conformational states and their lifetimes, as a function of ligand. While X-ray crystallography provides exquisitely detailed structures of lowest energy states associated with ligands, G proteins, and other proteins, NMR is able to validate such states, while providing insight into higher energy states that form part of the conformational landscape and are involved in activation. Through relaxation experiments spanning microseconds to seconds, lifetimes of these functional states can often be measured. By determining the effect of ligands on both equilibrium populations and rates of interconversion between states, it becomes possible to understand activation in terms of an ensemble description and in turn relate the ensemble to pharmaceutical phenomena.

Substitution of Co(2+) ions into CdS-based molecular clusters.

We report on the metal ion exchange between Co(2+) and CdS-based molecular clusters. These studies demonstrate that exchange into the smaller [Cd4(SPh)10](2-) clusters is facile compared to the larger [Cd10S4(SPh)16](4-) and [Cd17S4(SPh)28](2-) clusters. This trend correlates with the rate of benzenethiolate interconversion and μ-S(2-) and μ-SPh(-) content among the clusters.

Structure-biological function relationship extended to mitotic arrest-deficient 2-like protein Mad2 native and mutants-new opportunity for genetic disorder control.

Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN) and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2) to active closed (C-Mad2) conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20) or Mad1) were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR) method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided) and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53–0.65) and fitted correlation r2 (0.82–0.90). Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN.

Micelle-catalyzed domain swapping in the GlpG rhomboid protease cytoplasmic domain.

Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the Escherichia coli GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer-dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states. Catalysis involves the formation of a micelle-bound intermediate with a partially unfolded structure that is primed for domain swapping. Taken together, these results are the first to demonstrate true catalysis for domain swapping, by using micelles that work in a chaperonin-like fashion to unfold a kinetically trapped state and allow access to the domain-swapped form.

The study of separation of delapril’s s-cis/s-trans isomers and development and validation of an analytical method for the estimation of delapril by reversed-phase liquid chromatography

Delapril is an angiotensin converting enzyme (ACE) inhibitor, which exists as a mixture of rotational s-trans and s-cis isomers due to the rotation of the amide bond. Although these stereoisomers do interconvert by rotation about the amide bond, the rate of interconversion is slow, allowing separation of the isomers on the chromatographic time scale. The effect of the flow-rate, column temperature, pH, organic modifier, and counter-ion on the peak shape and the separation of the s-cis and s-trans conformers of delapril was investigated by HPLC. It was proved that the separation selectivity was improved by either a decrease of the flow-rate or an increase of the amount of the organic modifier or the addition of a positively charged counter ion. Delapril was then estimated by a reversed-phase liquid chromatographic method. This method was subsequently validated for specificity, linearity, accuracy and precision, according to the International Conference on Harmonization (ICH) guidelines. An NMR study indicates that the molecule exists as a mixture of rotational cis and trans isomers which confirms the results obtained by HPLC. The isomer ratio integrated from the obtained 1H NMR result was 75:25 in DMSO-d6 at 298 K, where the s-trans isomer was the major form.

Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam

Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between −10°C and −35°C, whereas for flunitrazepam on-column interconversion was observed at temperatures between −40°C and −66°C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At −20°C the enantiomerization barriers, ΔG≠, for diazepam, prazepam and tetrazepam were determined to be in the range 17.6–18.7kcal/mol. At −55°C ΔG≠ for flunitrazepam was determined to be in the 15.6–15.7kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases.