Conformational Energy Research Articles

Thermal adaptation of mRNA secondary structure: stability versus lability

Macromolecular function commonly involves rapidly reversible alterations in three-dimensional structure (conformation). To allow these essential conformational changes, macromolecules must possess higher order structures that are appropriately balanced between rigidity and flexibility. Because of the low stabilization free energies (marginal stabilities) of macromolecule conformations, temperature changes have strong effects on conformation and, thereby, on function. As is well known for proteins, during evolution, temperature-adaptive changes in sequence foster retention of optimal marginal stability at a species' normal physiological temperatures. Here, we extend this type of analysis to messenger RNAs (mRNAs), a class of macromolecules for which the stability-lability balance has not been elucidated. We employ in silico methods to determine secondary structures and estimate changes in free energy of folding (ΔGfold) for 25 orthologous mRNAs that encode the enzyme cytosolic malate dehydrogenase in marine mollusks with adaptation temperatures spanning an almost 60 °C range. The change in free energy that occurs during formation of the ensemble of mRNA secondary structures is significantly correlated with adaptation temperature: ΔGfold values are all negative and their absolute values increase with adaptation temperature. A principal mechanism underlying these adaptations is a significant increase in synonymous guanine + cytosine substitutions with increasing temperature. These findings open up an avenue of exploration in molecular evolution and raise interesting questions about the interaction between temperature-adaptive changes in mRNA sequence and in the proteins they encode.

Could the spanning of NAM-AD subsites by poly (ADP ribose) polymerase inhibitors potentiate their selective inhibitory activity in breast cancer treatment? Insight from biophysical computations

ABSTRACT Poly (ADP-ribose) polymerase (PARP) inhibition by pharmacological agents has been a useful cancer treatment in recent times. PARP inhibitors represent a category of anticancer drugs that function by blocking PARP catalytic activity. The design of highly selective and effective PARP inhibitors has significant therapeutic advantages and remains an integral part of several PARP-based drug discovery research. We employed conformational landscape and binding energy analyses to decipher the mechanisms underlying the inhibitory potencies and selectivity of Cpd81 towards PARP enzymes. Relatively, the thermodynamics analysis revealed that pp2-cpd81 (−55.38 kcal/mol) had a higher ΔGbind than pp1-cpd81 (−27.97 kcal/mol), indicating high selective binding activity of Cpd81 towards PARP-2. The binding activity of Cpd81 to PARP-2 was potentiated by the high effects of van der Waal. Moreso, the NAM-AD positioning of Cpd81 facilitated its optimal orientation for multiple high-affinity complementary interactions with key site residues. Resultantly, Cpd81 could span the two subsites of the PARP binding pocket effectively, as seen in the multiple interactions exhibited by the compound. These interactions serve as the basis for the selective inhibitory potential of Cpd81. We believe the findings here would pave the way for developing PARP inhibitors that are selective in breast cancer treatment.

Conformational Change in Molecular Crystals: Impact of Solvate Formation and Importance of Conformational Free Energies

Changes in molecular conformation and its relationship with crystal polymorphism have been well documented in previous work. To the best of our knowledge, however, the effect of solvate formation on molecular conformation has never been explored. Using the Cambridge Structural Database in combination with molecular modeling, we provide insights into the impact of solvate formation on the conformation adopted by a compound and whether such conformation is different to that found in its neat form(s). Typically, the more flexible a molecule is, the higher the chances that conformational change occurs upon solvate formation. There are no trends as to the relative stability of conformers and their likelihood to be observed in either the solvates or the neat forms. Typically, conformer energy differences in solvate-neat form pairs are small (<5 kJ/mol) and when larger energy differences are observed (>15 kJ/mol), these can be reduced significantly when both solvent and thermal effects are considered in the simulations. This highlights of the importance of computing thermal contributions in conformer energies as well as accounting for environmental effects. Overall, we find that conformational change in solvate-neat form pairs mirrors the behavior of conformational change in polymorphism.

Exploring the energy landscape of a SAM-I riboswitch

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg2+ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-l-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg2+ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated ‘hub’ state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription.

The reorganization energy of compounds upon binding to proteins, from dynamic and solvated bound and unbound states

The intramolecular reorganization energy (ΔEReorg) of compounds upon binding to proteins is a component of the binding free energy, which has long received particular attention, for fundamental and practical reasons. Understanding ΔEReorg would benefit the science of molecular recognition and drug design. For instance, the tolerable strain energy of compounds upon binding has been elusive. Prior studies found some large ΔEReorg values (e.g. > 10 kcal/mol), received with skepticism since they imply excessive opposition to binding. Indeed, estimating ΔEReorg is technically difficult. Typically, ΔEReorg has been approached by taking two energy-minimized conformers representing the bound and unbound states, and subtracting their conformational energy. This is a drastic oversimplification, liable to conformational collapse of the unbound conformer. Instead, the present work applies extensive molecular dynamics (MD) and the modern OPLS3 force-field to simulate compounds bound and unbound states, in explicit solvent under physically relevant conditions. The thermalized unbound compounds populate multiple conformations, not reducible to one or a few energy-minimized conformers. The intramolecular energies in the bound and unbound states were averaged over pertinent conformational ensembles, and the reorganization enthalpy upon binding (ΔHReorg) deduced by subtraction. This was applied to 76 systems, including 43 approved drugs, carefully selected for i) the quality of the bioactive X-ray structures and ii) the diversity of the chemotypes, their properties and protein targets. It yielded comparatively low ΔHReorg values (median = 1.4 kcal/mol, mean = 3.0 kcal/mol). A new finding is the observation of negative ΔHReorg values. Indeed, reorganization energies do not have to oppose binding, e.g. when intramolecular interactions stabilize preferentially the bound state. Conversely, even with competing water molecules, intramolecular interactions can occur predominantly in the unbound compound, and be replaced by intermolecular counterparts upon protein binding. Such disruption of intramolecular interactions upon binding gives rise to occasional larger ΔHReorg values. Such counterintuitive larger ΔHReorg values may be rationalized as a redistribution of interactions upon binding, qualitatively compatible with binding.

How to strike a conformational balance in protein force fields for molecular dynamics simulations?

AbstractMolecular dynamics (MD) simulation is a powerful tool for exploring the conformational energy landscape of proteins, and the reliability of MD results is crucially dependent on the underlying force field (FF). An accurate FF capable of producing balanced distributions of diverse conformations at multiple levels has been a long‐sought goal. Towards this, several decades of joint efforts have been made to address FF deficiencies, manifested by conformational biases at different levels (local conformations, secondary structures, and global extendedness of polypeptide chain). We first present the major FF biases, then review the strategies to address them separately. Specifically, both nonresidue‐specific and residue‐specific strategies for torsional parameter optimization have been applied to achieve local conformation and secondary structure balances. Significant improvements can be gained with residue‐specific torsional parameters especially when explicit dihedral couplings are considered. Further, the additional balance between protein–protein and protein–water interactions has been optimized via multiple ways to reproduce the global extendedness of polypeptide chains, especially for unfolded or disordered proteins. This review aims to summarize the most valuable experience and lessons gained from the past, which, we hope, can facilitate further improvements of both classical FFs and more sophisticated models such as polarizable FFs.This article is categorized under: Molecular and Statistical Mechanics &gt; Molecular Dynamics and Monte‐Carlo Methods Molecular and Statistical Mechanics &gt; Molecular Mechanics

Can ionic effects induce α-sheet conformation of Peptides?

We report coupled cluster, MP2 and DFT data on the relative energy and geometry of α-sheet and β-strand conformations of model peptides. We show that ionic effects have a strong effect on energy balance through formation of multiple cation-oxygen contacts in the α-sheet form. Such effects are markedly dependent on both sequence and ion: two peptides considered favour α-sheet in the presence of cations, whereas a third, non-polar one favours β-strand in the same conditions.

Boosting the conformational sampling by combining replica exchange with solute tempering and well-sliced metadynamics.

Methods that combine collective variable (CV) based enhanced sampling and global tempering approaches are used in speeding-up the conformational sampling and free energy calculation of large and soft systems with a plethora of energy minima. In this paper, a new method of this kind is proposed in which the well-sliced metadynamics approach (WSMTD) is united with replica exchange with solute tempering (REST2) method. WSMTD employs a divide-and-conquer strategy wherein high-dimensional slices of a free energy surface are independently sampled and combined. The method enables one to accomplish a controlled exploration of the CV-space with a restraining bias as in umbrella sampling, and enhance-sampling of one or more orthogonal CVs using a metadynamics like bias. The new hybrid method proposed here enables boosting the sampling of more slow degrees of freedom in WSMTD simulations, without the need to specify associated CVs, through a replica exchange scheme within the framework of REST2. The high-dimensional slices of the probability distributions of CVs computed from the united WSMTD and REST2 simulations are subsequently combined using the weighted histogram analysis method to obtain the free energy surface. We show that the new method proposed here is accurate, improves the conformational sampling, and achieves quick convergence in free energy estimates. We demonstrate this by computing the conformational free energy landscapes of solvated alanine tripeptide and Trp-cage mini protein in explicit water.

Efficient generation of self-avoiding, semiflexible rotational isomeric chain ensembles in bulk, in confined geometries, and on surfaces

We provide an efficient ready-to-run code gensaw that generates single or large ensembles of self-avoiding, flexible, semiflexible, rotationally isometric or helical chains in the bulk or subject to arbitrary confinement and tethering conditions, where we allow for arbitrary intramolecular bending and dihedral energy functions. The resulting configuration files are provided in various common formats and can be immediately used to do molecular simulations or statistical analysis. We work out analytic expressions for the mean squared end-to-end distance and gyration radius of the semiflexible, helical and rotational isomeric state models with a finite number of bonds and arbitrary interaction potentials for direct comparison and testing of the code in the limiting case of unconfined phantom chains. In addition to the graphics-free linux standalone batch version gensaw that creates configuration and other files for high throughput applications from the command line, we provide an interactive online version gensaw-visualization that serves as platform-independent graphical user interface, and animates the resulting conformations using a remote gensaw server. Program summaryProgram title:gensawCPC Library link to program files:https://doi.org/10.17632/8xfgf88f3w.1Licensing provisions: MITProgramming languages: fortran 90, perl (standalone batch version) or java (interactive online version)Required software:ifort 16+ or gfortran 4+, perl 5+ (standalone batch version), jre 1.8.0+ (interactive online version)Nature of problem: Efficient creation of self-avoiding, semiflexible multibead walks/chains of variable contour length, subject to arbitrary confinement and tethering conditions, characterized by a conformational energy that is composed of arbitrary bending and torsion contributions. The code must produce an ensemble of such chains, using a flexible output format. For the limiting case of chains with zero thickness, the code should reproduce analytic results for the ideal chains.Solution method: We use an adapted enrichment procedure that optimizes its parameters on the fly and derive all analytic results for semiflexible chains with arbitrary bending and torsion potentials for comparison with the created ensembles. While gensaw creates configuration and optionally other files from the command line, gensaw-visualization serves as a platform-independent graphical user interface and allows to visualize and animate the conformations.

The sequence–ensemble relationship in fuzzy protein complexes

Intrinsically disordered proteins (IDPs) interact with globular proteins through a variety of mechanisms, resulting in the structurally heterogeneous ensembles known as fuzzy complexes. While there exists a reasonable comprehension on how IDP sequence determines the unbound IDP ensemble, little is known about what shapes the structural characteristics of IDPs bound to their targets. Using a statistical thermodynamic model, we show that the target-bound ensembles are determined by a simple code that combines the IDP sequence and the distribution of IDP-target interaction hotspots. These two parameters define the conformational space of target-bound IDPs and rationalize the observed structural heterogeneity of fuzzy complexes. The presented model successfully reproduces the dynamical signatures of target-bound IDPs from the NMR relaxation experiments as well as the changes of interaction affinity and the IDP helicity induced by mutations. The model explains how the target-bound IDP ensemble adapts to mutations in order to achieve an optimal balance between conformational freedom and interaction energy. Taken together, the presented sequence-ensemble relationship of fuzzy complexes explains the different manifestations of IDP disorder in folding-upon-binding processes.

Docking and dynamic simulation study of Crizotinib and Temozolomide drug with Glioblastoma and NSCLC target to identify better efficacy of the drug

BackgroundCrizotinib and Temozolomide are the two major chemotherapy drugs used for the treatment of cancers. Crizotinib is used as a target chemotherapy drug in many cancers. It mainly binds on the ATP binding regions of receptor tyrosine kinases (RTKs) targets and inhibits protein phosphorylation, which has already been reported. Temozolomide drug is known as the alkylating agent. Its mechanism of action is the methylation of DNA and thereby inhibiting DNA replication. However, the Temozolomide drug with protein level interaction of Glioblastoma Multiforme (GBM) and Non-small-cell lung carcinoma (NSCLC) of RTKs targets has not been reported so far. In the proposed work, we investigated the molecular level interaction of the Temozolomide drug in C-MET, C-ROS1, and ALK RTKs targets of GBM and NSCLC using an in silico study. We performed comparative analysis studies in both drugs' docked complexes based on their drug properties and complex energy (CE) to identify the better efficacy of the drug.ResultsFrom the docking studies, we could identify that the Temozolomide drug bounded protein complexes showed the least complex energy. The most stable complexes were identified from these docking studies by Molecular Dynamic simulation. In the proposed study, we found that the docked complex attained a stable conformation and least energy via solid hydrogen bond interactions between the amino acid residues and the drug at the binding sites of the proteins. The least energy and the hydrogen bond interaction of Temozolomide drug with the amino acid residues of the protein complexes of C-MET, C-ROS1 and ALK protein with their id name are: 2WGJ is − 11305.0830 (PRO1158, MET1160), 3ZBF is − 11,659.6814 (MET2029, GLU2027), and 2XP2 is − 11,734.7565 (ARG1275, ASP 1160, GLU1167).ConclusionOur studies revealed that the Temozolomide drug bounded protein complex showed the least energy when compared to Crizotinib. So it will give better interaction on the binding sites of proteins and thereby provide better inhibition in the treatment of target therapy of GBM and NSCLC.

Entropy-Enthalpy Compensations Fold Proteins in Precise Ways.

Exploring the protein-folding problem has been a longstanding challenge in molecular biology and biophysics. Intramolecular hydrogen (H)-bonds play an extremely important role in stabilizing protein structures. To form these intramolecular H-bonds, nascent unfolded polypeptide chains need to escape from hydrogen bonding with surrounding polar water molecules under the solution conditions that require entropy-enthalpy compensations, according to the Gibbs free energy equation and the change in enthalpy. Here, by analyzing the spatial layout of the side-chains of amino acid residues in experimentally determined protein structures, we reveal a protein-folding mechanism based on the entropy-enthalpy compensations that initially driven by laterally hydrophobic collapse among the side-chains of adjacent residues in the sequences of unfolded protein chains. This hydrophobic collapse promotes the formation of the H-bonds within the polypeptide backbone structures through the entropy-enthalpy compensation mechanism, enabling secondary structures and tertiary structures to fold reproducibly following explicit physical folding codes and forces. The temperature dependence of protein folding is thus attributed to the environment dependence of the conformational Gibbs free energy equation. The folding codes and forces in the amino acid sequence that dictate the formation of β-strands and α-helices can be deciphered with great accuracy through evaluation of the hydrophobic interactions among neighboring side-chains of an unfolded polypeptide from a β-strand-like thermodynamic metastable state. The folding of protein quaternary structures is found to be guided by the entropy-enthalpy compensations in between the docking sites of protein subunits according to the Gibbs free energy equation that is verified by bioinformatics analyses of a dozen structures of dimers. Protein folding is therefore guided by multistage entropy-enthalpy compensations of the system of polypeptide chains and water molecules under the solution conditions.

Performance of small basis set Hartree–Fock methods for modeling non-covalent interactions

Non-covalent interactions (NCIs) play an essential role in (bio)chemistry. Wavefunction-based methods combined with large basis sets are able to accurately describe inter-and intra-molecular NCIs but are not practical for large molecular systems. Semi-empirical corrections have been developed recently that, when combined with Hartree–Fock (HF) and a small basis set, show promise in the ability to predict non-covalent binding and conformational energies over a wide range of systems. Compared to large-basis-set correlated wavefunction methods, small-basis-set HF methods significantly lower computational cost and are useful for modeling large molecular systems with sizes between many hundreds and a few thousand atoms. Using a large collection of non-covalent binding energies, conformational energies, and molecular deformation energies containing 105 880 entries, we provide a comprehensive evaluation of the performance of the minimal basis set (MINIX) HF method with three correction schemes: D3, 3c, and atom-centered potentials (ACPs). We also evaluate the performance of HF/6-31G* in combination with the D3 and ACP schemes. By comparing the three corrections, we analyze the strengths and weaknesses associated with each strategy in predicting NCIs. Our results show that D3 corrections alone do not offer significant improvements in the performance of HF/MINIX or HF/6-31G* and, in some cases, overestimate binding energies resulting in large errors when compared to the reference data. The correction strategies that offer the best reduction in the underlying errors of HF/MINIX and HF/6-31G* are shown to be 3c and ACP for HF/MINIX and ACP for HF/6-31G*.

Conformational energy (A-value) of the 4-phenyl-1,2,3-triazolyl group

Conformational energy (A-value) of the 4-phenyl-1,2,3- triazolyl group was estimated as 2.0 kcal mol–1 (8.5 kJ mol–1) using 1H NMR-based conformational study for a set of (4-phenyl-1,2,3-triazol-1-yl)cyclohexanes. This provides a reasonable estimation also for the conformational energy of 1,2,3-triazolyl group itself and its substituted analogues.

Effect of the powerful plasticity of the tert-butyl side chain on the conformational equilibrium of ascidiacyclamides.

Ascidiacyclamide [cyclo(-Ile1,5 -oxazoline2,6 -d-Val3,7 -thiazole4,8 -)2 ] is a cytotoxic cyclic peptide from ascidian. Through structural analyses using monosubstituted analogues (Xaa1 : Ala, 2-aminobutyric acid, Val, cyclohexylglycine, and phenylglycine), we previously demonstrated the conformational equilibrium between its square and folded forms. As the bulkiness of the Xaa1 residue side chain was reduced, spontaneous folding was promoted, and the cytotoxicity decreased accordingly. In the present study, five disubstituted analogues in which a tert-leucine residue (Tle) was incorporated at the 5-position of the abovementioned monosubstituted analogues were synthesized, after which their structures were analyzed using X-ray diffraction, circular dichroism (CD) spectral measurements, and 1 H NMR-based quantitative analysis. The side chains of the Tle and Ile residues are structural isomers of one another, and the Tle residue bearing the tert-butyl group can be expected to play a role as a building block. In fact, peptides incorporating Tle5 exhibited much less spontaneous folding than their Ile5 counterparts in both crystal and solution. Increases in enthalpy and entropy due to the tert-butyl group during the folding process resulted in increased conformational free energy (ΔG°). The powerful plasticity of the tert-butyl group would stabilize the square form relating with cytotoxicity.

Multiscale computational study of ligand binding pathways: Case of p38 MAP kinase and its inhibitors

Protein kinases are one of the most important drug targets in the past 10 years. Understanding the inhibitor association processes will profoundly impact new binder designs with preferred binding kinetics. However, after more than a decade of effort, a complete atomistic-level study of kinase inhibitor binding pathways is still lacking. As all kinases share a similar scaffold, we used p38 kinase as a model system to investigate the conformational dynamics and free energy transition of inhibitor binding toward kinases. Two major kinase conformations, Asp-Phe-Gly (DFG)-in and DFG-out, and three types of inhibitors, type I, II, and III, were thoroughly investigated in this work. We performed Brownian dynamics simulations and up to 340 μs Gaussian-accelerated molecular dynamics simulations to capture the inhibitor binding paths and a series of conformational transitions of the p38 kinase from its apo to inhibitor-bound form. Eighteen successful binding trajectories, including all types of inhibitors, are reported herein. Our simulations suggest a mechanism of inhibitor recruitment, a faster ligand association step to a pre-existing DFG-in/DFG-out p38 protein, followed by a slower molecular rearrangement step to adjust the protein-ligand conformation followed by a shift in the energy landscape to reach the final bound state. The ligand association processes also reflect the energetic favor of type I and type II/III inhibitor binding through ATP and allosteric channels, respectively. These different binding routes are directly responsible for the fast (type I binders) and slow (type II/III binders) kinetics of different types of p38 inhibitors. Our findings also echo the recent study of p38 inhibitor dissociation, implying that ligand unbinding could undergo a reverse path of binding, and both processes share similar metastates. This study deepens the understanding of molecular and energetic features of kinase inhibitor-binding processes and will inspire future drug development from a kinetic point of view.

Maximizing TADF via Conformational Optimization.

We investigate a new strategy to enhance thermally activated delayed fluorescence (TADF) in organic light-emitting diodes (OLEDs). Given that the TADF rate of a molecule depends on its conformation, we hypothesize that there exists a conformation that maximizes the TADF rate. To test this idea, we use time-dependent density functional theory (TDDFT) to simulate the TADF rates of several TADF emitters while varying their geometries in a select subspace of internal coordinates. We find that geometric changes in this subspace can increase the TADF rate up to 3 orders of magnitude with respect to the minimum energy conformation, and the simulated TADF rate can even be brought into the submicrosecond time scales under the right conditions. Furthermore, the TADF rate enhancement can be maintained with a conformational energy that might be within the reach of modern synthetic chemistry. Analyzing the maximum TADF conformation, we extract a number of structural motifs that might provide a useful handle on the TADF rate of a donor-acceptor (DA) system. The incorporation of conformational engineering into the TADF technology could usher in a new paradigm of OLEDs.

Correlated Response of Protein Side-Chain Fluctuations and Conformational Entropy to Ligand Binding.

The heterogeneous fast side-chain dynamics of proteins plays crucial roles in molecular recognition and binding. Site-specific NMR experiments quantify these motions by measuring the model-free order parameter (Oaxis2) on a scale of 0 (most flexible) to 1 (least flexible) for each methyl-containing residue of proteins. Here, we have examined ligand-induced variations in the fast side-chain dynamics and conformational entropy of calmodulin (CaM) using five different CaM-peptide complexes. Oaxis2 of CaM in the ligand-free (Oaxis,U2) and ligand-bound (Oaxis,B2) states are calculated from molecular dynamics trajectories and conformational energy surfaces obtained using the adaptive biasing force (ABF) method. ΔOaxis2 = Oaxis,B2 - Oaxis,U2 follows a Gaussian-like unimodal distribution whose second moment is a potential indicator of the binding affinity of these complexes. The probability for the binding-induced Oaxis,U2 → Oaxis,B2 transition decreases with increasing magnitude of ΔOaxis2, indicating that large flexibility changes are improbable for side chains of CaM after ligand binding. A linear correlation established between ΔOaxis2 and the conformational entropy change of the protein makes possible the determination of the conformational entropy of binding of protein-ligand complexes. The results not only underscore the functional importance of fast side-chain fluctuations but also highlight key motional and thermodynamic correlates of protein-ligand binding.

NMR and DFT analysis of the major diastereomeric degradation product of clopidogrel under oxidative stress conditions

In this work, we enriched the major oxidative degradation product of clopidogrel hydrogen sulfate with the addition of 2.0 equiv. of peroxymonosulfate and analyzed the diastereomers in situ with 1D NMR (1H and 13C) and 2D NMR (1H−1H COSY, 1H−13C HSQC and 1H−13C HMBC). We observed that the signal from the CH2 groups at the positions 4, 6 and 7 in the degradation products (S)-5-((S)-1-(2-chlorophenyl)-2‑methoxy-2-oxoethyl)-5‑hydroxy-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-5-ium and their analog diastereomer (5R,10S) are associated with the polarization of the NO bond in the piperidine ring. Depending on the conformation of this cyclic moiety, an intramolecular anisotropic effect between the phenyl ring and the methylene groups H-4 and H-6 affected the multiplicities and the chemical shifts of these oxidative degradation products in the 1H and 13C NMR spectra. We used DFT calculations to understand the pattern of the 1H NMR chemical shifts, the energies of diastereomers conformations and their intramolecular magnetic effects.

Subsecond Time-Resolved Mass Spectrometry in Dynamic Structural Biology

Life at the molecular level is a dynamic world, where the key players─proteins, oligonucleotides, lipids, and carbohydrates─are in a perpetual state of structural flux, shifting rapidly between local minima on their conformational free energy landscapes. The techniques of classical structural biology, X-ray crystallography, structural NMR, and cryo-electron microscopy (cryo-EM), while capable of extraordinary structural resolution, are innately ill-suited to characterize biomolecules in their dynamically active states. Subsecond time-resolved mass spectrometry (MS) provides a unique window into the dynamic world of biological macromolecules, offering the capacity to directly monitor biochemical processes and conformational shifts with a structural dimension provided by the electrospray charge-state distribution, ion mobility, covalent labeling, or hydrogen-deuterium exchange. Over the past two decades, this suite of techniques has provided important insights into the inherently dynamic processes that drive function and pathogenesis in biological macromolecules, including (mis)folding, complexation, aggregation, ligand binding, and enzyme catalysis, among others. This Review provides a comprehensive account of subsecond time-resolved MS and the advances it has enabled in dynamic structural biology, with an emphasis on insights into the dynamic drivers of protein function.