Conformational Interconversion Research Articles

Unraveling the Dynamic Molecular Motions of a Twin‐Cavity Cage with Slow Configurational but Rapid Conformational Interconversions

AbstractFeaturing diverse structural motions/changes, dynamic molecular systems hold promise for executing complex tasks. However, their structural complexity presents formidable challenge in elucidating their kinetics, especially when multiple structural motions are intercorrelated. We herein introduce a twin‐cavity cage that features interconvertible C3‐ and C1‐configurations, with each configuration exhibiting interchangeable P‐ and M‐conformations. This molecule is therefore composed of four interconnected chiral species (P)‐C3, (M)‐C3, (P)‐C1, (M)‐C1. We showcase an effective approach to decouple these sophisticated structural changes into two kinetically distinct pathways. Utilizing time‐dependent 1H NMR spectroscopy at various temperatures, which disregards the transition between mirror‐image conformations, we first determine the rate constant (kc) for the C3‐ to C1‐configuration interconversion, while time‐dependent circular dichroism spectroscopy at different temperatures quantifies the observed rate constant (kobs) of the ensemble of all the structural changes. As kobs kc, it allows us to decouple the overall molecular motions into a slow configurational transformation and rapid conformational interconversions, with the latter further dissected into two independent conformational interchanges, namely (P)‐C3 (M)‐C3 and (P)‐C1 (M)‐C1. This work, therefore, sheds light on the comprehensive kinetic study of complex molecular dynamics, offering valuable insights for the rational design of smart dynamic materials for applications of sensing, separation, catalysis, molecular machinery, etc.

Unraveling the Dynamic Molecular Motions of a Twin-Cavity Cage with Slow Configurational but Rapid Conformational Interconversions.

Featuring diverse structural motions/changes, dynamic molecular systems hold promise for executing complex tasks. However, their structural complexity presents formidable challenge in elucidating their kinetics, especially when multiple structural motions are intercorrelated. We herein introduce a twin-cavity cage that features interconvertible C3- and C1-configurations, with each configuration exhibiting interchangeable P- and M-conformations. This molecule is therefore composed of four interconnected chiral species (P)-C3, (M)-C3, (P)-C1, (M)-C1. We showcase an effective approach to decouple these sophisticated structural changes into two kinetically distinct pathways. Utilizing time-dependent 1H NMR spectroscopy at various temperatures, which disregards the transition between mirror-image conformations, we first determine the rate constant (kc) for the C3- to C1-configuration interconversion, while time-dependent circular dichroism spectroscopy at different temperatures quantifies the observed rate constant (kobs) of the ensemble of all the structural changes. As kobs kc, it allows us to decouple the overall molecular motions into a slow configurational transformation and rapid conformational interconversions, with the latter further dissected into two independent conformational interchanges, namely (P)-C3 (M)-C3 and (P)-C1 (M)-C1. This work, therefore, sheds light on the comprehensive kinetic study of complex molecular dynamics, offering valuable insights for the rational design of smart dynamic materials for applications of sensing, separation, catalysis, molecular machinery, etc.

Normal-to-Supercooled Liquid Transition in Molecular Glass-Formers: A Hidden Structural Transformation Fuelled by Conformational Interconversion.

Molecular dynamics and transport coefficients change significantly around the so-called Arrhenius crossover in glass-forming systems. In this article, we revisit the dynamic processes occurring in a glass-forming macrocyclic crown thiaether MeBzS2O above its glass transition, revealing two crossover temperatures: TB at 309 and TA at 333 K. We identify the second one as the Arrhenius crossover that is closely related to the normal-to-supercooled liquid transition in this compound. We show that the transformation occurring at this point goes far beyond molecular dynamics (where the temperature dependence of structural relaxation times changes its character from activation-like to super-Arrhenius), being reflected also in the internal structure and diffraction pattern. In this respect, we found a twofold local organization of the nearest-neighbor molecules via weak van der Waals forces, without the formation of any medium-range order or mesophases. The nearest surrounding of each molecule evolves structurally in time due to the ongoing fast conformational changes. We identify several conformers of MeBzS2O, demonstrating that its lowest-energy conformation is preferred mainly at lower temperatures, i.e., in the supercooled liquid state. Its increased prevalence modifies locally the short-range intermolecular order and promotes vitrification. Consequently, we indicate that the Arrhenius transition is fuelled rather by conformational changes in this glass-forming macrocyclic crown thiaether, which is a different scenario from the so-far existing concepts. Our studies combine broadband dielectric spectroscopy (BDS), X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations.

Taming Conformational Heterogeneity on Ion Racetrack to Unveil Principles that Drive Membrane Permeation of Cyclosporines.

Our study reveals the underlying principles governing the passive membrane permeability in three large N-methylated macrocyclic peptides (N-MeMPs): cyclosporine A (CycA), Alisporivir (ALI), and cyclosporine H (CycH). We determine a series of conformers required for robust passive membrane diffusion and those relevant to other functions, such as binding to protein targets or intermediates, in the presence of solvent additives. We investigate the conformational interconversions and establish correlations with the membrane permeability. Nuclear magnetic resonance (NMR) and cyclic ion-mobility spectrometry-mass spectrometry (cIMS-MS) are employed to characterize conformational heterogeneity and identify cis-amides relevant for good membrane permeability. In addition, ion mobility selected cIMS-MS and infrared (IR) multiple-photon dissociation (IRMPD) spectroscopy experiments are conducted to evaluate the energy barriers between conformations. We observe that CycA and ALI, both cyclosporines with favorable membrane permeabilities, display multiple stable and well-defined conformers. In contrast, CycH, an epimer of CycA with limited permeability, exhibits fewer and fewer stable conformers. We demonstrate the essential role of the conformational shift from the aqueous cis MeVal11-MeBmt1 state (A1) to the closed conformation featuring cis MeLeu9-MeLeu10 (C1) in facilitating membrane permeation. Additionally, we highlight that the transition from A1 to the all-trans open conformation (O1) is specifically triggered by the presence of CaCl2. We also capture a set of conformers with cis Sar3-MeLeu4, MeLeu9-MeLeu10, denoted as I. Conformationally selected cIMS-MS and IRMPD data of [CycA+Ca]2+ show immediate repopulation of the original population distribution, suggesting that CaCl2 smooths out the energy barriers. Finally, our work presents an improved sampling molecular dynamics approach based on a refined force field that not only consistently and accurately captures established conformers of cyclosporines but also exhibits strong predictive capabilities for novel conformers.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient), and state 2 (active, effector binding enabled). Here we use 31P NMR to delineate the differences in state 1 and state 2 populations present in wild-type (WT) and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analogue GppNHp. Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in the state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RBD (RAS Binding Domain) binding and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small molecule inhibitor, BBO-8956, which is effective against both GDP and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs the state 1 - state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1 RBD is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action (MOA) for this novel, active-conformation inhibitor.

Theoretical structural and spectroscopic characterization of peroxyacetic acid (CH3-CO-OOH): study of the far infrared region.

Peroxyacetic acid, a non-rigid oxygenated organic molecule which acts in the atmosphere as a reservoir of HOX and ROX radicals, is studied using highly correlated ab initio methods with the aim of its spectroscopic characterization in the gas phase. The study focuses on the far infrared region providing reliable rovibrational parameters such as energy levels and splittings. The molecule presents three conformers that inter-convert by internal rotation, drawing a potential energy surface of 12 minima. One of them shows prominent stability due to the formation of one weak intramolecular bond between the hydrogen atom of the hydroperoxy group and the oxygen atom of the carbonyl group. For the three minimum energy structures, rotational constants and centrifugal distortion constants are provided. It may be expected that the most stable conformer is the only one contributing to the spectral features in further measurements at low temperature. In this structure, the methyl torsional barrier has been found to be very low, V3 = 88.6 cm-1 producing a splitting of 2.262 cm-1 for the ground vibrational state. The study confirms that the ν20 torsional mode interacts strongly with the other two torsional modes ν19 and ν21, but slightly with the remaining vibrations. Then, a variational procedure in three dimensions allows the exploration of the low-frequency modes. The methyl torsional fundamental ν21 was found to be 49.1 cm-1 (Ai) and 33.4 cm-1 (E). The fundamentals of ν20 (C-O bond torsion) and ν19 (OH torsion) have been computed to be 216.7 cm-1 (A2) and 218.5 cm-1 (E) and 393.6 cm-1 (A2) and 394.1 cm-1. Since non-rigidity can have effects on the reactivity due to the conformer interconversion, and transitions involving low-lying levels can be observed with many spectroscopic techniques, this work can help kinetic studies and assignments of further spectroscopic studies needed for the detection in the gas phase of trace molecules.

Dimerized sesquiterpenoid [4 + 2] adducts with ferroptosis-promoting activity from Inula britannica Linn

Inubritanolides C and D (1 and 2), two exo sesquiterpenoid [4 + 2] adducts with unprecedented interconverting conformations of twist-chair and chair, together with two previously undescribed endo [4 + 2] dimers (3 and 4) were discovered from Inula britannica flowers. Dimers 1 and 2 have an undescribed carbon skeleton comprising of eudesmanolide and guaianolide units with the linkage mode of C-11/C-1ʹ and C-13/C-3ʹ via a Diels-Alder cycloaddition reaction. Their structures were elucidated using 1D/2D NMR, X-ray diffraction, ECD, and variable-temperature NMR experiments. Dimer 2 displayed a strong inhibitory effect on breast cancer cells by promoting lipid ROS production, showing its potential as ferroptosis inducer.

Studies of DNA 'Breathing' by Polarization-Sweep Single-Molecule Fluorescence Microscopy of Exciton-Coupled (iCy3)2 Dimer-Labeled DNA Fork Constructs.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA 'breathing') play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here, we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3)2] dimer probes. Previous work has shown that the constructs that we tested here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3)2 dimer has one symmetric (+) and one antisymmetric (-) exciton, with the respective transition dipole moments oriented perpendicular to one another. We excite single-molecule samples using a continuous-wave linearly polarized laser, with the polarization direction continuously rotated at the frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and antisymmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and the conformational interconversion dynamics of the (iCy3)2 probe. We find that at most construct positions that we examined, the (iCy3)2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome-regulatory proteins.

An NMR-based approach for the design of crystallization processes for rigid molecules and effects of molecular rigidity and impurities on crystallization pathways

A methodology based on NMR analysis to enable development of crystallization procedures for relatively rigid molecules is presented. The number of relatively stable conformers in solution and their relative proportions are determined from peak splitting of equivalent protons and their relative areas as measured by NMR. Experimental data indicated that slow conformational interconversion was limiting crystallization at low temperatures. Variable temperature NMR analysis was utilized to determine rotamers’ peak coalescence and identify a suitable crystallization solvent. This NMR-based approach led to the development of a manufacturing crystallization recipe of a relatively rigid molecule that was impossible to crystallize via a scalable process. Furthermore, prediction of relatively stable conformer in gas phase, determined by Molecular Dynamics (MD) calculations were in acceptable agreement with data obtained from NMR analyses. Finally, the effects of molecular rigidity and impurities on crystallization and oiling-out pathways are described and an approach to avoid oiling-out is presented.

The molecular basis for cellular function of intrinsically disordered protein regions.

Intrinsically disordered protein regions exist in a collection of dynamic interconverting conformations that lack a stable 3D structure. These regions are structurally heterogeneous, ubiquitous and found across all kingdoms of life. Despite the absence of a defined 3D structure, disordered regions are essential for cellular processes ranging from transcriptional control and cell signalling to subcellular organization. Through their conformational malleability and adaptability, disordered regions extend the repertoire of macromolecular interactions and are readily tunable by their structural and chemical context, making them ideal responders to regulatory cues. Recent work has led to major advances in understanding the link between protein sequence and conformational behaviour in disordered regions, yet the link between sequence and molecular function is less well defined. Here we consider the biochemical and biophysical foundations that underlie how and why disordered regions can engage in productive cellular functions, provide examples of emerging concepts and discuss how protein disorder contributes to intracellular information processing and regulation of cellular function.

Locking the Conformation of a Paddlewheel Rhodium Complex: Design, Synthesis, and Applications in Catalytic Nitrene Transfers

AbstractThe exponential proliferation of conformers makes it impossible to examine the entire population in most systems. Controlling conformational ensembles is thus pivotal in many areas of chemistry. Rh2(esp)2, a dicarboxylate‐derived paddlewheel rhodium complex, is one of the most effective catalysts for nitrene chemistry. Its enormous success has led to preparing many analogous complexes. However, there has been little consideration for the conformational dynamics of the parent catalyst. Herein, we report a new ligand modification principle that prevents conformer interconversion. The resulting complex comprises two isolable conformers, whose structures have been determined by X‐ray diffraction. Combined experimental and computational data has revealed similarities and dissimilarities between the conformationally confined and parent complexes. Three model cases have demonstrated the utility of conformational fixation in the development of stereoselective catalysts for nitrene transfer reactions. The design principle described in this study can be combined with other established modification strategies, serving as a springboard for further advancement of the chemistry of paddlewheel metal complexes.

Locking the Conformation of a Paddlewheel Rhodium Complex: Design, Synthesis, and Applications in Catalytic Nitrene Transfers.

The exponential proliferation of conformers makes it impossible to examine the entire population in most systems. Controlling conformational ensembles is thus pivotal in many areas of chemistry. Rh2(esp)2, a dicarboxylate-derived paddlewheel rhodium complex, is one of the most effective catalysts for nitrene chemistry. Its enormous success has led to preparing many analogous complexes. However, there has been little consideration for the conformational dynamics of the parent catalyst. Herein, we report a new ligand modification principle that prevents conformer interconversion. The resulting complex comprises two isolable conformers, whose structures have been determined by X-ray diffraction. Combined experimental and computational data has revealed similarities and dissimilarities between the conformationally confined and parent complexes. Three model cases have demonstrated the utility of conformational fixation in the development of stereoselective catalysts for nitrene transfer reactions. The design principle described in this study can be combined with other established modification strategies, serving as a springboard for further advancement of the chemistry of paddlewheel metal complexes.

When Phenyl and Cyclopropyl Rings Meet: Spectroscopic Shifts and Conformational Questions.

The electronic and vibrational spectra of cyclopropylbenzene (CPB) and 1,3-bromocyclopropylbenzene (BrCPB) in the gas phase were investigated using quantum chemical calculations in combination with resonance-enhanced multi-photon ionization REMPI techniques including 1c-R2PI, UV-UV holeburning, and IR-UV ion depletion in the CH stretch region. The electronic spectra revealed the presence of a single conformer for both species, with the absence of any perpendicular conformer attributed to low computed barriers to conformer interconversion. Assignment of CPB to the bisected conformer was made through interpreting distinctive CH stretch bands in the IR-UV spectrum in conjunction with quantum chemical calculations. A local anharmonic model based on DFT calculations was adapted to reproduce the cyclopropyl CH stretch spectrum successfully. It was not feasible to definitively assign which bisected conformer of BrCPB was observed using vibrational information alone due to the close similarity of their predicted IR spectra. However, conformational sensitivity of the S1 ← S0 transition dipole moment (TDM) alignments leads to simulated rotational contours that display stark differences, which prompted assignment to the "B1" bisecting conformer with the cyclopropyl ring directed away from the bromine atom. The absence of the energetically comparable "B2" conformer is unexpected. The analysis of the convolution of aromatic and aliphatic modes serves as a basis for assignment in constrained aliphatic systems.

Understanding the Conformational Interconversions of a Polymer Chain in a Liquid Environment at the Single-molecule Level

Understanding the Conformational Interconversions of a Polymer Chain in a Liquid Environment at the Single-molecule Level

Mutational and Environmental Effects on the Dynamic Conformational Distributions of Lys48-Linked Ubiquitin Chains.

In multidomain proteins, individual domains connected by flexible linkers are dynamically rearranged upon ligand binding and sensing changes in environmental factors, such as pH and temperature. Here, we characterize dynamic domain rearrangements of Lys48-linked ubiquitin (Ub) chains as models of multidomain proteins in which molecular surfaces mediating intermolecular interactions are involved in intramolecular domain-domain interactions. Using NMR and other biophysical techniques, we characterized dynamic conformational interconversions of diUb between open and closed states regarding solvent exposure of the hydrophobic surfaces of each Ub unit, which serve as binding sites for various Ub-interacting proteins. We found that the hydrophobic Ub-Ub interaction in diUb was reinforced by cysteine substitution of Lys48 of the distal Ub unit because of interaction between the cysteinyl thiol group and the C-terminal segment of the proximal Ub unit. In contrast, the replacement of the isopeptide linker with an artificial ethylenamine linker minimally affected the conformational distributions. Furthermore, we demonstrated that the mutational modification allosterically impacted the exposure of the most distal Ub unit in triUb. Thus, the conformational interconversion of Ub chains offers a unique design framework in Ub-based protein engineering not only for developing biosensing probes but also for allowing new opportunities for the allosteric regulation of multidomain proteins.

Elucidation of a dynamic interplay between a beta-2 adrenergic receptor, its agonist, and stimulatory G protein

G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (β2AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (Gs). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to β2AR by Gs protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from β2AR and the conformational interconversions of Gs between closed and open conformations in the NE(+)-β2AR-Gs ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter Gs α subunit (Gsα) conformational transitions. Our simulations showed that the interdomain movement and the stacking of Gsα α1 and α5 helices are significant for increasing the distance betweenthe Gsαand β2AR, which may indicate apartial dissociation ofGsαThe distance increase commences when Gsα is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of β2AR interacting with Gsα, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation.

Computational investigation of conformational fluctuations of Aβ42 monomers in aqueous ionic liquid mixtures

Aggregation of intrinsically disordered proteins (IDPs) is often triggered by protein–protein interaction, leading to several neurodegenerative diseases.In this study, we aim to understand the role of 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4] ionic liquid (IL) on altering secondary structure of the peptide and study the interactions responsible for their conformational modification. Here we report structural features of an ensemble of amyloid beta (Aβ42) peptide monomers employing all-atom molecular dynamics simulations in aqueous solution of IL at room temperature. Our calculations reveal, Aβ42 peptide exhibit rapidly inter-converting conformations adopting extended chain as well as collapsed coil-like structures with a propensity to form few transient α-helices and β-sheets at intermediate times. We have correlated this higher flexibility of the peptide with formation of less stable salt-bridge in presence of IL in aqueous solution. It is found that water molecules around the polar regions are more structured imparting structural rigidity to the peptide backbone than around the hydrophobic domains. Thus identifying the effect of electrostatic interaction of IL on expulsion of water molecules from the polar domains on the peptide surface can shed light on preventing the aggregation process. We have also determined the most energetically stable peptide conformations from various accessible intermediate states using metadynamics simulations that are characterized by the rugged free energy surface having multiple energy basins.

Simultaneous Tunneling Control in Conformer-Specific Reactions.

We present here a new example of chemical reactivity governed by quantum tunneling, which also highlights the limitations of the classical theories. The syn and anti conformers of a triplet 2-formylphenylnitrene, generated in a nitrogen matrix, were found to spontaneously rearrange to the corresponding 2,1-benzisoxazole and imino-ketene, respectively. The kinetics of both transformations were measured at 10 and 20 K and found to be temperature-independent, providing clear evidence of concomitant tunneling reactions (heavy-atom and H-atom). Computations confirm the existence of these tunneling reaction pathways. Although the energy barrier between the nitrene conformers is lower than any of the observed reactions, no conformational interconversion was observed. These results demonstrate an unprecedented case of simultaneous tunneling control in conformer-specific reactions of the same chemical species. The product outcome is impossible to be rationalized by the conventional kinetic or thermodynamic control.

Alternative RNA Conformations: Companion or Combatant.

RNA molecules, in one form or another, are involved in almost all aspects of cell physiology, as well as in disease development. The diversity of the functional roles of RNA comes from its intrinsic ability to adopt complex secondary and tertiary structures, rivaling the diversity of proteins. The RNA molecules form dynamic ensembles of many interconverting conformations at a timescale of seconds, which is a key for understanding how they execute their cellular functions. Given the crucial role of RNAs in various cellular processes, we need to understand the RNA molecules from a structural perspective. Central to this review are studies aimed at revealing the regulatory role of conformational equilibria in RNA in humans to understand genetic diseases such as cancer and neurodegenerative diseases, as well as in pathogens such as bacteria and viruses so as to understand the progression of infectious diseases. Furthermore, we also summarize the prior studies on the use of RNA structures as platforms for the rational design of small molecules for therapeutic applications.

Ring-System-Based Conformational Switches and their Applications in Sensing and Liposomal Drug Delivery

AbstractIn the past decades, a great number of stimuli-responsive systems have been developed to be used as drug-delivery systems with high sensitivity and selectivity in targeted therapy. Despite promising results, the current stimuli-responsive systems suffer from the complexity of preparation, as most novel stimuli-responsive systems are based on polymers. Small molecules have often been neglected as candidates for application for stimuli-responsive systems. Recently, structures based on six-membered ring molecules or bicyclic molecules have been developed into conformational switches working through conformational interconversion. These single conformational switches have significantly reduced the complexity of material preparation compared to polymers or copolymers. In this review, we focus on ring-system-based conformational switches that are involved in sensors and smart drug-delivery systems. We hope that this review will shed light on ring-system-based single conformational switches for use in the development of stimuli-responsive systems.1 Introduction2 Conformation Switches Based On Bispidine Derivatives3 Conformation Switches Based On Cycloalkanes4 Conformation Switches Based On Carbohydrates5 Conclusion