Rigid Conformation Research Articles

Manipulating noncovalent conformational lock via side‐chain engineering for luminescence at aggregate level

AbstractThe unfavorable photochemical processes at the molecular level have become a barrier limiting the use of aromatic amides as high‐performance luminescent materials. Herein, we propose a reliable strategy for manipulating noncovalent conformational lock (NCL) via side‐chain engineering to burst out eye‐catching luminescence at the aggregate level. Contrary to the invisible emission in dilute solutions, dyad OO with a three‐centered H‐bond gave the wondrous crystallization‐induced emission with a quantum yield of 66.8% and clusterization‐triggered emission, which were much brighter than those of isomers. Theoretical calculations demonstrate that crystallization‐induced planarized intramolecular charge transfer (PICT), conformation rigidification, and through‐space conjugation (TSC) are responsible for aggregate‐state luminescence. Robust NCL composed of intramolecular N‐H···O interactions could boost molecular rigidity and planarity, thus greatly facilitating PICT and TSC. This study would inspire researchers to design efficient luminescent materials at the aggregate level via rational conformational control.

Effect of Conformational Variability on the Drug Resistance of Candida auris ERG11p and FKS1.

Candida auris infection has been recognized as an urgent threat to antifungal drug resistance, and the Eagle effect of C. auris FKS1 (1,3-β-d-glucan synthase) wild-type isolates has also been noted. The Eagle effect, namely, where higher concentrations of antifungals reduce fungicidal activity relative to lower concentrations, is a confounding factor of apparent antifungal resistance, but the detailed mechanism remains unclear. Here, we present the conformational variability of mutation sites for ERG11p (lanosterol 14α-demethylase) and FKS1 from deep neural network-based prediction along with the reported X-ray crystallographic and cryo-electron microscopy (cryo-EM) structures of antifungals. The sequence variability maps provide valuable insights into the inconsistent correlation between azole resistance and the mysterious Eagle effect with the dispersion of minimal inhibitory concentration (MIC) for echinocandin resistance. The conformational variability prediction supports the hypothesis that mutations K143R of clade I, VF125AL of clade III, and Y132F of clade IV for C. auris ERG11p make the corresponding site variable and that an increased population of invisible variable conformations potentially contributes to triazole resistance. In contrast, the predicted rigid conformation by the S639F mutation of hot spot region 1 (HS1) for FKS1 suggests that caspofungin (CAS) is involved in an uncompetitive inhibition, and a decreased population of the CAS-bound state of FKS1 with Rho1 leads to drug resistance. The predicted variable HS1 region for FKS1 WT isolates and the rigid one for FKS1 S639F mutants support the in vivo drug response and the in vitro MIC dispersion. A plausible mechanism of the Eagle effect is hereby proposed, namely, that a high concentration of CAS with a high membrane affinity reduces the population of the CAS-bound state of FKS1 with Rho1, as well as accompanying events such as aggregation or association depending on the conformational variability of HS1.

BDNF- and VEGF-Responsive Stimulus to an NGF Mimic Cyclic Peptide with Copper Ionophore Capability and Ctr1/CCS-Driven Signaling.

Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu2+ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu2+ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu2+ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.

Strategies for the Construction of Multicyclic Phage Display Libraries.

Peptide therapeutics have gained great interest due to their multiple advantages over small molecule and antibody-based drugs. Peptide drugs are easier to synthesize, have the potential for oral bioavailability, and are large enough to target protein-protein interactions that are undruggable by small molecules. However, two major limitations have made it difficult to develop novel peptide therapeutics not derived from natural products, including the metabolic instability of peptides and the difficulty of reaching antibody-like potencies and specificities. Compared to linear and disulfide-monocyclized peptides, multicyclic peptides can provide increased conformational rigidity, enhanced metabolic stability, and higher potency in inhibiting protein-protein interactions. The identification of novel multicyclic peptide binders can be difficult, however, recent advancements in the construction of multicyclic phage libraries have greatly advanced the process of identifying novel multicyclic peptide binders for therapeutically relevant protein targets. This review will describe the current approaches used to create multicyclic peptide libraries, highlighting the novel chemistries developed and the proof-of-concept work done on validating these libraries against different protein targets.

Metal-Assisted Carbohydrate Assembly.

The sequence-controlled assembly of nucleic acids and amino acids into well-defined superstructures constitutes one of the most revolutionary technologies in modern science. The elaboration of such superstructures from carbohydrates, however, remains elusive and largely unexplored on account of their intrinsic constitutional and configurational complexity, not to mention their inherent conformational flexibility. Here, we report the bottom-up assembly of two classes of hierarchical superstructures that are formed from a highly flexible cyclo-oligosaccharide─namely, cyclofructan-6 (CF-6). The formation of coordinative bonds between the oxygen atoms of CF-6 and alkali metal cations (i) locks a myriad of flexible conformations of CF-6 into a few rigid conformations, (ii) bridges adjacent CF-6 ligands, and (iii) gives rise to the multiple-level assembly of three extended frameworks. The hierarchical superstructures present in these frameworks have been shown to modulate their nanomechanical properties. This research highlights the unique opportunities of constructing convoluted superstructures from carbohydrates and should encourage future endeavors in this underinvestigated field of science.

Magnetic nanoparticles immobilized thrombin ligand fishing to screen thrombin inhibitors in natural products

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking.

Clicked BODIPY-Fullerene-Peptide Assemblies: Studies of Electron Transfer Processes in Self-Assembled Monolayers on Gold Surfaces.

Two BODIPY-C60-peptide assemblies were synthesized by CuAAC reactions of BODIPY-C60 dyads and a helical peptide functionalized with a terminal alkyne group and an azide group, respectively. The helical peptide within these assemblies was functionalized at its other end by a disulfide group, allowing formation of self-assembled monolayers (SAMs) on gold surfaces. Characterizations of these SAMs, as well as those of reference molecules (BODIPY-C60-alkyl, C60-peptide and BODIPY-peptide), were carried out by PM-IRRAS and cyclic voltammetry. BODIPY-C60-peptide SAMs are more densely packed than BODIPY-C60-alkyl and BODIPY-peptide based SAMs. These findings were attributed to the rigid peptide helical conformation along with peptide-peptide and C60-C60 interactions within the monolayers. However, less dense monolayers were obtained with the target assemblies compared to the C60-peptide, as the BODIPY entity likely disrupts organization within the monolayers. Finally, electron transfer kinetics measurements by ultra-fast electrochemistry experiments demonstrated that the helical peptide is a better electron mediator in comparison to alkyl chains. This property was exploited along with those of the BODIPY-C60 dyads in a photo-current generation experiment by converting the resulting excited and/or charge separated states from photo-illumination of the dyad into electrical energy.

Template-Directed Synthesis of Strained meso-meso-Linked Porphyrin Nanorings.

Strained macrocycles display interesting properties, such as conformational rigidity, often resulting in enhanced π-conjugation or enhanced affinity for non-covalent guest binding, yet they can be difficult to synthesize. Here we use computational modeling to design a template to direct the formation of an 18-porphyrin nanoring with direct meso-meso bonds between the porphyrin units. Coupling of a linear 18-porphyrin oligomer in the presence of this template gives the target nanoring, together with an unexpected 36-porphyrin ring by-product. Scanning tunneling microscopy (STM) revealed the elliptical conformations and flexibility of these nanorings on a Au(111) surface.

Template‐Directed Synthesis of Strained meso‐meso‐Linked Porphyrin Nanorings

AbstractStrained macrocycles display interesting properties, such as conformational rigidity, often resulting in enhanced π‐conjugation or enhanced affinity for non‐covalent guest binding, yet they can be difficult to synthesize. Here we use computational modeling to design a template to direct the formation of an 18‐porphyrin nanoring with direct meso‐meso bonds between the porphyrin units. Coupling of a linear 18‐porphyrin oligomer in the presence of this template gives the target nanoring, together with an unexpected 36‐porphyrin ring by‐product. Scanning tunneling microscopy (STM) revealed the elliptical conformations and flexibility of these nanorings on a Au(111) surface.

Computational investigation of impact of Pb(II) and Ni(II) ions on hUNG enzyme: insights from molecular dynamics simulations

Human uracil DNA glycosylase (hUNG), a crucial player in the initiation of the base excision repair pathway, is susceptible to alterations in function and conformation induced by the accumulation of toxic metals. Despite the recognized impact of toxic metals on DNA repair enzymes, there exists a notable deficiency in theoretical investigations addressing this phenomenon. This study investigates the impact of toxic heavy metal ions, Pb(II) and Ni(II), on the stability of hUNG through molecular dynamics (MD) simulations. The initial analysis involved the identification of key cavities in the hUNG enzyme. Notably, the active site cavity emerged as a promising site for ligand binding. Subsequently, AutoDockTools software was employed to dock Pb(II) and Ni(II) onto the identified cavities, followed by extensive MD simulations. The MD analysis, encompassing parameters such as root mean square deviation, radius of gyration, solvent accessible surface area, hydrogen bond variations, Ramachandran plot, principal component analysis, and root mean square fluctuations, collectively revealed distinct alterations in the behavior of the enzyme upon complexation with Pb(II) and Ni(II). Interestingly, the enzyme exhibited enhanced structural stability, reduced flexibility, and modified hydrogen bonding patterns in the presence of these toxic metal ions. The observed limitation in structural flexibility implies a more rigid and stable conformation when the enzyme complex with Pb(II) and Ni(II) compared to its free form. This structural alteration may lead to a potential reduction in enzymatic activity, suggesting that toxic metal ions influence the functional dynamics of hUNG. These computational findings offer valuable insights into the molecular interactions between metal ions and enzymes. Communicated by Ramaswamy H. Sarma

An Autocatalytic Peptide Cyclase Improves Fidelity and Yield of Circular Peptides In Vivo and In Vitro.

Peptide cyclization improves conformational rigidity, providing favorable pharmacological properties, such as proteolytic resistance, target specificity, and membrane permeability. Thus, many synthetic and biosynthetic peptide circularization strategies have been developed. PatG and related natural macrocyclases process diverse peptide sequences, generating millions of cyclic derivatives. However, the application of these cyclases is limited by low yields and the potential presence of unwanted intermediates. Here, we designed a covalently fused G macrocyclase with substrates that efficiently and spontaneously release cyclic peptides. To increase the fidelity of synthesis, we developed an orthogonal control mechanism enabling precision synthesis in Escherichia coli. As a result, a library comprising 4.8 million cyclic derivatives was constructed, producing an estimated 2.6 million distinct cyclic peptides with an improved yield and fidelity.

Tetraasteranes as homologues of cubanes: effective scaffolds for drug discovery.

Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market for a variety of applications, but one notable omission is that of tetraasteranes, which are homologues of cubanes belonging to a class of polycyclic hydrocarbon cage compounds. Tetraasteranes exhibit potential as scaffolds in drug discovery due to their identical cyclobutane structures and rigid conformation resembling cubanes. Based on the studies of the physical and chemical properties of tetraasteranes by density functional theory, three series of compounds were designed as homologues of cubanes by the substitution of cubane scaffolds in pharmaceuticals with tetraasteranes. Their potential for pharmaceutical applications was evaluated in silico by molecular docking and dynamics simulations. Their pharmacokinetic and physicochemical properties were studied by the ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis. The results indicate that tetraasteranes may be scaffolds as novel bioisosteres of cubanes, as well as hydrogen bond donors or acceptors, which enhance the affinity between ligands and receptors with more stable binding behavior and feasible tolerability in ADMET. All these findings provide new opportunities for tetraasteranes to serve as effective pharmaceutical scaffolds for drug discovery and to accelerate the drug discovery process by repurposing both new and old commercial compounds.

Thermodynamics of Mitochondrial Aspartate Aminotransferases in the Presence of Nanoparticles

Background and objectives. The nanoparticles are extensively used in the fields of biotechnology, the medical biochemistry and nanomedicne for enhancement and improvement drugs targeting in the chemotherapy. This study was conducted firstly, to understand the underlying mechanisms and potential applications of the thermo-inactivation kinetics of mitochondrial aspartate aminotransferase (mAspAT) in the presence of various nanoparticles, specifically TiO2 and Fe3O4, as well as gold nanoparticles (AuNP), secondly, to determine the impacts of nanoparticle concentration on the aggregation process and the influence the conformational stability and thermal behavior of mAspAT. Methods. The mAspAT was isolated from the pig heart, and SDS electrophoresis was used to determine the level of the protein's purity. A solution of native and modified dextran sulfate nanoparticles was prepared and incubated with mAspAT at various temperatures. The thermo-Inactivation was carried out at different temperatures (45, 50, 55, 60, 65, and 70 °C) and the aggregation of mAspAT was studied at 320 nm using spectrophotometry. The output data was manipulated (Calculations) and analyzed by Software Stadia 6.0. Results. TiO2 and Fe3O4 nanoparticles, both native and dextran sulfate-coated, demonstrated thermoprotective effects on mAspAT at temperatures above 60 °C. The interaction of mAspAT with TiO2 nanoparticles resulted in a decrease in thermoinactivation energy (Ea), indicating increased conformational stability. Therefore, constant thermoinactivation rate (kin) of mAspAT was significantly decreased in the presence of TiO2 nanoparticles. The dextran sulfate modification further enhanced this effect. Conclusion. The study concludes that the interaction of mAspAT with NP results in an increase in the conformation rigidity of the enzyme, which is primarily determined by the nature of NP and can be increased by coating the surface of NP with a polymer dextran sulfate. Also, Gold nanoparticles could potentially be used to increase the stability of mAspAT, preventing it from thermoaggregation.

A Rigid Multiple Resonance Thermally Activated Delayed Fluorescence Core Toward Stable Electroluminescence and Lasing.

The investigation of organic light-emitting diodes (OLEDs) and organic laser devices with thermally activated delayed fluorescence (TADF) molecules is emerging due to the potential of harnessing triplets. In this work, a boron/nitrogen multiple-resonance TADF polycyclic framework fusing carbazole units (CzBNPh) was proposed. CzBNPh exhibited a narrowband emission (<30 nm), a unity photoluminescence quantum yield, and a fast radiative rate. Consequently, CzBNPh demonstrated a low distributed feedback (DFB) lasing threshold of 0.68 μJ cm-2 . Furthermore, the stimulated emission zone of CzBNPh was effectively separated from its singlet and triplet absorption, thereby minimizing the singlet-triplet annihilation under long-pulsed excitation ranging from 20 μs to 2.5 ms. Significantly, the enhanced rigid molecular conformation, thermal stability, and photo-stability resulted in improved lasing and electroluminescence stability compared to that of 5,9-diphenyl-5,9-diaza-13b-boranaphtho[3,2,1-de]anthracene (DABNA)-core. These findings indicate the potential of CzBN-core as a promising framework for achieving long-pulsed wave and electrically-pumped lasing in the future.

A Rigid Multiple Resonance Thermally Activated Delayed Fluorescence Core Toward Stable Electroluminescence and Lasing

AbstractThe investigation of organic light‐emitting diodes (OLEDs) and organic laser devices with thermally activated delayed fluorescence (TADF) molecules is emerging due to the potential of harnessing triplets. In this work, a boron/nitrogen multiple‐resonance TADF polycyclic framework fusing carbazole units (CzBNPh) was proposed. CzBNPh exhibited a narrowband emission (&lt;30 nm), a unity photoluminescence quantum yield, and a fast radiative rate. Consequently, CzBNPh demonstrated a low distributed feedback (DFB) lasing threshold of 0.68 μJ cm−2. Furthermore, the stimulated emission zone of CzBNPh was effectively separated from its singlet and triplet absorption, thereby minimizing the singlet‐triplet annihilation under long‐pulsed excitation ranging from 20 μs to 2.5 ms. Significantly, the enhanced rigid molecular conformation, thermal stability, and photo‐stability resulted in improved lasing and electroluminescence stability compared to that of 5,9‐diphenyl‐5,9‐diaza‐13b‐boranaphtho[3,2,1‐de]anthracene (DABNA)‐core. These findings indicate the potential of CzBN‐core as a promising framework for achieving long‐pulsed wave and electrically‐pumped lasing in the future.

The Conformational Change of the L3 Loop Affects the Structural Changes in the Substrate Binding Pocket Entrance of β-Glucosidase

β-glucosidase (Bgl) hydrolyzes cellobiose to glucose, thereby releasing non-reducing terminal glucosyl residues. Bgl is an essential enzyme belonging to the biomass-degrading enzyme family, which plays a vital role in enzymatic saccharification during biofuel production. The four loops above the Bgl substrate-binding pocket undergo a conformational change upon substrate recognition. However, the structural dynamism of this loop and how it is conserved among Bgl family members remain unknown. Herein, to better understand the four loops above the substrate-binding pocket of Bgl, four Bgl crystal structures in Thermoanaerobacterium saccharolyticum (TsaBgl) were determined at 1.5–2.1 Å. The L1, L2, and L4 loops of TsaBgl showed a rigid conformation stabilized by their neighboring residues via hydrogen bonds and hydrophobic interactions. The TsaBgl L3 loop showed relatively high flexibility and two different N-terminal region conformations. The conformational change in the TsaBgl L3 loop induced a change in charge and shaped at the substrate-binding pocket entrance. The amino acid sequences and structures of the TsaBgl L1–4 loops were compared with other 45 Bgl proteins, and a diversity of the L2 and L3 loops was observed. Differences in amino acids and lengths of Bgls L2–L3 loop induced differences in the conformation and structure of the Bgls substrate-binding pocket entrance. These findings expand our knowledge on the molecular function of the loops in the Bgl enzyme family.

Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-β structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-β structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like β-structures in HEWL, retaining most of the cross-β structures. In contrast, λ-carrageenan even increased the content of amyloid cross-β structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-β structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

Conformational properties of heterogeneous arabinoxylan protein gums from corn bran and distillers grains in comparison with gum arabic

The molecular structure and conformation of arabinoxylan-protein gum, commonly referred as corn fiber gum (CFG) were analyzed by high-performance size-exclusion chromatography (HPSEC) coupled with RI, UV, light scattering and viscometer detectors. CFG had a heterogeneous structure. The detailed conformation of CFG at different molecular weights was compared with that of hemicellulose fiber gum (HFG) from dried distiller's grains with solubles and gum arabic. The CFG molecules mainly had random coil conformation; only 10 % of them exhibited rigid rod conformation. Approximately 80 % of the CFG had a molecular weight between 105 and 105.4 Da, while the other 20 % of molecules were between 105.4 and 1.5 × 107.7 Da. The overall conformational properties of CFG and HFG were closer but differed from that of gum arabic. The intrinsic viscosity and radius of gyration of both CFG and HFG were greater than those of gum arabic although the average molecular weight of CFG and HFG was lower. The protein and carbohydrate were covalently linked in CFG molecules as shown by the HPSEC-multiple detectors combined with partial acid hydrolysis. Based on the detailed conformation of CFG and the methylation analysis, 1D and 2D NMR spectroscopy results, the molecular structure of CFG was proposed.

Synthesis of biologically active spiro oxindole through multicomponent reaction and it’s physico-chemical studies

Indole-2,3-dione has been utilized in many multicomponent reactions that produce complex spiro compounds with readily available starting materials and considered as an importantbuilding block in organic synthesis. Spiro compounds in which two cyclic rings are fused at a common carbon atom are promising interesting skeletal systems in drug discovery due to their unique conformational features, structural complexity and rigidity. Here we have synthesized a spiro-oxindole which has been synthesized from indole-2,3-dione, dimedone and 2-aminobenzimidazole using nickel-zinc ferrite silicate nano composite as catalyst, and characterization of synthesized spiro-oxindole compound were carried. Computationalstudies of the synthesized compound were performed using DFT method at the CAM-B3LYP/6–311++G (d, p) level. Geometry optimization, FMO studies, vibrational and NBO analysis reveals the molecular structure, electronic transitions and intramolecular charge delocalization of the Spiro oxindole.Molecular docking studies of the synthesized compound was evaluated for their antimicrobial activities against three bacteria viz., Staphylococcus aureus, E-Coli, Klebsiella species andfungus viz., Aspergillus Niger. The spiro-oxindole compound showed very good invitro DPPH scavenging antioxidantproperty.

An NMR Spectroscopy View on London Dispersion in Catalysis: Detection, Quantification, and Application in Ion Pair and Transition Metal Catalysis.

ConspectusThe energetic contribution of London dispersion (LD) can cover a broad range from very few to hundreds of kJ mol-1 for extended interaction interfaces due to its pairwise additivity. However, for a designed and successful application of LD in chemical catalysis, there are still many obstacles and questions that remain. In principle, LD can be regarded as the attractive part of the van der Waals potential. Thus, considering the whole van der Waals potential, including the repulsive part (steric repulsion), the ideal solution to the problem in catalysis would be to design compatible interaction interfaces at exactly the correct distance. In the case of a self-assembled, flexible structure arrangement, entropic contributions and solvent interactions might be detrimental. In the case of a rigid catalyst pocket, steric hindrance might not allow for large substituents that are usually applied as dispersion energy donors (DEDs). For a working catalytic system, the following question arises: how is it possible to dissect the complex interaction interfaces in terms of energetic contributions? Usually, the energetic contribution of LD to catalysis is addressed by using calculations. However, adequately computing the correct energetic contributions can be extremely challenging for a vast conformational space with all kinds of intermolecular interactions. Thus, experimental data are essential for comparison or benchmarking.Therefore, in this Account, we describe our quest for detailed experimental data obtained via NMR spectroscopy to experimentally dissect and quantify LD in catalytic systems. In addition, we address the question of whether bulky substituents used as DEDs can be used in confined catalytic pockets. With the example of Pd phosphoramidite complexes, we show how it is possible to experimentally dissect and quantify the contribution of individual interaction areas in complicated transition metal complexes. Furthermore, a correlation between conformational rigidity and heterodimer preference clearly reveals that LD can only unfold its full potential in cases where entropic contributions are minimized. This finding can also explain the small contribution of LD in flexible and solvent-exposed molecular balances. In the field of Brønsted acid catalysis, we demonstrated that LD has a strong influence on the structures, stability, and populations of confined catalytic intermediates. LD is key for populating higher aggregates such as dimers. In addition, offsets between the experimental and computational results were observed and attributed to solvent-solute dispersion interactions. We studied the delicate interplay of attractive and repulsive interactions by adding bulky DED substituents onto a substrate, which can function as a molecular balance system. Intriguingly, the effect of LD on the free substrate was straightforwardly transferred onto the highly confined intermediates. Furthermore, this effect could even be read out in the enantioselectivities of the underlying reaction. This conceptualized a general approach regarding how LD can be used beneficially in catalysis to convert from moderate/good to excellent stereoselectivities. It showcased that bulky groups such as tert-butyl must not only be regarded as occupied volumes.