Conformational Interconversion Research Articles

Evidence for dynamic in vivo interconversion of the conformational states of IscU during iron-sulfur cluster biosynthesis.

IscU is a central component of the ISC machinery and serves as a scaffold for de novo assembly of Fe-S clusters. The dedicated chaperone system composed of the Hsp70-chaperone HscA and the J-protein cochaperone HscB synergistically interacts with IscU and facilitates cluster transfer from IscU to recipient apo-proteins. Here, we report that the otherwise essential roles of HscA and HscB can be bypassed in vivo by a number of single amino acid substitutions in IscU. CD spectroscopic studies of the variant IscU proteins capable of this bypass activity revealed dynamic interconversion between two conformations: the denatured (D) and the structured (S) state in the absence and presence of Zn2+ , respectively, which was far more prominent than interconversion observed in wild-type IscU. Furthermore, we found that neither the S-shifted (more structured) variants of IscU nor the perpetually denatured variants could perform their in vivo role regardless of whether the chaperone system was present or not. The present study thus provides for the first time evidence that an in vivo D-state of IscU exists and implies that conformational interconversion between the S- and D-states of the scaffolding protein is a fundamental requirement for the assembly and transfer of the Fe-S cluster.

INTERRELATION OF SERUM ALBUMIN CONFORMATIONAL AND PHASE STATES IN SALT SOLUTIONS AS STUDIED BY EPR SPIN LABELING

The use of serum albumins (SA) in model systems in the study of the biological activity of chemical compounds and nanomaterials, as well as the physicochemical properties, in particular, phase properties of biological fluids, including the nucleoplasm and cytoplasm of living cells, requires a deeper understanding of the relationship of conformational (structural-dynamic) states of protein molecules with phase states of protein dispersions in a wide range of temperatures and compositions. The joint plotting and analysis of the protein stability curve and phase diagrams of protein solutions commonly used in the study of this relationship are significantly complicated by this protein’s elevated tendency to aggregate. In this paper, an experimental-theoretical approach, based on the application of the electron spin resonance (ESR) of spin labels (maleimido-TEMPO and dichlortriasine-TEMPO spin labels) covalently bound to the protein and sensitive to changes in both the structural-dynamic state of protein molecules and the phase state of protein dispersions, is proposed for studying this relationship. Data on the mobility characteristics of the spin labels (correlation times, state functions, equilibrium constants), indicating the state of intra- and intermolecular interactions of SA molecules, are presented. Transitions in temperature dependences of these characteristics on the temperature and concentration of NaCl, CaCl2, (NH4)2SO4, sucrose, polyethylene glycol, and heavy water have been shown to reflect the interconversions of protein conformers and their aggregates as well as liquid-liquid phase transitions, including those of a reentrant type. Based on the results, a phase diagram of the SA dispersion describing liquid-liquid phase transitions in the temperature range of cold and thermal denaturation and taking into account the role of native protein conformers, their aggregates and transitions between them in the physiological interval has been proposed.

Rapid and accurate determination of atomistic RNA dynamic ensemble models using NMR and structure prediction

Biomolecules form dynamic ensembles of many inter-converting conformations which are key for understanding how they fold and function. However, determining ensembles is challenging because the information required to specify atomic structures for thousands of conformations far exceeds that of experimental measurements. We addressed this data gap and dramatically simplified and accelerated RNA ensemble determination by using structure prediction tools that leverage the growing database of RNA structures to generate a conformation library. Refinement of this library with NMR residual dipolar couplings provided an atomistic ensemble model for HIV-1 TAR, and the model accuracy was independently supported by comparisons to quantum-mechanical calculations of NMR chemical shifts, comparison to a crystal structure of a substate, and through designed ensemble redistribution via atomic mutagenesis. Applications to TAR bulge variants and more complex tertiary RNAs support the generality of this approach and the potential to make the determination of atomic-resolution RNA ensembles routine.

Self-Assembly of Switchable Protein Nanocages via Allosteric Effect

Protein cages have promising applications in highly efficient gene transportation. Many methods of engineering protein–interface interactions have been developed to build such viral mimics. Missing...

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

Atropisomerism in Diarylamines: Structural Requirements and Mechanisms of Conformational Interconversion.

In common with other hindered structures containing two aromatic rings linked by a short tether, diarylamines may exhibit atropisomerism (chirality due to restricted rotation). Previous examples have principally been tertiary amines, especially those with cyclic scaffolds. Little is known of the structural requirement for atropisomerism in structurally simpler secondary and acyclic diarylamines. In this paper we describe a systematic study of a series of acyclic secondary diarylamines, and we quantify the degree of steric hindrance in the ortho positions that is required for atropisomerism to result. Through a detailed experimental and computational analysis, the role of each ortho‐substituent on the mechanism and rate of conformational interconversion is rationalised. We also present a simple predictive model for the design of configurationally stable secondary diarylamines.

Atropisomerism in Diarylamines: Structural Requirements and Mechanisms of Conformational Interconversion

AbstractIn common with other hindered structures containing two aromatic rings linked by a short tether, diarylamines may exhibit atropisomerism (chirality due to restricted rotation). Previous examples have principally been tertiary amines, especially those with cyclic scaffolds. Little is known of the structural requirement for atropisomerism in structurally simpler secondary and acyclic diarylamines. In this paper we describe a systematic study of a series of acyclic secondary diarylamines, and we quantify the degree of steric hindrance in the ortho positions that is required for atropisomerism to result. Through a detailed experimental and computational analysis, the role of each ortho‐substituent on the mechanism and rate of conformational interconversion is rationalised. We also present a simple predictive model for the design of configurationally stable secondary diarylamines.

NMR Characterization of Conformational Interconversions of Lys48-Linked Ubiquitin Chains.

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.

Total synthesis and conformational study of ovalifoliolatin B

The diaryletherheptanoid natural product, ovalifoliolatin B, was synthesized. The synthesis features a regioselective reduction of a conjugated dienone and an intramolecular Ullmann etherification. The conformation of the natural product was studied by dynamic NMR methods and by X-ray crystallography. Although the natural product structure is strained, it has a relatively low barrier for interconversion of enantiomeric conformations.

3-Nitrene-2-formylthiophene and 3-Nitrene-2-formylfuran: Matrix Isolation, Conformation, and Rearrangement Reactions.

Two new heteroarylnitrenes, 3-nitrene-2-formylthiophene (15/15') and 3-nitrene-2-formylfuran (16/16'), in the triplet ground state have been generated in solid Ar (10.0 K) and N2 (15.0 K) matrices by the 266 nm laser photolysis of 3-azido-2-formylthiophene (13) and 3-azido-2-formylfuran (14), respectively. According to the characterization with matrix-isolation IR spectroscopy and quantum chemical calculations at the B3LYP/6-311++G(3df,3pd) level, both nitrenes exhibit two conformations depending on the orientation of the formyl groups. Upon subsequent green-light irradiation (532 nm), both the nitrenes 15/15' and 16/16' undergo ring closure to form 3,2-thienoisoxazole (17) and 3,2-furoisoxazole (18), respectively. Traces of 3-imino-4,5-dihydrothiophene-2-ketene (19), formally formed through the intramolecular 1,4-H shift in the corresponding nitrenes 15/15', have been also identified among the laser photolysis products of the azide 13. In sharp contrast to the photochemistry, the high-vacuum flash pyrolysis (HVFP) of the azide 13 at ca. 1000 K mainly yields imino ketene in two conformations 19/19' together with traces of isoxazole 17. In addition to the reversible conformational interconversion in the imino ketene 19 ↔ 19', the photoisomerization from isoxazole 17 to imino ketene 19 has also been observed. The HVFP of the azide 14 at ca. 1000 K results in complete dissociation to HCN, C2H2, CO, CO2, H2O, and N2. Unlike the recently disclosed hydrogen-atom tunneling (HAT) in the transformation from the structurally related 2-formyl phenylnitrene (2) to imino ketene 3 in a cryogenic Ar-matrix, the absence of HAT in nitrenes 15 and 16 can be reasonably explained by the higher barrier heights and also larger barrier widths in the isomerization reactions.

Toward the origin of life over feldspar surfaces: Adsorption of amino acids and catalysis of conformational interconversions

AbstractFeldspars are a group of tectosilicate minerals terminated with a full layer of hydroxyl groups and have been regarded as “hotbed” for birth of life. In this study, periodic density functional theory calculations are conducted to address two associated issues, as adsorption of amino acids and conformational interconversions over feldspar surfaces. Distribution of glycine isomers depends strongly on the number of interacted surface silanols (n), and canonical isomers exist exclusively for n ≤ 2 while zwitterions predominate for n = 3. Adsorption of amino acids is significantly affected by sidechains, especially those forming H‐bonds with surfaces; however, sidechain contacts with feldspar surfaces disfavor zwitterion stabilization, and zwitterions become preferred without sidechain contacts. For all amino acids, conformational interconversions undergo four elementary steps, and proton transfer between two carboxylic‐O atoms (step 2) is always rate‐decisive. Step 2 is greatly accelerated with catalysis of surface silanols and activation barriers depend significantly on sidechains. All amino acids have moderate activation barriers and conformational interconversions (in both forward and reverse directions) proceed favorably at ambient conditions. Sidechains of amino acids exhibit influences through sidechain contacts with surfaces rather than as substituent effect.

Conformational potential energy surfaces and cationic structure of 3,4-dihydro-2H-pyran by VUV-MATI spectroscopy and Franck-Condon fitting.

Ring conformations of 3,4-dihydro-2H-pyran (34DHP) have attracted considerable interest owing to their structural similarity to cyclohexene, an important molecule in stereochemistry. In this study, we investigated the conformational interconversion of 34DHP in both the neutral (S0) and the cationic (D0) ground states. High-resolution vacuum ultraviolet mass-analyzed threshold ionization (VUV-MATI) spectroscopy was utilized to obtain information regarding the adiabatic ionic transition between the S0 and the D0 states. Based on the 0-0 band in the VUV-MATI spectrum supported by the VUV-photoionization efficiency curve, the adiabatic ionization energy of 34DHP was accurately determined to be 8.3355 ± 0.0005 eV (67 230 ± 4 cm-1). To identify the conformer corresponding to this measured value, two-dimensional potential energy surfaces (2D PESs) associated with conformational interconversion in the S0 and the D0 states were constructed at the B3LYP/aug-cc-pVTZ level. It was revealed that in the S0 state, the twisted conformers undergo interconversion through the asymmetric bent conformation on the pseudorotational pathway, whereas in the D0 state, the half-bent conformers directly undergo interconversion via the planar conformation at the saddle point of 2D PES. The change in the conformational interconversion pathway upon ionization is attributed to electron removal from the highest occupied molecular orbital, which consists of a π orbital in the 2C-3C double bond interacting with a nonbonding orbital in the oxygen atom of 34DHP. Then, vibrational assignment of the observed spectrum could be achieved through Franck-Condon fitting for ionic transitions between the neutral twisted and the cationic half-bent conformers. The strong promotion of the ring bending and the 1O-2C-3C asymmetric stretching modes in the adiabatic ionic transitions confirmed the determined cationic structure of 34DHP.

The chemical gymnastics of enterocin: evidence for stereodivergence in Nature.

Stereodivergence in Nature encapsulates both enzymatic (biosynthetic) and non-enzymatic (chemical) diversification of natural product scaffolds arising from a single biosynthetic pathway. Herein, we report a fascinating example of stereodivergence for the bacterial polyketide enterocin, which we observed to undergo a series of facile skeletal rearrangements in solution, leading to four distinct isomeric structures. The final distribution of the four isomers was found to be highly sensitive to the conditions used, including solvent, temperature and pH. In this study, we have investigated the kinetics of these isomeric conversions, and using a combination of DFT and thermochemical calculations, were able to establish a mechanism detailing a concerted rearrangement and an unusual "gymnastic" sequence of pseudo-chair-boat conformational interconversions. In addition to these kinetic and mechanistic studies, we also performed a semisynthetic study aimed at stabilising the enterocin scaffold. In total, seven analogues of enterocin were synthesised and investigated for their stability and in vitro activity against a panel of bacteria, fungi, plants and mammalian cells.

Determination of the cationic conformational structure of tetrahydrothiophene by one-photon MATI spectroscopy and Franck-Condon fitting.

The conformers of tetrahydrothiophene (THT) in the neutral (S0) and cationic (D0) ground states have attracted significant attention in terms of the conformational interconversion through pseudorotation. Herein, these conformers were explored by utilising one-photon mass-analysed threshold ionization (MATI) spectroscopy using the coherently tunable vacuum ultraviolet laser pulse generated by four-wave difference-frequency mixing in Kr medium, which allowed the acquisition of the vibrational spectrum of the corresponding cation. To identify the conformer corresponding to the measured MATI spectrum, the potential energy surfaces associated with pseudorotation in the S0 and D0 states were constructed at the B3LYP/cc-pVTZ level, where the twisted conformer with C2 symmetry in both states lies at the global minimum, while the Cs and C2v conformations were located at the saddle points. Although most of the peaks observed in the spectrum could be assigned as the ionic transitions between the twisted conformers (C2 symmetry) in the S0 and D0 states, distinct nontotally symmetric modes could not be assigned to any allowed vibration. Hence, Franck-Condon fitting was applied for the vibrational assignments in the observed spectrum. This revealed that the cationic conformer had a bent-like twist conformation of C1 symmetry instead of C2 symmetry. Furthermore, the geometrical changes induced by the removal of an electron from the non-bonding orbital of the sulfur atom gave prominent overtones and combination bands of the ring out-of-plane modes associated with pseudorotation as well as the stretching of 2C-1S-3C in the ring.

Large-Scale Conformational Changes and Protein Function: Breaking the in silico Barrier.

Large-scale conformational changes are essential to link protein structures with their function at the cell and organism scale, but have been elusive both experimentally and computationally. Over the past few years developments in cryo-electron microscopy and crystallography techniques have started to reveal multiple snapshots of increasingly large and flexible systems, deemed impossible only short time ago. As structural information accumulates, theoretical methods become central to understand how different conformers interconvert to mediate biological function. Here we briefly survey current in silico methods to tackle large conformational changes, reviewing recent examples of cross-validation of experiments and computational predictions, which show how the integration of different scale simulations with biological information is already starting to break the barriers between the in silico, in vitro, and in vivo worlds, shedding new light onto complex biological problems inaccessible so far.

Theoretical Studies of Structures and Conformational Analysis of Anthracene-2,7-diyl Cyclic Oligomers

Abstract The structures and conformation of anthracene-2,7-diyl cyclic oligomers were investigated by DFT calculations. The energy minimum and transition state structures of the oligomers ranging from pentamer to nonamer were optimized at the M05-2X/6-31G(d) level. The mechanisms of conformational interconversion were analyzed on the basis of the calculated structures and the thermodynamic energies. The number of structures and the conformational flexibility rapidly increased with increasing ring size. In the stable structures, the dihedral angles between the anthracene units were ca. ±40° or ±140°, and the obtuse angles appeared only in large oligomers. We estimated the cavity size of the cyclic oligomers from the calculated structures and their strain energies by thermochemical calculations of the homodesmotic reactions.

Drivers of α-Sheet Formation in Transthyretin under Amyloidogenic Conditions.

Amyloid diseases make up a set of fatal disorders in which proteins aggregate to form fibrils that deposit in tissues throughout the body. Amyloid-associated diseases are challenging to study because amyloid formation occurs on time scales that span several orders of magnitude and involve heterogeneous, interconverting protein conformations. The development of more effective technologies to diagnose and treat amyloid disease requires both a map of the conformations sampled during amyloidogenesis and an understanding of the molecular mechanisms that drive this process. In prior molecular dynamics simulations of amyloid proteins, we observed the formation of a nonstandard type of secondary structure, called α-sheet, that we proposed is associated with the pathogenic conformers in amyloid disease, the soluble oligomers. However, the detailed molecular interactions that drive the conversion to α-sheet remain elusive. Here we use molecular dynamics simulations to interrogate a critical event in transthyretin aggregation, the formation of aggregation-competent, monomeric species. We show that conformational changes in one of the two β-sheets in transthyretin enable solvent molecules and polar side chains to form electrostatic interactions with main-chain peptide groups to facilitate and modulate conversion to α-sheet secondary structure. Our results shed light on the early conformational changes that drive transthyretin toward the α-sheet structure associated with toxicity. Delineation of the molecular events that lead to aggregation at atomic resolution can aid strategies to target the early, critical toxic soluble oligomers.

Synthesis of Forsythenethoside A, a Neuroprotective Macrocyclic Phenylethanoid Glycoside, and NMR Analysis of Conformers.

Forsythenethoside A (1) is a structurally unique macrocyclic phenylethanoid glycoside, which was isolated from Forsynthia suspensa and displayed considerable neuroprotective activities. Here, we report its first chemical synthesis via a longest linear sequence of 14 steps in 5% overall yield wherein intramolecular oxidative coupling was successfully employed to realize the pivotal macrocyclization. NMR analysis revealed the existence of an unexpected conformational interconversion of the congested macrocycles.

Review and Modeling of Crystal Growth of Atropisomers from Solutions

In this paper, theories on anisotropic crystal growth and crystallization of atropisomers are reviewed and a model for anisotropic crystal growth from solution containing slow inter-converting conformers is presented. The model applies to systems with growth-dominated crystallization from solutions and assumes that only one conformation participates in the solute integration step and is present in the crystal lattice. Other conformers, defined as the wrong conformers, must convert to the right conformer before they can assemble to the crystal lattice. The model presents a simple implicit method for evaluating the growth inhibition effect by the wrong conformers. The crystal growth model applies to anisotropic growth in two main directions, namely a slow-growing face and a fast-growing face and requires the knowledge of solute crystal face integration coefficients in both directions. A parameter estimation algorithm was derived to extract those coefficients from data about temporal concentration and crystal size during crystallization and was designed to have a short run time, while providing a high-resolution estimation. The model predicts a size-dependent growth rate and simulations indicated that for a given seed size and solvent system and for an isothermal anti-solvent addition crystallization, the seed loading and the supersaturation at seeding are the main factors impacting the final aspect ratio. The model predicts a decrease of the growth inhibition effect by the wrong conformer with increasing temperature, likely due to faster equilibration between conformers and/or a decrease of the population of the wrong conformer, if of low energy, at elevated temperatures. Finally, the model predicts that solute surface integration becomes the rate-limiting mechanism for high solute integration activation energies, resulting in no impact of the WC on the overall crystal growth process.

Synthesis and anion binding properties of phthalimide-containing corona[6]arenes.

Functionalized O6-corona[3]arene[3]tetrazines were synthesized efficiently and conveniently by means of a macrocyclic condensation reaction between N-functionalized 3,6-dihydroxyphthalimides and 3,6-dichlorotetrazine under mild conditions in a one-pot reaction manner. The novel macrocycles exist as a mixture of rapidly interconvertible conformers in solution while in the solid state they adopt the conformation in which three phthalimide units are cis,trans-orientated. Acting as electron-deficient macrocyclic hosts, the synthesized O6-corona[3]arene[3]tetrazines self-regulated conformational structures to complex anions in the gas phase and in the solid state owing to the anion–π noncovalent interactions between anions and the tetrazine rings.