Heme pocket modulates protein conformation and diguanylate cyclase activity of a tetrameric globin coupled sensor.

Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O2-dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O2 binding modulates activity of diguanylate cyclase-containing GCS proteins.

Molecular insights into the interaction between a disordered protein and a folded RNA.

Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, SERF. At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 TAR RNA (TAR) with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation.

Characterization of recombinant human lactoferrin expressed in Komagataella phaffii.

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system.

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3ß3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by ß-ß interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the ß subunit interfaces, with subsequent targeted improvements of the subunits.

A Piecewise Design Approach to Engineering a Miniature ACE2 Mimic to Bind SARS-CoV-2.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues its global spread, the exploration of novel therapeutic and diagnostic strategies is still needed. The virus enters host cells by binding the angiotensin-converting enzyme 2 (ACE2) receptor through the spike protein. Here, we develop an engineered, small, stable, and catalytically inactive version of ACE2, termed miniature ACE2 (mACE2), designed to bind the spike protein with high affinity. Employing a magnetic nanoparticle-based assay, we harnessed the strong binding affinity of mACE2 to develop a sensitive and specific platform for the detection or neutralization of SARS-CoV-2. Our findings highlight the potential of engineered mACE2 as a valuable tool in the fight against SARS-CoV-2. The success of developing such a small reagent based on a piecewise molecular design serves as a proof-of-concept approach for the rapid deployment of such agents to diagnose and fight other viral diseases.

Disordered regions in proteusin peptides guide post-translational modification by a flavin-dependent RiPP brominase.

To biosynthesize ribosomally synthesized and post-translationally modified peptides (RiPPs), enzymes recognize and bind to the N-terminal leader region of substrate peptides which enables catalytic modification of the C-terminal core. Our current understanding of RiPP leaders is that they are short and largely unstructured. Proteusins are RiPP precursor peptides that defy this characterization as they possess unusually long leaders. Proteusin peptides have not been structurally characterized, and we possess scant understanding of how these atypical leaders engage with modifying enzymes. Here, we determine the structure of a proteusin peptide which shows that unlike other RiPP leaders, proteusin leaders are preorganized into a rigidly structured region and a smaller intrinsically disordered region. With residue level resolution gained from NMR titration experiments, the intermolecular peptide-protein interactions between proteusin leaders and a flavin-dependent brominase are mapped onto the disordered region, leaving the rigidly structured region of the proteusin leader to be functionally dispensable. Spectroscopic observations are biochemically validated to identify a binding motif in proteusin peptides that is conserved among other RiPP leaders as well. This study provides a structural characterization of the proteusin peptides and extends the paradigm of RiPP modification enzymes using not only unstructured peptides, but also structured proteins as substrates.

Oxygen-selective regulation of cyclic di-GMP synthesis by a globin coupled sensor with a shortened linking domain modulates Shewanella sp. ANA-3 biofilm.

Bacteria utilize heme proteins, such as globin coupled sensors (GCSs), to sense and respond to oxygen levels. GCSs are predicted in almost 2000 bacterial species and consist of a globin domain linked by a central domain to a variety of output domains, including diguanylate cyclase domains that synthesize c-di-GMP, a major regulator of biofilm formation. To investigate the effects of middle domain length and heme edge residues on GCS diguanylate cyclase activity and cellular function, a putative diguanylate cyclase-containing GCS from Shewanella sp. ANA-3 (SA3GCS) was characterized. Binding of O2 to the heme resulted in activation of diguanylate cyclase activity, while NO and CO binding had minimal effects on catalysis, demonstrating that SA3GCS exhibits greater ligand selectivity for cyclase activation than many other diguanylate cyclase-containing GCSs. Small angle X-ray scattering analysis of dimeric SA3GCS identified movement of the cyclase domains away from each other, while maintaining the globin dimer interface, as a potential mechanism for regulating cyclase activity. Comparison of the Shewanella ANA-3 wild type and SA3GCS deletion (ΔSA3GCS) strains identified changes in biofilm formation, demonstrating that SA3GCS diguanylate cyclase activity modulates Shewanella phenotypes.

Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer.

Technologically critical rare-earth elements are notoriously difficult to separate, owing to their subtle differences in ionic radius and coordination number1-3. The natural lanthanide-binding protein lanmodulin (LanM)4,5 is a sustainable alternative to conventional solvent-extraction-based separation6. Here we characterize a new LanM, from Hansschlegelia quercus (Hans-LanM), with an oligomeric state sensitive to rare-earth ionic radius, the lanthanum(III)-induced dimer being >100-fold tighter than the dysprosium(III)-induced dimer. X-ray crystal structures illustrate how picometre-scale differences in radius between lanthanum(III) and dysprosium(III) are propagated to Hans-LanM's quaternary structure through a carboxylate shift that rearranges a second-sphere hydrogen-bonding network. Comparison to the prototypal LanM from Methylorubrum extorquens reveals distinct metal coordination strategies, rationalizing Hans-LanM's greater selectivity within the rare-earth elements. Finally, structure-guided mutagenesis of a key residue at the Hans-LanM dimer interface modulates dimerization in solution and enables single-stage, column-based separation of a neodymium(III)/dysprosium(III) mixture to >98% individual element purities. This work showcases the natural diversity of selective lanthanide recognition motifs, and it reveals rare-earth-sensitive dimerization as a biological principle by which to tune the performance of biomolecule-based separation processes.

Enteroviral 2C protein is an RNA-stimulated ATPase and uses a two-step mechanism for binding to RNA and ATP.

The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here, we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state. 2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the α and ß phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase.

The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg2+ under conditions that mimic the Escherichia coli cytoplasm. We determined Mg2+ binding constants for the top 15 E. coli metabolites, comprising 80% of the metabolome by concentration at physiological pH and monovalent ion concentrations. These data were used to inform the development of an artificial cytoplasm that mimics in vivo E. coli conditions, which we term "Eco80". We empirically determined that the mixture of E. coli metabolites in Eco80 approximated single-site binding behavior toward Mg2+ in the biologically relevant free Mg2+ range of ∼0.5 to 3 mM Mg2+, using a Mg2+-sensitive fluorescent dye. Effects of Eco80 conditions on the thermodynamic stability, chemical stability, structure, and catalysis of RNA were examined. We found that Eco80 conditions lead to opposing effects on the thermodynamic and chemical stabilities of RNA. In particular, the thermodynamic stability of RNA helices was weakened by 0.69 ± 0.12 kcal/mol, while the chemical stability was enhanced ∼2-fold, which can be understood using the speciation of Mg2+ between weak and strong Mg2+-metabolite complexes in Eco80. Overall, the use of Eco80 reflects RNA function in vivo and enhances the biological relevance of mechanistic studies of RNA.

Adaptation-proof SARS-CoV-2 vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through 'epitope grafting,' in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of 'epitope grafting' in rational vaccine design.

Adaptation-proof SARS-CoV-2 vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through 'epitope grafting,' in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of 'epitope grafting' in rational vaccine design.

Heme-Edge Residues Modulate Signal Transduction within a Bifunctional Homo-Dimeric Sensor Protein.

Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.

Bio-inspired strategies for next-generation perovskite solar mobile power sources.

Smart electronic devices are becoming ubiquitous due to many appealing attributes including portability, long operational time, rechargeability and compatibility with the user-desired form factor. Integration of mobile power sources (MPS) based on photovoltaic technologies with smart electronics will continue to drive improved sustainability and independence. With high efficiency, low cost, flexibility and lightweight features, halide perovskite photovoltaics have become promising candidates for MPS. Realization of these photovoltaic MPS (PV-MPS) with unconventionally extraordinary attributes requires new 'out-of-box' designs. Natural materials have provided promising designing solutions to engineer properties under a broad range of boundary conditions, ranging from molecules, proteins, cells, tissues, apparatus to systems in animals, plants, and humans optimized through billions of years of evolution. Applying bio-inspired strategies in PV-MPS could be biomolecular modification on crystallization at the atomic/meso-scale, bio-structural duplication at the device/system level and bio-mimicking at the functional level to render efficient charge delivery, energy transport/utilization, as well as stronger resistance against environmental stimuli (e.g., self-healing and self-cleaning). In this review, we discuss the bio-inspired/-mimetic structures, experimental models, and working principles, with the goal of revealing physics and bio-microstructures relevant for PV-MPS. Here the emphasis is on identifying the strategies and material designs towards improvement of the performance of emerging halide perovskite PVs and strategizing their bridge to future MPS.

Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme.

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

Maize unstable factor for orange1 is essential for endosperm development and carbohydrate accumulation.

Maize (Zea mays L.) Ufo1-1 is a spontaneous dominant mutation of the unstable factor for orange1 (ufo1). We recently cloned ufo1, which is a Poaceae-specific gene highly expressed during seed development in maize. Here, we have characterized Ufo1-1 and a loss-of-function Ds insertion allele (ufo1-Dsg) to decipher the role of ufo1 in maize. We found that both ufo1 mutant alleles impact sugars and hormones, and have defects in the basal endosperm transfer layer (BETL) and adjacent cell types. The Ufo1-1 BETL had reduced cell elongation and cell wall ingrowth, resulting in cuboidal shaped transfer cells. In contrast, the ufo1-Dsg BETL cells showed a reduced overall size with abnormal wall ingrowth. Expression analysis identified the impact of ufo1 on several genes essential for BETL development. The overexpression of Ufo1-1 in various tissues leads to ectopic phenotypes, including abnormal cell organization and stomata subsidiary cell defects. Interestingly, pericarp and leaf transcriptomes also showed that as compared with wild type, Ufo1-1 had ectopic expression of endosperm development-specific genes. This study shows that Ufo1-1 impacts the expression patterns of a wide range of genes involved in various developmental processes.

Small Glycols Discover Cryptic Pockets on Proteins for Fragment-Based Approaches.

Cryptic pockets are visible in ligand-bound protein structures but are occluded in unbound structures. Utilizing these pockets in fragment-based drug-design provides an attractive option for proteins not tractable by classical binding sites. However, owing to their hidden nature, they are difficult to identify. Here, we show that small glycols find cryptic pockets on a diverse set of proteins. Initial crystallography experiments serendipitously revealed the ability of ethylene glycol, a small glycol, to identify a cryptic pocket on the W6A mutant of the RBSX protein (RBSX-W6A). Explicit-solvent molecular dynamics (MD) simulations of RBSX-W6A with the exposed state of the cryptic pocket (ethylene glycol removed) revealed closure of the pocket reiterating that the exposed state of cryptic pockets in general are unstable in the absence of ligands. Also, no change in the pocket was observed for simulations of RBSX-W6A with the occluded state of the cryptic pocket, suggesting that water molecules are not able to open the cryptic pocket. "Cryptic-pocket finding" potential of small glycols was then supported and generalized through additional crystallography experiments, explicit-cosolvent MD simulations, and protein data set construction and analysis. The cryptic pocket on RBSX-W6A was found again upon repeating the crystallography experiments with another small glycol, propylene glycol. Use of ethylene glycol as a probe molecule in cosolvent MD simulations led to the enhanced sampling of the exposed state of experimentally observed cryptic sites on a test set of two proteins (Niemann-Pick C2, Interleukin-2). Further, analyses of protein structures with validated cryptic sites showed that ethylene glycol molecules bind to sites on proteins (Bcl-xL, G-actin, myosin II, and glutamate receptor 2), which become apparent upon binding of biologically relevant ligands. Our study thus suggests potential application of the small glycols in experimental and computational fragment-based approaches to identify cryptic pockets in apparently undruggable and/or difficult targets, making these proteins amenable to drug-design strategies.

Cocrystalline Polymer Films Exhibiting Second-Order Nonlinear Optical Properties.

Tailored polymer materials exhibiting high-glass transition temperatures, cross-linked matrices, and/or strong intermolecular interactions containing electric-field poled nonlinear optical (NLO) chromophores are promising materials for applications in optical telecommunication, high-performance computing, and data transmission. Although the current design parameters have led to significant advances in NLO materials, we introduce an alternative, yet highly effective, approach in which a NLO chromophore is cocrystallized with a polymer, forming a noncentrosymmetric hybrid host-guest complex. Specifically, poly(ethylene oxide) (PEO) and 2-chloro-4-nitroaniline (CNA) will cocrystallize and exhibit second harmonic generation (SHG) activity due to the formation of a noncentrosymmetric cocrystalline unit cell where the chromophore exhibits acentric alignment. Furthermore, the hybrid PEO/CNA films exhibit interesting SHG activity at elevated temperature in which SHG intensity decreases to zero when the cocrystal orientation randomizes due to sample melting. Aligning and maintaining a cocrystalline domain orientation via the formation of hybrid host-guest complexes, while imparting SHG properties, is an innovative approach for creating materials exhibiting SHG properties.