Structural and Electronic Evolution of Ethanolamine upon Microhydration: Insights from Hyperfine Resolved Rotational Spectroscopy.

Ethanolamine hydrates containing from one to seven water molecules were identified via rotational spectroscopy with the aid of accurate quantum chemical methods considering anharmonic vibrational corrections. Ethanolamine undergoes significant conformational changes upon hydration to form energetically favorable hydrogen bond networks. The final structures strongly resemble the pure (H2O)3-9 complexes reported before when replacing two water molecules by ethanolamine. The 14N nuclear quadrupole coupling constants of all the ethanolamine hydrates have been determined and show a remarkable correlation with the strength of hydrogen bonds involving the amino group. After addition of the seventh water molecule, both hydrogen atoms of the amino group actively contribute to hydrogen bond formation, reinforcing the network and introducing approximately 21-27% ionicity towards the formation of protonated amine. The findings highlight the critical role of microhydration in altering the electronic environment of ethanolamine, enhancing our understanding of amine hydration dynamics.

The Importance of Structured Water in HDAC8 for Correct Binding Pose Applied for Drug Design of Anticancer Molecules.

AIMS: Validating the docking procedure and maintaining the structural water molecules at HDAC8 catalytic site. BACKGROUND: Molecular docking simulations play a significant role in Computer-Aided Drug Design, contributing to the development of new molecules. To ensure the reliability of these simulations, a validation process called "Re-docking" is employed, focusing on the binding mode of a ligand co-crystallized with the protein of interest. OBJECTIVE: In this study, several molecular docking studies were conducted using five X-ray structures of HDAC8-ligand complexes from the PDB. METHOD: Ligands initially complexed with HDAC8 were removed and re-docked onto the free protein, revealing a poor reproduction of the expected binding mode. In response to this, we observed that most HDAC8-ligand complexes contained one to two water molecules in the catalytic site, which were crucial for maintaining the cocrystallized ligand. RESULT: These water molecules enhance the binding mode of the co-crystallized ligand by stabilizing the proteinligand complex through hydrogen bond interactions between ligand and water molecules. Notably, these interactions are lost if water molecules are removed, as is often done in classical docking methodologies. Considering this, molecular docking simulations were repeated, both with and without one or two conserved water molecules near Zn+2 in the catalytic cavity. Simulations indicated that replicating the native binding pose of co-crystallized ligands on free HDAC8 without these water molecules was challenging, showing greater coordinate displacements (RMSD) compared to those including conserved water molecules from crystals. CONCLUSION: The study highlighted the importance of conserved water molecules within the active site, as their presence significantly influenced the successful reproduction of the ligands' native binding modes. The results suggest an optimal molecular docking procedure for validating methods suitable for filtering new HDAC8 inhibitors for future experimental assays.

Alternate conformations found in protein structures implies biological functions: A case study using cyclophilin A.

Protein dynamics linked to numerous biomolecular functions, such as ligand binding, allosteric regulation, and catalysis, must be better understood at the atomic level. Reactive atoms of key residues drive a repertoire of biomolecular functions by flipping between alternate conformations or conformational substates, seldom found in protein structures. Probing such sparsely sampled alternate conformations would provide mechanistic insight into many biological functions. We are therefore interested in evaluating the instance of amino acids adopted alternate conformations, either in backbone or side-chain atoms or in both. Accordingly, over 70000 protein structures appear to contain alternate conformations only 'A' and 'B' for any atom, particularly the instance of amino acids that adopted alternate conformations are more for Arg, Cys, Met, and Ser than others. The resulting protein structure analysis depicts that amino acids with alternate conformations are mainly found in the helical and ß-regions and are often seen in high-resolution X-ray crystal structures. Furthermore, a case study on human cyclophilin A (CypA) was performed to explain the pre-existing intrinsic dynamics of catalytically critical residues from the CypA and how such intrinsic dynamics perturbed upon Ser99Thr mutation using molecular dynamics simulations on the ns-µs timescale. Simulation results demonstrated that the Ser99Thr mutation had impaired the alternate conformations or the catalytically productive micro-environment of Phe113, mimicking the experimentally observed perturbation captured by X-ray crystallography. In brief, a deeper comprehension of alternate conformations adopted by the amino acids may shed light on the interplay between protein structure, dynamics, and function.

Subatomic structure of orthorhombic thaumatin at 0.89 Å reveals that highly flexible conformations are crucial for thaumatin sweetness.

Thaumatin is a sweet-tasting protein that elicits a sweet taste at a threshold of approximately 50 nM. Structure-sweetness relationships in thaumatin suggest that the basicity of two amino acids residues, Arg82 and Lys67, are particularly responsible for sweetness. Using tetragonal crystals, our structural analysis suggested that flexible sidechain conformations of these two residues play an important role in sweetness. However, in tetragonal crystals, Arg82 is adjacent to symmetry-related residues, and its flexibility is relatively restrained by the crystal packing. To reduce and diminish these symmetry-related effects, orthorhombic crystals were prepared, and their structures were successfully determined at a resolution of 0.89 Å. Within the orthorhombic lattice, two alternative conformations were more clearly visible at Lys67 than in a tetragonal system. Interestingly, for the first time, three alternative conformations at Arg82 were only found in an orthorhombic system. These results suggest the importance of flexible conformations in sweetness determinants. Such subtle structural variations might serve to adjust the complementarity of the electrostatic potentials of sweet receptors, thereby eliciting the potent sweet taste of thaumatin.

On the Swelling of Polymer Network Strands.

Large scale computer simulations are employed to analyze the conformations of network strands in polymer networks at preparation conditions (characterized by a polymer volume fraction of ϕ0) and when swollen to equilibrium (characterized by a polymer volume fraction ϕ < ϕ0). Network strands in end-linked model networks are weakly stretched and partially swollen at preparation conditions as compared to linear polymers in the same solvent at ϕ0. Equilibrium swelling causes non-ideal chain conformations characterized by an effective scaling exponent approaching 7/10 on intermediate length scales for increasing overlap of the chains. The chain size in a network consists of a fluctuating and a time average "elastic" contribution. The elastic contribution swells essentially affinely ∝(ϕ0/ϕ)2/3, whereas the swelling of the fluctuating part lies between the expected swelling of the entanglement constraints and the swelling of non-cross-linked chains in a comparable semi-dilute solution. The total swelling of chain size results from the changes of both fluctuating and non-fluctuating contributions.

The catalytic domain of free or ligand bound histone deacetylase 4 occurs in solution predominantly in closed conformation.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors.

Structural Antagonism-Aided Conformational Regulation Enables an Aptamer-Loop G-Quadruplex Modular Sensor of ß-Lactoglobulin.

A simple, reliable method for identifying ß-lactoglobulin (ß-LG) in dairy products is needed to protect those with ß-LG allergies. A common, practical strategy for target detection is designing simplified nucleic acid nanodevices by integrating functional components. This work presents a label-free modular ß-LG aptasensor consisting of an aptamer-loop G-quadruplex (G4), the working conformation of which is regulated by conformational antagonism to ensure respective module functionality and the related signal transduction. The polymorphic conformations of the module-fused sequence are systematically characterized, and the cause is revealed as shifting antagonistic equilibrium. Combined with conformational folding dynamics, this helped regulate functional conformations by fine-tuning the sequences. Furthermore, the principle of specific ß-LG detection by parallel G4 topology is examined as binding on the G4 aptamer loop by ß-LG to reinforce the G4 topology and fluorescence. Finally, a label-free, assembly-free, succinct, and turn-on fluorescent aptasensor is established, achieving excellent sensitivity across five orders of magnitude, rapidly detecting ß-LG within 22-min. This study provides a generalizable approach for the conformational regulation of module-fused G4 sequences and a reference model for creating simplified sensing devices for a variety of targets.

Sequence-Dependent Conformational Transitions of Disordered Proteins During Condensation.

Intrinsically disordered proteins (IDPs) can form biomolecular condensates through phase separation. It is recognized that the conformation of IDPs in the dense and dilute phases as well as at the interfaces of condensates can critically impact the resulting properties associated with their functionality. However, a comprehensive understanding of the conformational transitions of IDPs during condensation remains elusive. In this study, we employ a coarse-grained polyampholyte model, comprising an equal number of oppositely charged residues-glutamic acid and lysine-whereby conformations and phase behavior can be readily tuned by altering the protein sequence. By manipulating the sequence patterns from perfectly alternating to block-like, we obtain chains with ideal-like conformations to semi-compact structures in the dilute phase, while in the dense phase, the chain conformation is approximately that of an ideal chain, irrespective of the protein sequence. By performing simulations at different concentrations, we find that the chains assemble from the dilute phase through small oligomeric clusters to the dense phase, accompanied by a gradual swelling of the individual chains. We further demonstrate that these findings are applicable to several naturally occurring proteins involved in the formation of biological condensates. Concurrently, we delve deeper into the chain conformations within the condensate, revealing that chains at the interface show a strong sequence dependence, but remain more collapsed than those in the bulk-like dense phase. This study addresses critical gaps in our knowledge of IDP conformations within condensates as a function of protein sequence.

Understanding the Influence of API Conformations on Amorphous Dispersion Formation Potential Predictions using the R3m Molecular Descriptor.

The R3m molecular descriptor (R-GETAWAY third-order autocorrelation index weighted by the atomic mass) has previously been shown to encode molecular attributes that appear to be physically and chemically relevant to grouping diverse active pharmaceutical ingredients (API) according to their potential to form persistent amorphous solid dispersions (ASDs) with polyvinylpyrrolidone-vinyl acetate copolymer (PVPVA). The initial R3m dispersibility model was built by using a single three-dimensional (3D) conformation for each drug molecule. Since molecules in the amorphous state will adopt a distribution of conformations, molecular dynamics simulations were performed to sample conformations that are probable in the amorphous form, which resulted in a distribution of R3m values for each API. Although different conformations displayed R3m values that differed by as much as 0.4, the median of each R3m distribution and the value predicted from the single 3D conformation were very similar for most structures studied. The variability in R3m resulting from the distribution of conformations was incorporated into a logistic regression model for the prediction of ASD formation in PVPVA, which resulted in a refinement of the classification boundary relative to the model that only incorporated a single conformation of each API.

Deep learning for protein structure prediction and design-progress and applications.

Proteins are the key molecular machines that orchestrate all biological processes of the cell. Most proteins fold into three-dimensional shapes that are critical for their function. Studying the 3D shape of proteins can inform us of the mechanisms that underlie biological processes in living cells and can have practical applications in the study of disease mutations or the discovery of novel drug treatments. Here, we review the progress made in sequence-based prediction of protein structures with a focus on applications that go beyond the prediction of single monomer structures. This includes the application of deep learning methods for the prediction of structures of protein complexes, different conformations, the evolution of protein structures and the application of these methods to protein design. These developments create new opportunities for research that will have impact across many areas of biomedical research.

A comparative study of influenza A M2 protein conformations in DOPC/DOPS liposomes and in native E. coli membranes.

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IAM2) protein reconstituted at pH 7.4 in DOPC/DOPS bilayers to those in isolated E. coli membranes, having preserved its native proteins and lipids. IAM2 is a single-pass transmembrane protein known to assemble into homo-tetrameric proton channel. To represent this channel, we made a construct containing the IAM2's TMD region flanked by the juxtamembrane residues. The single cysteine substitute, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the ESR (electron spin resonance) study. We compared the conformations of the spin-labeled IAM2 residing in DOPC/DOPS and native E. coli membranes using continuous-wave (CW) ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400⩦ The CW ESR spectra corresponded to a nearly rigid limit spin label dynamics in both environments. In all cases, the DEER data were reconstructed into the distance distributions showing well-resolved peaks at 1.68 nm and 2.37 nm. The peak distance ratio was 1.41±0.2 and the amplitude ratio was 2:1. This is what one expects from four nitroxide spin-labels located at the corners of a square, indicative of an axially symmetric tetramer. Distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IAM2 tetramer in agreement with the NMR model. Thus, we can conclude that IAM2 TMD has similar conformations in model and native E. coli membranes of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Capillary electrophoresis-mass spectrometry for protein analyses under native conditions: Current progress and perspectives.

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigration separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out.

Isotropic liquid state of cocoa butter.

The complex crystal structure of coca butter (CB) is responsible for the unique melting behavior, surface gloss, and mechanical properties of chocolate. While most studies concentrated on the crystalline state of CB, few studied the isotropic liquid state, which has a major impact on the crystallization process and the characteristics of the resulting crystals. In this study, the molecular organizations of the main CB triacylglycerols (TAGs; 1,3-dipalmitoyl-2-oleoylglycerol, palmitoyl-oleoyl-stearoylglycerol, POS, and 1,3-distearoyl-2-oleoylglycerol) were studied. The findings revealed the tunning-fork (Tf) conformation, commonly found in the crystalline state, is the least abundant in the isotropic liquid state of CB and pure TAGs. Notably, POS was found to interact with itself in CB, while its molecules with Tf conformation, although in small amounts in the mixture, tend to pair with each other at lower temperatures. These results highlight the significance of POS in CB crystallization and provide insights for developing CB alternatives.

Synthesis and Characterization of Stimuli-Responsive Polymer Brushes in Nanofluidic Channels.

Nanochannels with controllable gating behavior are attractive features in a wide range of nanofluidic applications including viral detection, particle sorting, and flow regulation. Here, we use selective sidewall functionalization of nanochannels with a polyelectrolyte brush to investigate the channel gating response to variations in solution pH and ionic strength. The conformational and structural changes of the interfacial brush layer within the channels are interrogated by specular and off-specular neutron reflectometry. Simultaneous fits of the specular and off-specular signals, using a dynamical theory model and a fitting optimization protocol, enable detailed characterization of the brush conformations and corresponding channel geometry under different solution conditions. Our results indicate a collapsed brush state under basic pH, equivalent to an open gate, and an expanded brush state representing a partially closed gate upon decreasing the pH and salt concentration. These findings open new possibilities in noninvasive in situ characterization of tunable nanofluidics and lab-on-chip devices with advanced designs and improved functionality.

The pseudotorsional space of RNA.

The characterization of the conformational landscape of the RNA backbone is rather complex due to the ability of RNA to assume a large variety of conformations. These backbone conformations can be depicted by pseudotorsional angles linking RNA backbone atoms, from which Ramachandran-like plots can be built. We explore here different definitions of these pseudotorsional angles, finding that the most accurate ones are the traditional η (eta) and θ (theta) angles, which represent the relative position of RNA backbone atoms P and C4'. We explore the distribution of η - θ in known experimental structures, comparing the pseudotorsional space generated with structures determined exclusively by one experimental technique. We found that the complete picture only appears when combining data from different sources. The maps provide a quite comprehensive representation of the RNA accessible space, which can be used in RNA-structural predictions. Finally, our results highlight that protein interactions lead to significant changes in the population of the η - θ space, pointing toward the role of induced-fit mechanisms in protein-RNA recognition.

Control of Substrate Conformation by Hydrogen Bonding in a Retaining ß-Endoglycosidase.

Bacterial ß-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as ß-cellulose, ß-glucans and ß-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed 4 C1 conformation to a distorted 1 S3 /1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OH⋅⋅⋅nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells.

The envelope (Env) glycoproteins on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAbs) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes, including V2i, the gp120-g41 interface, and gp41-MPER, are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by the pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where the virus-mAb mix was pre-incubated/not pre-incubated for 1 hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein mediates viral entry and is the sole target of neutralizing antibodies. Our data suggest that antibody epitopes including V2q (e.g., PG9, PGT145), CD4bs (e.g., VRC01, 3BNC117), and V3 (2219, 2557) are masked on HIV-1 particles. The PG9 and 2219 epitopes became accessible for binding after conformational unmasking was induced by the pre-binding of select mAbs. Attempts to understand the masking mechanism led to the revelation that interaction between virus and host cells is needed to sensitize the virions for neutralization by broadly neutralizing antibodies (bNAbs). These data provide insight on how bNAbs may gain access to these occluded epitopes to exert their neutralization effects and block HIV-1 infection. These findings have important implications for the way we evaluate the neutralizing efficacy of antibodies and can potentially guide vaccine design.

Ternary Mixed Micelle Hexadecyltrimethylammonium Bromide-Dodecyltrimethylammonium Bromide-Sodium Deoxycholate: Gibbs Free Energy of Mixing and Excess Gibbs Energy of Mixing.

Pharmaceutical, food, and cosmetic formulations often contain binary or ternary surfactant mixtures with synergistic interactions amongst micellar building blocks. Here, a ternary mixture of the surfactants hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, and sodium deoxycholate is examined to see if the molar fractions of the surfactants in the ternary mixed micellar pseudophase are determined by the interaction coefficients between various pairs of the surfactants or by their propensity to self-associate. Critical micelle concentrations (CMC) of the analyzed ternary mixtures are determined experimentally (spectrofluorimetrically using pyrene as the probe molecule). Thermodynamic parameters of ternary mixtures are calculated from CMC values using the Regular Solution protocol. The tendency for monocomponent surfactants to self-associate (lower value of CMC) determines the molar fractions of surfactant in the mixed micelle if there is no issue with the packing of the micelle building units of the ternary mixed micelle. If a more hydrophobic surfactant is incorporated into the mixed micelle, the system (an aqueous solution of surfactants) is then the most thermodynamically stabilized.

Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.

The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.

Regio- and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X-ray crystallography and computational modeling.

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25-hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio- and stereoselective manner to 16ß-hydroxytestosterone. To reveal the structural determinants governing the regio- and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme-iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon-hydrogen bond was taken into account. Overall, the first-time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site-directed mutagenesis toward improved enzymatic properties of this enzyme.