Binding Signatures Research Articles

Structural basis for high specificity of octopine binding in the plant pathogen Agrobacterium tumefaciens

Agrobacterium pathogens of octopine- and nopaline-types force host plants to produce either octopine or nopaline compounds, which they use as nutrients. Two Agrobacterium ABC-transporters and their cognate periplasmic binding proteins (PBPs) OccJ and NocT import octopine and nopaline/octopine, respectively. Here, we show that both octopine transport and degradation confer a selective advantage to octopine-type A. tumefaciens when it colonizes plants. We report the X-ray structures of the unliganded PBP OccJ and its complex with octopine as well as a structural comparison with NocT and the related PBP LAO from Salmonella enterica, which binds amino acids (lysine, arginine and ornithine). We investigated the specificity of OccJ, NocT and LAO using several ligands such as amino acids, octopine, nopaline and octopine analogues. OccJ displays a high selectivity and nanomolar range affinity for octopine. Altogether, the structural and affinity data allowed to define an octopine binding signature in PBPs and to construct a OccJ mutant impaired in octopine binding, a selective octopine-binding NocT and a non-selective octopine-binding LAO by changing one single residue in these PBPs. We proposed the PBP OccJ as a major trait in the ecological specialization of octopine-type Agrobacterium pathogens when they colonize and exploit the plant host.

Crystal structure and thermodynamic dissection of chitin oligosaccharide binding to the LysM module of chitinase-A from Pteris ryukyuensis

We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 Å. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (ΔGr°) for binding of (GlcNAc)6, (GlcNAc)5, and (GlcNAc)4 to PrLysM2 were determined to be −5.4, −5,4 and −4.6 kcal mol−1, respectively, by ITC. Thermodynamic dissection of the binding energetics of (GlcNAc)6 revealed that the driving force is the enthalpy change (ΔHr° = −11.7 ± 0.2 kcal/mol) and the solvation entropy change (−TΔSsolv° = −5.9 ± 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module.

Generic Schemes for Single-Molecule Kinetics. 2: Information Content of the Poisson Indicator.

Recently, we described a pathway analysis technique (paper 1) for analyzing generic schemes for single-molecule kinetics based upon the first-passage time distribution. Here, we employ this method to derive expressions for the Poisson indicator, a normalized measure of stochastic variation (essentially equivalent to the Fano factor and Mandel's Q parameter), for various renewal (i.e., memoryless) enzymatic reactions. We examine its dependence on substrate concentration, without assuming all steps follow Poissonian kinetics. Based upon fitting to the functional forms of the first two waiting time moments, we show that, to second order, the non-Poissonian kinetics are generally underdetermined but can be specified in certain scenarios. For an enzymatic reaction with an arbitrary intermediate topology, we identify a generic minimum of the Poisson indicator as a function of substrate concentration, which can be used to tune substrate concentration to the stochastic fluctuations and to estimate the largest number of underlying consecutive links in a turnover cycle. We identify a local maximum of the Poisson indicator (with respect to substrate concentration) for a renewal process as a signature of competitive binding, either between a substrate and an inhibitor or between multiple substrates. Our analysis explores the rich connections between Poisson indicator measurements and microscopic kinetic mechanisms.

Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity.

Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. We used these binding signatures to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. Taken together, we identify enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity.

Abstract 4493: The co-chaperone UNC45A controls cancer cell proliferation through Nek7 and centrosomal separation

Abstract Recent findings have shown that the Heat Shock Protein 90 (Hsp90) co-chaperone UNC45A is overexpressed in ovarian and breast cancers. Previously, we have shown that UNC45A is a centrosomal protein essential for cervical tumor cell growth through activation of the checkpoint kinase 1 (ChK1). In this report, we further examined the role of UNC45A in breast tumorigenesis using a variety of biochemical and cell biology techniques and animal models. We confirmed that UNC45A is highly overexpressed in human breast-infiltrating ductal carcinomas as compared to adjacent normal tissues. Silencing UNC45A in vitro blocked the proliferation of all breast cancer subtypes and drastically reduced tumor growth of the triple negative MDA-MB-231 cell line implanted in mammary fat pads of NOD/SCID mice. However, loss of UNC45A did not affect the proliferation of normal mammary cells. Remarkably, UNC45A becomes more nuclear in human cancer tissues and cancer cell lines as compared to normal tissues and non-transformed Hs578Bst and HME mammary cell lines, respectively. This suggests an important nuclear function for UNC45A during tumorigenesis. Microarray analysis of mRNA from Hs578T cells showed that loss of UNC45A alters the expression of 121 genes, involved in cancer and cellular development and growth networks. Relevant to cell proliferation, we found that Nek7 gene was significantly repressed upon silencing UNC45A, which was validated by RTqPCR and Western blot analyses in multiple breast cancer cell lines. Nek7 is a member of the NIMA (never in mitosis, gene A) family of serine/threonine kinases. It plays a key role in centrosomal separation during mitosis. This correlates neatly with our observation that loss of UNC45A causes a centrosomal separation defect, cell proliferation arrest and death of breast cancer cell lines. ChIP experiments showed that UNC45A binds to the promoter of the Nek7 gene, suggesting direct transcriptional regulation. Interestingly, the UNC45A sequence contains four LxxLL motifs, which are thought to be signatures for co-activator binding to nuclear receptors. Furthermore, computational analysis identified two glucocorticoid response elements (GRE) consensus sequences in the Nek7 promoter, suggesting its transcriptional regulation by the glucocorticoid receptor (GR). This hypothesis was further strengthened by a significant decrease in the mRNA and protein levels of Nek7 upon silencing GR. Thus, our data suggest that UNC45A functions as a GR co-activator to control Nek7 gene transcription. Consistent with this, immunoprecipitation experiments confirmed that UNC45A and GR form endogenous complexes, and treatment of Hs578T and MCF7 cell lines with dexamethasone upregulates Nek7 mRNA and protein levels. In conclusion our data strongly support the premise that UNC45A promotes Nek7 transcription through activation of GR, and thus controls centrosomal separation and cancer cell proliferation. Citation Format: Yasmeen Jilani, Nada H. Eisa, Kashish Kainth, Sumin Lu, Nehal M. Elsherbiny, Laila A. Eissa, Mamdouh M. Elshishtawy, Hasan Korkaya, Abdeljabar El Andaloussi, Ahmed Chadli. The co-chaperone UNC45A controls cancer cell proliferation through Nek7 and centrosomal separation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2017-4493

Abstract 5626: Investigation of T cell activation by anti-human PD-1 antibodies Nivolumab, Pembrolizumab and BGB-A317 using tumor-infiltrating lymphocytes (TILs) from colorectal cancer and colorectal liver metastasis patients

Abstract Blockade of the PD-1 pathway with anti-PD-1 antibody, such as nivolumab (nivo) and pembrolizumab (pembro), has led to remarkable clinical responses in patients with many different cancer types. In this study, we investigated the activation of tumor-infiltrating lymphocytes (TILs) from patients with colorectal cancer (CRC) and colorectal liver metastasis (CLM) by nivo, pembro and BGB-A317, a novel humanized IgG4 anti-PD-1 antibody under clinical development. BGB-A317 has a unique binding signature to PD-1 with high affinity. Additionally, it is engineered to remove Fc gamma receptor (FcγR) binding, including FcγRI, FcγRIIA, FcγRIIB and FcγRIIIA. In our studies, all three anti-PD-1 antibodies showed significant increases in IFN-γ and TILs proliferation in a 3D tumor spheroid model. In both CRC and CLM patients, BGB-A317 treatment at concentration levels of 0.1, 1 and 10 μg/mL led to higher IFN-γ production than that in nivo and pembro treated groups. The enhanced TILs function was associated with a high density of CD8+ T cells, but inversely correlated with the percentage of CD11b+ myeloid cells in CRC tumors. Of interest, BGB-A317 showed better activation of TILs in the liver metastasis tumor microenvironment (TME) where macrophages were more abundant. This observation is consistent with the hypothesis that removal of FcγR binding might offer advantage to PD-1 mAbs in tumor microenvironment enriched with macrophages. Citation Format: Lusong Luo, Xiaoran Wu, Tong Zhang, Chunyan Fu, Yanjuan Zhang, Amy Guo, Dongping Zhou, Lianhai Zhang, Kun Wang, Baocai Xing, Jiafu Ji, Lai Wang, Kang Li. Investigation of T cell activation by anti-human PD-1 antibodies Nivolumab, Pembrolizumab and BGB-A317 using tumor-infiltrating lymphocytes (TILs) from colorectal cancer and colorectal liver metastasis patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5626. doi:10.1158/1538-7445.AM2017-5626

Doxorubicin kinetics and effects on lung cancer cell lines using in vitro Raman micro-spectroscopy: binding signatures, drug resistance and DNA repair.

Raman micro-spectroscopy is a non-invasive analytical tool, whose potential in cellular analysis and monitoring drug mechanisms of action has already been demonstrated, and which can potentially be used in pre-clinical and clinical applications for the prediction of chemotherapeutic efficacy. To further investigate such potential clinical application, it is important to demonstrate its capability to differentiate drug mechanisms of action and cellular resistances. Using the example of Doxorubicin (DOX), in this study, it was used to probe the cellular uptake, signatures of chemical binding and subsequent cellular responses, of the chemotherapeutic drug in two lung cancer cell lines, A549 and Calu-1. Multivariate statistical analysis was used to elucidate the spectroscopic signatures associated with DOX uptake and subcellular interaction. Biomarkers related to DNA damage and repair, and mechanisms leading to apoptosis were also measured and correlated to Raman spectral profiles. Results confirm the potential of Raman spectroscopic profiling to elucidate both drug kinetics and pharmacodynamics and differentiate cellular drug resistance associated with different subcellular accumulation rates and subsequent cellular response to DNA damage, pointing towards a better understanding of drug resistance for personalised targeted treatment.

Characterization of the differential coregulator binding signatures of the Retinoic Acid Receptor subtypes upon (ant)agonist action

Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARβ/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARβ) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to >60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARβ compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.

The thermodynamic signature of ligand binding to histone deacetylase-like amidohydrolases is most sensitive to the flexibility in the L2-loop lining the active site pocket

BackgroundThe analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied. MethodsDirected mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases. ResultsThe general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation. ConclusionsThe study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes. General significanceThe flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness.

Water‐Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase

AbstractThis study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein–ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water‐mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy‐driven or entropy‐driven. Our findings highlight a possible asymmetry in protein–ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.

Water‐Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase

This study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein-ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water-mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy-driven or entropy-driven. Our findings highlight a possible asymmetry in protein-ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM min−1 and 68.49 s−1 respectively and 0.693 mM, 105.32 mM min−1 and 89.57 s−1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.

DRP-1 is required for BH3 mimetic-mediated mitochondrial fragmentation and apoptosis

The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs, such as Navitoclax and Venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend on MCL-1 for survival, which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far, while demonstrating early promise in vitro, fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects, we developed a rapid differential scanning fluorimetry (DSF)-based assay, and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested, only A-1210477 induced rapid, concentration-dependent apoptosis, which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-XL, A-1210477 exhibited marked synergy with A-1331852, a BCL-XL specific inhibitor, to induce cell death. Despite this selectivity and potency, A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression, suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However, A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1, and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands, and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis.

Coordination-Dependent Spectroscopic Signatures of Divalent Metal Ion Binding to Carboxylate Head Groups: H2- and He-Tagged Vibrational Spectra of M2+·RCO2¯ (M = Mg and Ca, R = -CD3, -CD2CD3) Complexes.

We explore the intramolecular distortions present in divalent metal ion-carboxylate ion pairs using vibrational spectroscopy of the cryogenically cooled, mass-selected species isolated in the gas phase. The spectral signatures of the C-O stretching modes are identified using the perdeutero isotopologues of the acetate and propionate anions to avoid congestion arising from the CH2 fundamentals. Both Ca2+ and Mg2+ are observed to bind in a symmetrical, so-called "bidentate" arrangement to the -CO2¯ group. The very strong deformations of the head groups displayed by the binary complexes dramatically relax when either neutral water molecules or counterions are attached to the Mg2+RCO2¯ cation. These results emphasize the critical role that local coordination plays when using the RCO2¯ bands to deduce the metal ion complexation motif in condensed media.

AML associated oncofusion proteins PML-RARA, AML1-ETO and CBFB-MYH11 target RUNX/ETS-factor binding sites to modulate H3ac levels and drive leukemogenesis.

Chromosomal translocations are one of the hallmarks of acute myeloid leukemia (AML), often leading to gene fusions and expression of an oncofusion protein. Over recent years it has become clear that most of the AML associated oncofusion proteins molecularly adopt distinct mechanisms for inducing leukemogenesis. Still these unique molecular properties of the chimeric proteins converge and give rise to a common pathogenic molecular mechanism. In the present study we compared genome-wide DNA binding and transcriptome data associated with AML1-ETO, CBFB-MYH11 and PML-RARA oncofusion protein expression to identify unique and common features. Our analyses revealed targeting of oncofusion binding sites to RUNX1 and ETS-factor occupied genomic regions. In addition, it revealed a highly comparable global histone acetylation pattern, similar expression of common target genes and related enrichment of several biological pathways critical for maintenance of AML, suggesting oncofusion proteins deregulate common gene programs despite their distinct binding signatures and mechanisms of action.

Rational Design of Thermodynamic and Kinetic Binding Profiles by Optimizing Surface Water Networks Coating Protein-Bound Ligands.

A previously studied congeneric series of thermolysin inhibitors addressing the solvent-accessible S2' pocket with different hydrophobic substituents showed modulations of the surface water layers coating the protein-bound inhibitors. Increasing stabilization of water molecules resulted in an enthalpically more favorable binding signature, overall enhancing affinity. Based on this observation, we optimized the series by designing tailored P2' substituents to improve and further stabilize the surface water network. MD simulations were applied to predict the putative water pattern around the bound ligands. Subsequently, the inhibitors were synthesized and characterized by high-resolution crystallography, microcalorimetry, and surface plasmon resonance. One of the designed inhibitors established the most pronounced water network of all inhibitors tested so far, composed of several fused water polygons, and showed 50-fold affinity enhancement with respect to the original methylated parent ligand. Notably, the inhibitor forming the most perfect water network also showed significantly prolonged residence time compared to the other tested inhibitors.

Structural Basis for High Specificity of Amadori Compound and Mannopine Opine Binding in Bacterial Pathogens

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,β-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria.

Temperature Independence of Ultrafast Photoisomerization in Thermophilic Rhodopsin: Assessment versus Other Microbial Proton Pumps.

Primary photochemical events in the unusually thermostable proton pumping rhodopsin of Thermus thermophilus bacterium (TR) are reported for the first time. Internal conversion in this protein is shown to be significantly faster than in bacteriorhodopsin (BR), making it the most rapidly isomerizing microbial proton pump known. Internal conversion (IC) dynamics of TR and BR were recorded from room temperature to the verge of thermal denaturation at 70 °C and found to be totally independent of temperature in this range. This included the well documented multiexponential nature of IC in BR, suggesting that assignment of this to ground state structural inhomogeneity needs revision. TR photodynamics were also compared with that of the phylogenetically more similar proton pump Gloeobacter rhodopsin (GR). Despite this similarity GR has poor thermal stability, and the excited state decays significantly more slowly and exhibits very prominent stretched exponential behavior. Coherent torsional wave-packets induced by impulsive photoexcitation of TR and GR show marked resemblance to each other in frequency and amplitude and differ strikingly from similar signatures in pump-probe data of BR and other microbial retinal proteins. Possible correlations between IC rates and thermal stability and the promise of using torsional coherence signatures for understanding chromophore protein binding in microbial retinal proteins are discussed.

Regular Behaviours with Names

Nominal sets provide a framework to study key notions of syntax and semantics such as fresh names, variable binding and α-equivalence on a conveniently abstract categorical level. Coalgebras for endofunctors on nominal sets model, e.g., various forms of automata with names as well as infinite terms with variable binding operators (such as λ-abstraction). Here, we first study the behaviour of orbit-finite coalgebras for functors \(\bar F\) on nominal sets that lift some finitary set functor F. We provide sufficient conditions under which the rational fixpoint of \(\bar F\), i.e. the collection of all behaviours of orbit-finite \(\bar F\)-coalgebras, is the lifting of the rational fixpoint of F. Second, we describe the rational fixpoint of the quotient functors: we introduce the notion of a sub-strength of an endofunctor on nominal sets, and we prove that for a functor G with a sub-strength the rational fixpoint of each quotient of G is a canonical quotient of the rational fixpoint of G. As applications, we obtain a concrete description of the rational fixpoint for functors arising from so-called binding signatures with exponentiation, such as those arising in coalgebraic models of infinitary λ-terms and various flavours of automata.

Recognition of Specific Uridines in tRNA Substrates by Dihydrouridine Synthases

The reduction of specific uridines to dihydrouridine is one of the most abundant modifications found in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Different subfamilies of dihydrouridine synthases (Dus) selectively reduce distinct uridines, located at spatially unique positions of folded tRNA. Because the catalytic center of all Dus enzymes is conserved, it was unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site.Unexpectedly, crystal structures of U16-specific E.coli Dus in complex with tRNA_Phe and tRNA_Trp [1] show that the U16-specific subfamily binds their respective tRNA substrates in an almost reverse orientation to the U20-specific subfamily [2]. The tRNA docking orientation appears to be guided by subfamily-specific clusters of ‘binding signatures’ together with differences in the shape of the positively charged tRNA-binding surfaces. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule.Interestingly, the X-ray structure of the type-2 human Dus reveals differences in the recognition domain and a unique beta-sheet insertion into the catalytic domain [3] which may influence tRNA recognition.