Solution Conformation Research Articles

Synthesis, Characterization, Conformation in Solution, and Thermoresponsiveness of Polymer Brushes of methoxy[oligo (propylene glycol)-block-oligo(ethylene glycol)]methacrylate and N-[3-(dimethylamino)propyl]methacrylamide Obtained via RAFT Polymerization

The thermo- and pH-responsive polymer brushes based on methoxy[oligo(propyleneglycol)8-block-oligo(ethyleneglycol)8]methacrylate with different concentrations of N-[3-(dimethylamino)propyl]methacrylamide (from 0% to 20%) were synthesized via RAFT polymerization. The "grafting-through" approach was used to prepare the low-molar-mass dispersion samples (Mw/Mn ≈ 1.3). Molar masses and hydrodynamic characteristics were obtained using static and dynamic light scattering and viscometry. The solvents used were acetonitrile, DMFA, and water. The molar masses of the prepared samples ranged from 40,000 to 60,000 g·mol-1. The macromolecules of these polymer brushes were modeled using a prolate revolution ellipsoid or a cylinder with spherical ends. In water, micelle-like aggregates were formed. Critical micelle concentrations decreased with the content of N-[3-(dimethylamino)propyl]methacrylamide. Molecular brushes demonstrated thermo- and pH-responsiveness in water-salt solutions. It was shown that at a given molecular mass and at close pH values, the increase in the number of N-[3-(dimethylamino)propyl]methacrylamide units led to an increase in phase separation temperatures.

Coiled-coil chains with Cis-conformations of Poly(7,7,8,8-tetrakis(ethoxycarbonyl)-p-quinodimethane) in solution on the basis of 1D monomer assemblies

Specific 1D columnar assemblies of 7,7,8,8-tetrakis(ethoxycarbonyl)-p-quinodimethane (1b(Et)) were found to cause three kinds of polymeric chains with different conformations in solution. The known alternating copolymers of 1b(Et) with molecular oxygen present random chains with random conformations, while the other ones with 7,7,8,8-tetracyanoquinodimethane (2) do linear chains with cis-conformations. In contrast, this article describes homopolymers of 1b(Et) involving coiled-coil chains with cis-conformations. Thus, 1b(Et) polymerized in benzene and melt with radical initiators to afford the soluble homopolymers in chloroform. Their characteristic 1H NMR spectra and their simulations by geometry optimization and spectroscopic simulations proved that they have coiled-coil chains with cis-conformations. This finding prompts us to propose a polymerization mechanism involving specific supramolecular one-dimensional (1D) columns for providing cis-addition polymerizations in solution and melt states.

Single-Molecule Electrical and Spectroscopic Profiling Protein Allostery Using a Gold Plasmonic Nanopore.

Direct structural and dynamic characterization of protein conformers in solution is highly desirable but currently impractical. Herein, we developed a single molecule gold plasmonic nanopore system for observation of protein allostery, enabling us to monitor translocation dynamics and conformation transition of proteins by ion current detection and SERS spectrum measurement, respectively. Allosteric transition of calmodulin (CaM) was elaborately probed by the nanopore system. Two conformers of CaM were well-resolved at a single-molecule level using both the ion current blockage signal and the SERS spectra. The collected SERS spectra provided structural evidence to confirm the interaction between CaM and the gold plasmonic nanopore, which was responsible for the different translocation behaviors of the two conformers. SERS spectra revealed the amino acid residues involved in the conformational change of CaM upon calcium binding. The results demonstrated that the excellent spectral characterization furnishes a single-molecule nanopore technique with an advanced capability of direct structure analysis.

Stretching Peptides to Generate Small Molecule β-Strand Mimics.

Advances in the modulation of protein-protein interactions (PPIs) enable both characterization of PPI networks that govern diseases and design of therapeutics and probes. The shallow protein surfaces that dominate PPIs are challenging to target using standard methods, and approaches for accessing extended backbone structures are limited. Here, we incorporate a rigid, linear, diyne brace between side chains at the i to i+2 positions to generate a family of low-molecular-weight, extended-backbone peptide macrocycles. NMR and density functional theory studies show that these stretched peptides adopt stable, rigid conformations in solution and can be tuned to explore extended peptide conformational space. The diyne brace is formed in excellent conversions (>95%) and amenable to high-throughput synthesis. The minimalist structure-inducing tripeptide core (<300 Da) is amenable to further synthetic elaboration. Diyne-braced inhibitors of bacterial type 1 signal peptidase demonstrate the utility of the technique.

Stereochemical Properties of Two Schiff-Base Transition Metal Complexes and Their Ligand by Using Multiple Chiroptical Spectroscopic Tools and DFT Calculations.

Two transition metal complexes were synthesized with Ni(II) and Cu(II) using a tetradentate Schiff-base ligand, (R,R) and (S,S)-N,N'-Bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediamine. The stereochemical properties of the ligand and the metal complexes were investigated using a combined experimental and theoretical approach. Multiple spectroscopic techniques, which include IR, vibrational circular dichroism (VCD), UV-Vis and electronic circular dichroism (ECD), as well as Raman and the newly discovered ECD-circularly polarized Raman (i.e., eCP-Raman) spectroscopies were utilized. The good agreement achieved between the experimental and simulated IR, VCD, UV-Vis and ECD spectra of the ligand allowed one to identify the presence of three main ligand conformers in solution, thanks, especially to the high VCD sensitivity to the conformations associated with the tertbutyl groups. The helicity of the metal complexes was identified to be M and P for those with the (R,R) and (S,S) ligands, respectively. Furthermore, eCP-Raman measurements were carried out for the two metal complexes under (near) resonance. Their induced solvent chiral Raman features were explained, and the potential application of eCP-Raman was discussed.

Nonstructural N- and C-tails of Dbp2 confer the protein full helicase activities

Human DDX5 and its yeast ortholog Dbp2 are ATP-dependent RNA helicases that play a key role in normal cell processes, cancer development, and viral infection. The crystal structure of the RecA1-like domain of DDX5 is available but the global structure of DDX5/Dbp2 subfamily proteins remains to be elucidated. Here, we report the first X-ray crystal structures of the Dbp2 helicase core alone and in complex with ADP at 3.22Å and 3.05Å resolutions, respectively. The structures of the ADP-bound post-hydrolysis state and apo-state demonstrate the conformational changes that occur when the nucleotides are released. Our results showed that the helicase core of Dbp2 shifted between open and closed conformation in solution but the unwinding activity was hindered when the helicase core was restricted to a single conformation. A small-angle X-ray scattering experiment showed that the disordered amino (N) tail and carboxy (C) tails are flexible in solution. Truncation mutations confirmed that the terminal tails were critical for the nucleic acid binding, ATPase, and unwinding activities, with the C-tail being exclusively responsible for the annealing activity. Furthermore, we labeled the terminal tails to observe the conformational changes between the disordered tails and the helicase core upon binding nucleic acid substrates. Specifically, we found that the nonstructural terminal tails bind to RNA substrates and tether them to the helicase core domain, thereby conferring full helicase activities to the Dbp2 protein. This distinct structural characteristic provides new insight into the mechanism of DEAD-box RNA helicases.

Characterization of a novel LTA/LPS-binding antimicrobial and anti-inflammatory temporin peptide from the skin of Fejervary limnocharis (Anura: Ranidae)

Septic shock caused by Gram-positive bacteria continues to be a major cause of morbidity and mortality in intensive care units globally. Most Temporins are excellent growth inhibitors of gram-positive bacteria and candidates for developing antimicrobial treatments due to their biological action and small molecular weight. In this study, a novel Temporin peptide from the skin of Fejervarya limnocharis frog, named as Temporin-FL, was characterized. Temporin-FL was found to adopt typical α-helical conformation in SDS solution and to exhibit selective antibacterial activity against Gram-positive bacteria through a membrane destruction mechanism. Accordingly, Temporin-FL showed protective effects against Staphylococcus aureus-induced sepsis in mice. Finally, Temporin-FL was demonstrated to exert anti-inflammatory effects by neutralizing the action of LPS/LTA and by inhibiting MAPK pathway activation. Therefore, Temporin-FL represents a novel candidate for moleculartherapy of Gram-positive bacterial sepsis.

Effect of polyaspartic acid on the setting time and mechanical properties of α-hemihydrate gypsum

Polyaspartic acid (PASP) was an environment-friendly and biodegradable polymer, with a protein-like structure composed of L-aspartic acid residues. In this work, PASP with different molecular weights (2000, 4000–5000 and 6000–8000) was utilized as the retarder for α-hemihydrate gypsum (α-HH), and the effect of PASP on the coagulation, hydration and crystallization process of gypsum were investigated. Results showed that the addition of PASP with a low dosage of 0.01% efficiently delayed the hydration of α-HH and prolonged the setting process. Among three polyaspartic acids, PASP with molecular weight of 4000–5000 (PASP 4–5) exhibited the best performance not only in postponing the setting time to 95 min, but also in decreasing the negative impact of retarder on the strength of gypsum. Significantly, the slightest loss of 2.65% in compressive strength was obtained here when compared with other retarders previously reported, under the lowest dosage of 0.01% PASP 4–5. Consequently, the effect of PASP chain length on the retardation of α-HH was analyzed in detail. The conductivity, heat of hydration, X-ray diffraction (XRD), EDTA titration method and scanning electron microscopy (SEM) characterization found that PASP prevented the dissolution of α-HH. Total organic carbon analyzer (TOC) and X-ray photoelectron spectroscopy (XPS) measurements showed that the carboxyl groups of PASP adsorbed on the surface of α-HH to hinder the dissolution of α-HH. The molecular dynamic simulation of PASP further exhibited that three kinds of PASPs presented different conformations in supersaturated calcium sulfate solution due to the interaction between PASP and Ca2+ ions, and affected their solvent accessible surface area (SASA). In particular PASP 4–5 exhibited the smallest SASA and the strongest interaction with Ca2+ ions, causing the longest setting time of α-HH.

Site-specific labeling of SBDS to monitor interactions with the 60S ribosomal subunit.

The Shwachman-Diamond syndrome (SDS) is a rare inherited ribosomopathy that is predominantly caused by mutations in the Shwachman-Bodian-Diamond Syndrome gene (SBDS). SBDS is a ribosomal maturation factor that is essential for the release of eukaryotic translation initiation factor 6 (eIF6) from 60S ribosomal subunits during the late stages of 60S maturation. Release of eIF6 is critical to permit inter-subunit interactions between the 60S and 40S subunits and to form translationally competent 80S monosomes. SBDS has three key domains that are highly flexible and adopt varied conformations in solution. To better understand the domain dynamics of SBDS upon binding to 60S and to assess the effects of SDS-disease specific mutations, we aimed to site-specifically label individual domains of SBDS. Here we detail the generation of a fluorescently labeled SBDS to monitor the dynamics of select domains upon binding to 60S. We describe the incorporation of 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid in human SBDS. Site-specific labeling of SBDS using fluorophore and assessment of 60S binding activity are also described. Such labeling approaches to capture the interactions of individual domains of SBDS with 60S are also applicable to study the dynamics of other multi-domain proteins that interact with the ribosomal subunits.

Acidic polysaccharide from corn silk: Structural & conformational properties and hepatoprotective activity

This study aimed to investigate the structural characterization, conformational properties, and hepatoprotective activity of corn silk acidic polysaccharide (CSP-50E). CSP-50E with molecular weights of 1.93 × 105 g/mol was composed of Gal, Glc, Rha, Ara, Xyl, Man and uronic acid with a weight ratio of 12:25:1:2:2:5:21. Structural analysis with methylation indicated that CSP-50E mainly contained T-Manp, 4-substituted-D-Galp/GalpA, and 4-substituted-D-Glcp. CSP-50E presented random coils conformation in an aqueous solution based on the analysis of HPSEC. In vitro experiments showed that CSP-50E exhibited significant hepatoprotective effects, CSP-50E reduce IL-6, TNF-α content, and AST, ALT activity to protect ethanol-induced damage liver cells (HL-7702), while the polysaccharide functioned mainly through the caspase cascade and mediate the mitochondrial apoptosis pathway. In this study, we describe a novel acidic polysaccharide from corn silk with hepatoprotective activity that facilitates the development and utilization of corn silk resources.

Probing the E1o-E2o and E1a-E2o Interactions in Binary Subcomplexes of the Human 2-Oxoglutarate Dehydrogenase and 2-Oxoadipate Dehydrogenase Complexes by Chemical Cross-Linking Mass Spectrometry and Molecular Dynamics Simulation

The human 2-oxoglutarate dehydrogenase complex (hOGDHc) is a key enzyme in the tricarboxylic acid cycle and is one of the main regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. Evidence was obtained for formation of a hybrid complex between the hOGDHc and its homologue the 2-oxoadipate dehydrogenase complex (hOADHc) in the L-lysine metabolic pathway, suggesting a crosstalk between the two distinct pathways. Findings raised fundamental questions about the assembly of hE1a (2-oxoadipate-dependent E1 component) and hE1o (2-oxoglutarate-dependent E1) to the common hE2o core component. Here we report chemical cross-linking mass spectrometry (CL-MS) and molecular dynamics (MD) simulation analyses to understand assembly in binary subcomplexes. The CL-MS studies revealed the most prominent loci for hE1o-hE2o and hE1a-hE2o interactions and suggested different binding modes. The MD simulation studies led to the following conclusions: (i) The N-terminal regions in E1s are shielded by, but do not interact directly with hE2o. (ii) The hE2o linker region exhibits the highest number of H-bonds with the N-terminus and α/β1 helix of hE1o, yet with the interdomain linker and α/β1 helix of hE1a. (iii) The C-termini are involved in dynamic interactions in complexes, suggesting the presence of at least two conformations in solution.

Unveiling mutation effects on the structural dynamics of the main protease from SARS-CoV-2 with hybrid simulation methods

The main protease of SARS-CoV-2 (called Mpro or 3CLpro) is essential for processing polyproteins encoded by viral RNA. Several Mpro mutations were found in SARS-CoV-2 variants, which are related to higher transmissibility, pathogenicity, and resistance to neutralization antibodies. Macromolecules adopt several favored conformations in solution depending on their structure and shape, determining their dynamics and function. In this study, we used a hybrid simulation method to generate intermediate structures along the six lowest frequency normal modes and sample the conformational space and characterize the structural dynamics and global motions of WT SARS-CoV-2 Mpro and 48 mutations, including mutations found in P.1, B.1.1.7, B.1.351, B.1.525 and B.1.429+B.1.427 variants. We tried to contribute to the elucidation of the effects of mutation in the structural dynamics of SARS-CoV-2 Mpro. A machine learning analysis was performed following the investigation regarding the influence of the K90R, P99L, P108S, and N151D mutations on the dimeric interface assembling of the SARS-CoV-2 Mpro. The parameters allowed the selection of potential structurally stable dimers, which demonstrated that some single surface aa substitutions not located at the dimeric interface (K90R, P99L, P108S, and N151D) are able to induce significant quaternary changes. Furthermore, our results demonstrated, by a Quantum Mechanics method, the influence of SARS-CoV-2 Mpro mutations on the catalytic mechanism, confirming that only one of the chains of the WT and mutant SARS-CoV-2 Mpros are prone to cleave substrates. Finally, it was also possible to identify the aa residue F140 as an important factor related to the increasing enzymatic reactivity of a significant number of SARS-CoV-2 Mpro conformations generated by the normal modes-based simulations.

Structure, anticoagulant and cytotoxic activity of a sulfated polysaccharide from green seaweed Chaetomorpha linum

In this article, chemical structure and conformation in an aqueous solution of a new sulfated polysaccharide, PCL, extracted from green seaweed Chaetomorpha linum were elucidated by SEC-MALL, IR, NMR and SAXS. The results indicated that the obtained polysaccharide is a sulfated arabinogalactan with a molecular weight of 223 kDa, and is mainly composed of →3,6)-α-D-Galp4S→ and →2)-α-L-Araf→ connecting together through 1→3 glycoside linkages. It has a broken rod-like conformation in solution with Rgc estimated as 0.43 nm from SAXS measurements. The polysaccharide exhibited a notable anticoagulant activity measured by the assays of activated partial thromboplastintime, thrombintime and prothrombine time as well as a significant cytotoxic activity against hepatocellular, human breast cancer, and cervical cancer cell lines.

Enzyme-like polyene cyclizations catalyzed by dynamic, self-assembled, supramolecular fluoro alcohol-amine clusters

Terpene cyclases catalyze one of the most powerful transformations with respect to efficiency and selectivity in natural product (bio)synthesis. In such polyene cyclizations, structurally highly complex carbon scaffolds are built by the controlled ring closure of linear polyenes. Thereby, multiple C,C bonds and stereocenters are simultaneously created with high precision. Structural pre-organization of the substrate carbon chain inside the active center of the enzyme is responsible for the product- and stereoselectivity of this cyclization. Here, we show that in-situ formed fluorinated-alcohol-amine supramolecular clusters serve as artificial cyclases by triggering enzyme-like reactivity and selectivity by controlling substrate conformation in solution. Because of the dynamic nature of these supramolecular assemblies, a broad range of terpenes can be produced diastereoselectively. Mechanistic studies reveal a finely balanced interplay of fluorinated solvent, catalyst, and substrate as key to establishing nature’s concept of a shape-selective polyene cyclization in organic synthesis.

Position of Geminal Substitution of γ Amino Acid Residues Modulates Their Ability to Form Isolated Non‐Helical C12 β‐turn Mimics

AbstractWe have attempted the construction of isolated αγ non‐helical C12 β‐turn mimics in Boc‐Val‐DPro‐γx,x‐Leu‐NHMe (x,x=2/2, 3/3, 4/4) peptides by the incorporation of differentially dimethyl substituted γ amino acid residues at the (i+2) position of the turn. Solution conformation was probed in detail using NMR and CD spectroscopy. Irrespective of the solvent polarity, peptide containing γ2,2 amino acid residues failed to form the isolated tight β‐turn mimetic while peptides containing γ3,3 and γ4,4 residues successfully adopted isolated non‐helical C12 β‐turn mimic structure. The conformations adopted in solution were corroborated with DFT calculations. Such difference in the stereochemical ability of these di‐substituted γ amino acid residues arose from the difference in position of the di‐substitution along the backbone of the residue. Thus prudent choice of the position of di‐substitution in ω amino acid residues might be used as a strong peptidomimetic handle to control the structural design and generate complex structures.

Stable Enzymatic Nanoparticles from Nucleases, Proteases, Lipase and Antioxidant Proteins with Substrate-Binding and Catalytic Properties.

Limited membrane permeability and biodegradation hamper the intracellular delivery of the free natural or recombinant enzymes necessary for compensatory therapy. Nanoparticles (NP) provide relative protein stability and unspecific endocytosis-mediated cellular uptake. Our objective was the fabrication of NP from 7 biomedicine-relevant enzymes, including DNase I, RNase A, trypsin, chymotrypsin, catalase, horseradish peroxidase (HRP) and lipase, the analysis of their conformation stability and enzymatic activity as well as possible toxicity for eukaryotic cells. The enzymes were dissolved in fluoroalcohol and mixed with 40% ethanol as an anti-solvent with subsequent alcohol evaporation at high temperature and low pressure. The shapes and sizes of NP were determined by scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Enzyme conformations in solutions and in NP were compared using circular dichroism (CD) spectroscopy. The activity of the enzymes was assayed with specific substrates. The cytotoxicity of the enzymatic NP (ENP) was studied by microscopic observations and by using an MTT test. Water-insoluble ENP of different shapes and sizes in a range 50-300 nm consisting of 7 enzymes remained stable for 1 year at +4 °C without any cross-linking. CD spectroscopy of the ENP permitted us to reveal changes in proportions of α-helixes, β-turns and random coils in comparison with fresh enzyme solutions in water. Despite the minor conformation changes of the proteins in the ENP, the enzymes retained their substrate-binding and catalytic properties. Among the studied bioactive ENP, only DNase NP were highly toxic for 3 cell lines with granulation in 1 day posttreatment, whereas other NP were less toxic (if any). Taken together, the enzymes in the stable ENP retained their catalytic activity and might be used for intracellular delivery.

Altered conformational landscape upon sensing guanine nucleotides in a disease mutant of Elongation Factor-like 1 (EFL1) GTPase.

Ribosome biogenesis is a highly regulated process that requires the participation of several accessory proteins that participates in the cytoplasmic last maturation step before entering the pool of translating ribosomes. One of these accessory proteins is the GTPase Elongation Factor-like 1 (EFL1), a homologue of the elongation factor 2 (EF-2). EFL1, together with SBDS (yeast Sdo1), evict the anti-association factor eIF6 (yeast Tif6) from the surface of the 60S subunit, allowing mature ribosome assembly. Defects in the function of any of these two proteins lead to a disease called Shwachman-Diamond Syndrome (SDS). Mutations prevent the release of eIF6 from the 60S subunit, with the consequent imbalance of mature 60S subunits entering the pool of active translating 80S ribosomes and a decrease in global translation. EFL1 is a molecular motor that adopts at least four distinct conformations in solution regulated by SBDS and guanine nucleotides whose malfunction in the SDS has been scarcely studied. The malfunction of the non-synonymous mutations in EFL1 has been scarcely studied, with little effect on its catalytic activity or fold. We used Isothermal Titration Calorimetry to gain further insights into the malfunction of Efl1 mutations. We characterised the binding energetics of S. cerevisiae Efl1 R1086Q equivalent to EFL1 R1095Q reported in patients with SDS. The data presented exhibits a different structural and energetic response in the yeast mutant Efl1 R1086Q compared to that previously reported Efl1 wild type. The heat capacity change (ΔCp) and conformational binding entropy of the complexes formed between the mutant and the wild type proteins showed opposite values suggesting they adopt different conformations. We conclude that mutation R1086Q profoundly disrupts the internal rearrangements necessary to elicit a similar conformational change as that in native EFL1.

Exploring the conformational landscape of the rabies virus glycoprotein using HDX-MS.

Rabies virus, a member of the lyssavirus family, causes on the order of 60,000 deaths per year, disproportionately affecting historically exploited regions of the world. While prophylactic measures such as vaccines and immunoglobulin therapies are very effective at preventing rabies upon exposure, these treatments are very expensive and require cold-chain logistics, making them inaccessible to much of the world. Furthermore, there is no cure for rabies after the onset of symptoms, making it a death sentence for people who do not seek treatment because either they lack access or they do not suspect the animal they were bitten by was infectious. The rabies virus glycoprotein (RABV-G) is densely displayed on the surface of the virion, making it an attractive target for vaccine and drug development, yet this protein remains poorly understood. RABV-G is a highly dynamic protein that is known to reversibly sample its pre- and post-fusion conformations in equilibrium at physiological pH, making it difficult to study using many in vitro methods. Hydrogen deuterium exchange monitored by mass spectrometry (HDX-MS) is an ideal technique to probe the conformational landscape of proteins that occupy more than one conformation in solution. This work uses HDX-MS to measure the local stability of the protein when biased to both the prefusion and postfusion conformation as well as quantify the kinetics of the conformational change between the two conformations. This work will be useful in exploring how antibodies interact with the different conformations of rabies G in order to design better therapeutic strategies to combat this lethal virus.

Modelling solution structures of cyclic peptides - How good are we?

Cyclic peptides (CPs) are macrocycles made from amino acids that can bind to classically undruggable drug targets. Controlling pharmacokinetic properties of these macrocyclic peptides is an open question, limited by having precise knowledge of the underlying solution structures. CP solution dynamics are complex: individual structures are often separated by large energy barriers; the presence of conformers is environment dependent and solution conformers can be different from bioactive structures. Specialised simulation methods for CPs have previously been designed to reproduce available x-ray structures, but it is often unknown how well solution structures are reproduced. Here, we report the largest cyclic peptide solution NMR dataset “MacroConf”, which we built from existing literature. We use a range of molecular dynamics methods and specialised cheminformatics methods to model the solution structures of over 30 cyclic peptides from this dataset. We explore (a) how different methods perform relative to one another and to the experimental reference structures and (b) whether computationally cheaper methods can reach the accuracy of gold-standard accelerated molecular dynamics simulations to allow for fast prediction of solution ensembles. By providing automated computational workflows, this work is easily extendable, fully reproducible, and is built as a reference point to guide development of future conformer generators for cyclic peptides.

Understanding the Cis-Trans Amide Bond Isomerization of N,N'-Diacylhydrazines to Develop Guidelines for A Priori Prediction of Their Most Stable Solution Conformers.

N,N'-diacylhydrazines (R1CO-NR3-NR4-COR2) are a class of small molecules with a wide range of applications in chemistry and biology. They are structurally unique in the sense that their two amide groups are connected via a N-N single bond, and as a result, these molecules can exist in eight different isomeric forms. Four of these are amide isomers [trans-trans (t-t), trans-cis (t-c), cis-trans (c-t), and cis-cis (c-c)] arising from C-N bond restricted rotation. In addition, each of these amide isomers can exist in two different isomeric forms due to N-N bond restricted rotation, especially when R3 and R4 groups are relatively bigger. Herein, we have systematically investigated the conformations of 55 N,N'-diacylhydrazines using a combination of solution NMR spectroscopy, X-ray crystallography, and density functional theory calculations. Our data suggest that when the substituents R3 and R4 on the nitrogen atoms are both hydrogens. These molecules prefer twisted trans-trans (t-t) (>90%) geometries (H-N-C═O ∼ 180°), whereas the N-alkylated and N,N'-dialkylated molecules prefer twisted trans-cis (t-c) geometries. Herein, we have analyzed the stabilization of the various isomers of these molecules in light of steric and stereoelectronic effects. We provide a guideline to a priori predict the most stable conformers of the N,N'-diacylhydrazines just by examining their substituents (R1-R4).