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An automated pipeline for comprehensive calculation of intermolecular interaction energies based on molecular force-fields using the Tinker molecular modelling package is presented. Starting with non-optimized chemically intuitive monomer structures, the pipeline allows the approximation of global minimum energy monomers and dimers, configuration sampling for various monomer-monomer distances, estimation of coordination numbers by molecular dynamics simulations, and the evaluation of differential pair interaction energies. The latter are used to derive Flory-Huggins parameters and isotropic particle-particle repulsions for Dissipative Particle Dynamics (DPD). The computational results for force fields MM3, MMFF94, OPLS-AA and AMOEBA09 are analyzed with Density Functional Theory (DFT) calculations and DPD simulations for a mixture of the non-ionic polyoxyethylene alkyl ether surfactant C 10 E 4 with water to demonstrate the usefulness of the approach.Scientific ContributionTo our knowledge, there is currently no open computational pipeline for differential pair interaction energies at all. This work aims to contribute an (at least academically available, open) approach based on molecular force fields that provides a robust and efficient computational scheme for their automated calculation for small to medium-sized (organic) molecular dimers. The usefulness of the proposed new calculation scheme is demonstrated for the generation of mesoscopic particles with their mutual repulsive interactions., (© 2024. The Author(s).)

3β-hydroxy-Δ5-steroid dehydrogenases (3βHSDs) are supposed to be involved in 5β -cardenolide biosynthesis. Here, a novel 3βHSD ( Dl3βHSD2 ) was isolated from Digitalis lanata shoot cultures and expressed in E. coli . Recombinant Dl 3βHSD1 and Dl 3βHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl 3βHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3βHSD1, Dl3βHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3βHSDs was realised by Agrobacterium -mediated transfer of Dl3βHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S: Dl3βHSD1 and 35S: Dl3βHSD2 ) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S: Dl3βHSD1 lines than in the controls. In the 35S: Dl3βHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3βHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3β-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3βHSD1 knockdown. These results can be taken as the first direct proof that Dl 3βHSD1 is indeed involved in 5β -cardenolide biosynthesis., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Objectives: Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide., Methods: The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70 289-306 , citrullinated CILP 982-996 and galactosylated Col2 259-273 were determined on cocrystallisation. T cells specific for Col2 259-273 were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2 259-273 tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) α-chains and β-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells., Results: The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2 259-273 were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2 259-273 were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2 259-273 -specific TCR complexed with DRB1*04:01/Col2 259-273 provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264., Conclusions: The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis., Competing Interests: Competing interests: NS, BX, SW, RH and HB are listed as inventors on Patent EP2020072287 (https://www.onscope.com/ipowner/en/ip/ptwo/EP2020072287.html). SW, N-ND, RH and HB are lasted as inventors on Patent EP2020072280 (https://www.onscope.com/ipowner/en/ip/ptwo/EP2020072280.html). The owner of both patents is Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung E.V. (Germany). SW is listed on these patents under her maiden name of Sylvia Cienciala., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Three putative 21-hydroxypregnane 21-O-malonyltransferases (21MaT) from Digitalis lanata were partially purified. Two of them were supposed to be BAHD-type enzymes. We were unable to purify them in quantities necessary for reliable sequencing. We identified two genes in A. thaliana coding for substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases (AtPMaT1, AtPMaT2) and docked various 21-hydroxypregnanes into the substrate-binding site of a homology model built on the BAHD template 2XR7 (NtMaT1 from N. tabacum). Recombinant forms of Atpmat1 and Atpmat2 were expressed in E. coli and the recombinant enzymes characterized with regard to their substrate preferences. They were shown to malonylate various 21-hydroxypregnanes. The Atpmat1 sequence was used to identify candidate genes in Digitalis lanata (Dlmat1 to Dlmat4). Dlmat1 and Dlmat2 were also expressed in E. coli and shown to possess 21-hydroxypregnane 21-O-malonyltransferase activity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Targeted structural modifications have led to a novel type of buprenorphine-derived opioid receptor ligand displaying an improved selectivity profile for the μ-OR subtype. On this basis, it is shown that phenylazocarboxamides may serve as useful bioisosteric replacements for the widely occurring cinnamide units, without loss of OR binding affinity or subtype selectivity. This study further includes functional experiments pointing to weak partial agonist properties of the novel μ-OR ligands, as well as docking and metabolism experiments. Finally, the unique bifunctional character of phenylazocarboxylates, herein serving as precursors for the azocarboxamide subunit, was exploited to demonstrate the accessibility of an 18 F-fluorinated analogue., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Fibulin-4 is a 50 kDa glycoprotein of elastic fibers and plays an important role in development and function of elastic tissues. Fibulin-4 consists of a tandem array of five calcium-binding epidermal growth factor-like modules flanked by N- and C-terminal domains. Mutations in the human fibulin-4 gene EFEMP2 have been identified in patients affected with various arteriopathies including aneurysm, arterial tortuosity, or stenosis, but the molecular basis of most genotype-phenotype correlations is unknown. Here we present biochemical and computer modelling approaches designed to gain further insight into changes in structure and function of two fibulin-4 mutations (E126K and D203A), which are potentially involved in Ca 2+ binding in the EGF2 and EGF4 domain, respectively. Using recombinantly produced fibulin-4 mutant and wild type proteins we show that both mutations introduced additional protease cleavage sites, impaired extracellular assembly into fibers, and affected binding to to fibrillin-1, latent TGF-β-binding proteins, and the lysyl oxidase LOXL2. Molecular dynamics studies indicated that the E126K and D203A mutations do not necessarily result in a direct loss of the complexed Ca 2+ ion after 500 ns simulation time, but in significantly enhanced fluctuations within the connecting loop between EGF3 and EGF4 domains and other conformational changes. In contrast, intentionally removing Ca 2+ from EGF4 (D203A ΔCa) predicted dramatic changes in the protein structure. These results may explain the changes in protease cleavage sites, reduced secretion and impaired extracellular assembly of the E126K and D203A fibulin-4 mutants and provide further insight into understanding the molecular basis of the associated clinical phenotypes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly. The pathogenetic mechanisms how these mutations translate into the clinical phenotype are, however, poorly understood. In order to elucidate these mechanisms, we expressed fibulin-4 mutants recombinantly in HEK293 cells, purified the proteins in native forms and analyzed alterations in protein synthesis, secretion, matrix assembly, and interaction with other proteins in relation to wild type fibulin-4. Our studies show that different mutations affect these properties in multiple ways, resulting in fibulin-4 deficiency and/or impaired ability to form elastic fibers. The substitutions E126K and C267Y impaired secretion of the protein, but not mRNA synthesis. Furthermore, the E126K mutant showed less resistance to proteases, reduced binding to collagen IV and fibrillin-1, as well as to LTBP1s and LTBP4s. The A397T mutation introduced an extra O-glycosylation site and deleted binding to LTBP1s. We show that fibulin-4 binds stronger than fibulin-3 and -5 to LTBP1s, 3, and 4s, and to the lysyl oxidases LOX and LOXL1; the binding of fibulin-4 to the LOX propeptide was strongly reduced by the mutation E57K. These findings show that different mutations in the fibulin-4 gene result in different molecular defects affecting secretion rates, protein stability, LOX-induced cross-linking, or binding to other ECM components and molecules of the TGF-β pathway, and thus illustrate the complex role of fibulin-4 in connective tissue assembly., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Vein Patterning 1 (VEP1)-encoded progesterone 5β-reductases/iridoid synthases (PRISE) belong to the short-chain dehydrogenase/reductase superfamily of proteins. They are characterized by a set of highly conserved amino acids in the substrate-binding pocket. All PRISEs are capable of reducing the activated C=C double bond of various enones enantioselectively and therefore have a potential as biocatalysts in bioorganic synthesis. Here, recombinant forms of PRISEs of Arabidopsis thaliana and Digitalis lanata were modified using site-directed mutagenesis (SDM). In rDlP5βR, a set of highly conserved amino acids in the vicinity of the catalytic center was individually substituted for alanine resulting in considerable to complete loss of enone reductase activity. F153 and F343, which can be found in most PRISEs known, are located at the outer rim of the catalytic cavity and seem to be involved in substrate binding and their role was addressed in a series of SDM experiments. The wild-type PRISE accepted progesterone (large hydrophobic 1,4-enone) as well as 2-cyclohexen-1-one (small hydrophilic 1,4-enone), whereas the double mutant rAtP5βR_F153A_F343A converted progesterone much better than the wild-type enzyme but almost lost its capability of reducing 2-cyclohexen-1-one. Recombinant Draba aizoides P5βR (rDaP5βR) has a second pair of phenylalanines at position 156 and 345 at the rim of the binding site. These two phenylalanines were introduced into rAtP5βR_F153A_F343A and the resulting quadruple mutant rAtP5βR_F153A_F343A_V156F_V345F partly recovered the ability to reduce 2-cyclohexen-1-one. These results can best be explained by assuming a trapping mechanism in which phenylalanines at the rim of the substrate-binding pocket are involved. The dynamic behavior of individual P5βRs and mutants thereof was investigated by molecular dynamics simulations and all calculations supported the 'gatekeeper' role of phenylalanines at the periphery of the substrate-binding pocket. Our findings provide structural and mechanistic explanations for the different substrate preferences seen among the natural PRISEs and help to explain the large differences in catalytic efficiency found for different types of 1,4-enones.

A 4.1 μs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the 'traditional' nuclear receptor ligand-binding site. A NR4A1/bis-indole ligand complex at this novel site has been found to be stable over 1 μs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators.

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gαi-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy.

Based on the recently described crystal structure of the β2 adrenergic receptor--Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound β2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-β1 and α4-β6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations.

Introduction: Nurse-to-nurse relationships are a key component in determining the health of a work setting. Because of the impact that such relationships can have on patients, health care organizations, nurses, and the nursing profession, it is imperative to understand how nurses relate to each other., Purpose: The purpose of this article is to present findings from a study that explored how direct-care nurses relate to each other in everyday interactions on patient care units., Method: This study used a mixed method research design. Both quantitative and qualitative data were collected, but only qualitative findings are presented. Eighty-two staff registered nurses responded to a researcher-developed online questionnaire. A qualitative content analysis was conducted. Data were categorized according to questions then analyzed for key thoughts. Common themes were determined., Findings: Findings revealed participants had considered leaving the profession, and had considered leaving or had left a nursing unit because of poor nurse relationships. Participants identified environmental characteristics necessary for good relations. The crucial role nurse managers play in establishing good nurse relations was highlighted. Participants cited new graduate actions that could enhance relationships with expert nurses. Participants also noted missing factors on many nursing units that are essential for good nurse-to-nurse relationships., Conclusion: Positive nursing relationships are the key to establishing a healthy place of work. Only when positive nurse-to-nurse relationships are established can healthy work environments be created and sustained for nurses in the future.

G-protein-coupled receptors have extraordinary therapeutic potential as targets for a broad spectrum of diseases. Understanding their function at the molecular level is therefore essential. A variety of crystal structures have made the investigation of the inactive receptor state possible. Recently released X-ray structures of opsin and the β(2)-adrenergic receptor (β(2)AR) have provided insight into the active receptor state. In addition, we have contributed to the crystal structure of an irreversible agonist-β(2) adrenoceptor complex. These extensive studies and biophysical investigations have revealed that agonist binding leads to a low-affinity conformation of the active state that is suggested to facilitate G-protein binding. The high-affinity receptor state, which promotes signal transduction, is only formed in the presence of both agonist and G-protein. Despite numerous crystal structures, it is not yet clear how ligands tune receptor dynamics and G-protein binding. We have now used molecular dynamics simulations to elucidate the distinct impact of agonist and inverse agonist on receptor conformation and G-protein binding by investigating the influence of the ligands on the structure and dynamics of a complex composed of β(2)AR and the C-terminal end of the Gα(s) subunit (GαCT). The simulations clearly showed that the agonist isoprenaline and the inverse agonist carazolol influence the ligand-binding site and the interaction between β(2)AR and GαCT differently. Isoprenaline induced an inward motion of helix 5, whereas carazolol blocked the rearrangement of the extracellular part of the receptor. Moreover, in the presence of isoprenaline, β(2)AR and GαCT form a stable interaction that is destabilized by carazolol., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Single-chain Fv triplebodies (sctb), consisting of a single polypeptide chain with 3 single-chain antibody variable fragments connected in tandem, were generated as antileukemic agents. A CD19-specific sctb of this format has previously been shown to be superior to a bispecific single-chain Fv antibody fragment (bsscFv) for the elimination of leukemic B-lineage cells, but corresponding targeted agents for the treatment of acute myeloid leukemia are still lacking. For this purpose, both a bsscFv and a sctb specific for CD33 and the trigger molecule CD16 (FcgammaRIII) were produced. The sctb displayed 3.5-fold greater avidity for CD33 than the bsscFv 33xds16, whereas both had close to equal affinity for CD16. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, both the bsscFv 33xds16 and the sctb induced lysis of tumor cells with half maximum effective concentrations (EC50) in the low picomolar range. It is interesting to note that the sctb promoted equal lysis of human leukemia-derived cell lines at 10 to 200-fold lower concentrations than the bsscFv. Both molecules mediated ADCC of primary patient cells. In conclusion, both the bsscFv 33xds16 and the sctb 33xds16x33 eliminated acute myeloid leukemia cells in ADCC reactions, but the novel sctb format showed significantly greater specific activity.

Synthesis, biological investigations and molecular docking studies of nonantibiotic and nontetracyclic inducers that feature a minimal key motif of the natural lead tetracycline are presented. The diarylpropane-1,3-dione motif was identified as the minimal substructure responsible for TetR induction by tetracyclines. The first nontetracyclic surrogates of the natural tetracyclines displayed significant inducing effects for TetR(BD)S135L, whereby the chlorohydroxyphenyl-substituted beta-diketone 31 displayed the highest activity. Interestingly, antibiotic activity could not be detected for 31. Homology modeling based on the X-ray structure of 7-chlorotetracycline bound to TetR indicated analogous binding modes for the natural inducer and the synthetic diarylpropane-1,3-dione derivatives.

Extensive molecular-dynamics simulations show that the distance between the centers of gravity of the two equivalent helices 3 in the DNA-binding heads of the dimer of the tetracycline-repressor protein (TetR) can be used as a reliable diagnostic of induction. This is not, however, true for X-ray structures, but only for molecular-dynamics simulations. This is suggested to be because TetR is inherently flexible along the coordinate of the allosteric change (as is always likely to be the case for allosteric proteins), so that crystal-packing forces can determine the conformation of the protein. However, the time scale of the allosteric rearrangement in the absence of DNA-complexation is found to be of the order of tens of nanoseconds, so that rearrangements can be observed reproducibly in 100 ns simulations. Metastable (pre-equilibrium) conformations of TetR have been observed for up to 60 ns. The likely equilibrium processes and key features of the TetR system are discussed., (2009 Wiley-Liss, Inc.)

Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D4, D2, and D3, respectively. To complete this set, the methylthio analogues 2b and 3b exceeding the affinity of 2a and 3a by one order of magnitude and the structural intermediate 1b were synthesized. These chemically similar but biologically divergent target compounds served as molecular probes for radioligand displacement experiments, mutagenesis, and docking studies on homology models based on the recent crystal structure of the beta2-adrenergic receptor. Specific interactions with the highly conserved amino acids Asp3.32 and His6.55 and less conserved residues at positions 2.61, 2.64, 3.28, and 3.29 were identified. Inclusion of a carefully modeled extracellular loop 2 displayed two nonconserved residues in EL2 that differently contribute to ligand binding. Obviously, subtype selectivity is caused by nonconserved but frequently mediated by conserved amino acids.

Collagen type II (CII) is a cartilage-specific target of pathologic humoral autoimmune responses in rheumatoid arthritis as well as in the collagen-induced arthritis model. The aim of the present study is to investigate the critical amino acid residues conferring CII epitope specificity of the prototypic arthritogenic murine mAb CIIC1. A homology model of the CIIC1 single-chain antibody fragment (CIIC1scFv) in complex with its triple helical epitope was established. In silico predictions based on extensive molecular dynamics simulations were experimentally tested by the recombinant expression and functional analysis of CIIC1scFv containing alanine replacements allowing the identification of crucial CII-binding sites in the CDR2 and CDR3 regions of both heavy and light chains. Since the conversion of the CIIC1scFv sequence into the respective germline at all 13 somatically mutated positions did not affect its CII binding, our data indicate that potentially harmful cartilage-specific humoral autoimmunity could be germline encoded. The molecular modeling further demonstrates that the rigid collagen triple helix restricts the likelihood of molecular interactions with the CDR regions of the antibody considerably compared with globular antigens. These sterical constraints provide an explanation as to why somatic mutations in the arthritogenic autoantibody have no obvious impact on CII recognition.

In recent decades, new less-invasive, nonlinear optical methods have been proposed and optimized for monitoring fast physiological processes in biological cells. One of these methods allows the action potential (AP) in cardiomyocytes or neurons to be monitored by means of second-harmonic generation (SHG). We now present the first, to our knowledge, simulations of the dependency of the intensity of the second harmonic (I(SHG)) on variations of the transmembrane potential (TMP) in a cardiomyocyte during an action potential (AP). For this, an amphiphilic potential-sensitive styryl dye molecule with nonlinear optical properties was embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer, replacing one of the phospholipid molecules. External electrical fields with different strengths were applied across the membrane to simulate the AP of a heart-muscle cell. We used a combined classical/quantum mechanical approach to model the structure and the spectroscopic properties of the embedded chromophore. Two 10 ns molecular dynamics (MD) simulations provided input geometries for semiempirical molecular orbital (QM/MM) single-point configuration interaction (CI) calculations, which were used to calculate the wavelengths and oscillator strengths of electronic transitions in the di-8-ANEPPS dye molecule. The results were then used in a sum-over-states treatment to calculate the second-order hyperpolarizability. The square of the hyperpolarizability scales with the intensity of the second harmonic, which is used to monitor the action potentials of cardiomyocytes experimentally. Thus, we computed changes in the intensity of the second harmonic (DeltaI(SHG)) as function of TMP changes. Our results agree well with experimental measurements.

Proline-derived peptide mimetics have become an area of paramount importance in peptide and protein chemistry. Since protein crystal structures frequently display Psi angles of 140-170 degrees for prolyl moieties, our intention was to design a completely novel series of 2,3-fused-proline-derived lactams covering this particular conformational space. Extending our recently described toolset of spirocyclic reverse-turn mimetics, we synthesized pyrrolidinyl-fused seven-, eight-, and nine-membered unsaturated lactam model peptides taking advantage of Grubbs' ring-closing metathesis. Investigating the seven-membered lactam 3a by means of IR and NMR spectroscopy and semiempirical molecular dynamics simulations, we could not observe a U-turn conformation; however, increasing the ring size to give eight- and nine-membered congeners revealed moderate and high type IotaIota beta-turn inducing properties. Interestingly, the conformational properties of our model systems depend on both the ring size of the fused dehydro-Freidinger lactam and the position of the endocyclic double bond. Superior reverse-turn inducing properties could be observed for the fused azacyclononenone 3e. According to diagnostic transanular NOEs, a discrete folding principle of the lactam ring strongly deviating from the regioisomeric lactams 3c,f explains the conformational behavior. Hence, we were able to establish a molecular building kit that allows adjustments of a wide range of naturally occurring proline Psi angles and thus can be exploited to probe molecular recognition and functional properties of biological systems.

The binding specificity of alpha7beta1 integrins for different laminin isoforms is defined by the X1 and X2 splice domains located in the beta-propeller domain of the alpha7 subunit. In order to gain insight into the mechanism of specific laminin-integrin interactions, we defined laminin-binding epitopes of the alpha7X1 and -X2 domains by single amino acid substitutions and domain swapping between X1 and X2. The interaction of mutated, recombinantly prepared alpha7X1beta1 and alpha7X2beta1 heterodimers with various laminin isoforms was studied by surface plasmon resonance and solid phase binding assays. The data show that distinct clusters of surface-exposed acidic residues located in different positions of the X1 and the X2 loops are responsible for the specific recognition of laminins. These residues are conserved between the respective X1 or X2 splice domains of the alpha7 chains of different species, some also in the corresponding X1/X2 splice domains of alpha6 integrin. Interestingly, ligand binding was also modulated by mutating surface-exposed hydrophobic residues (alpha7X1L205, alpha7X2Y208) at positions corresponding to the fibronectin binding synergy site in alpha5beta1 integrin. Mutations in X1 that affected binding to laminin-1 also affected binding to laminin-8 and -10, but not to the same extent, thus allowing conclusions on the specific role of individual surface epitopes in the selective recognition of laminin-1 versus laminins -8 and -10. The role of the identified epitopes was confirmed by molecular dynamics simulations of wild-type integrins and several inactivating mutations. The analysis of laminin isoform interactions with various X1/X2 chimaera lend further support to the key role of negative surface charges and pointed to an essential contribution of the N-terminal TARVEL sequence of the X1 domain for recognition of laminin-8 and -10. In conclusion, specific surface epitopes containing charged and hydrophobic residues are essential for ligand binding and define specific interactions with laminin isoforms.

Molecular-dynamics simulations have been used to investigate the mechanism of induction of a mutant (revTetR) of the tetracycline repressor protein (TetR) that shows the reverse phenotype (i.e., it is induced in the absence of tetracyclines and not in their presence). Low-frequency, normal-mode analyses demonstrate that the reverse phenotype is reproduced by the simulations on the basis of criteria established for wild-type TetR. The reverse phenotype is caused by the fact that the DNA-binding heads in revTetR are closer than the ideal distance needed for DNA-binding when no inducer is present. This distance increases on binding an inducer. Whereas this distance increase makes the interhead distance too large in wild-type TetR, it increases to the ideal value in revTetR. Thus, the mechanism of induction is the same for the two proteins, but the consequences are reversed because of the smaller interhead distance in revTetR when no inducer is present.

A combination of structures, energies, and spectral data calculated using density functional theory (DFT) with experimental NMR data has been used to assign conformational equilibria for tetracycline and 5a,6-anhydrotetracycline in water at pH 1, 7, and 10 and in chloroform (5a,6-anhydrotetracycline) and methanol (tetracycline). The results suggest that tetracycline always prefers the extended conformation but that 5a,6-anhydrotetracycline exists in water as a mixture of the two conformers and in chloroform exclusively in the twisted conformation. The conformational equilibria are also shown to be pH dependent.

A series of novel 2,4-diaminopyrimido[4,5-b]indol-6-ols has been synthesized and the in vitro cytotoxic activities were evaluated against four human cancer cell lines originating from solid tumors. An increase in activity was observed when a heteroaromatic ring was annulated on side g of the pyrimido[4,5-b]indole system to give compounds with activities comparable to ellipticine and cisplatin. To understand the experimental cytotoxic activities, QSAR investigations were performed, which showed a very good linearity between the experimental and predicted IC(50).

Semiempirical molecular orbital theory has been used for a systematic scan of the binding positions for a Mg2+ ion with 5a,6-anhydrotetracycline taking both conformational flexibility and possible different tautomeric forms into account. The magnesium ion has been calculated alone and with four or five complexed water molecules in order to simulate the experimental situation more closely. The results are analyzed by comparing the behavior of the title compound with that of tetracycline itself and possible causes for the stronger induction of the Tetracycline Receptor (TetR) by 5a,6-anhydrotetracycline than by tetracycline are considered. Energetically favored 3D-structure of the zwitteranionic 5a,6-anhydrotetracycline magnesium complex in solution.

Molecular dynamics simulations on the tetracycline-repressor (TetR) protein, both in the absence of an inducer and complexed with the inducers tetracycline and 5a,6-anhydrotetracycline, show significant differences in the structures and dynamics of the induced and non-induced forms of the protein. Calpha-density-difference plots, low-frequency normal vibrations and inter-residue interaction energies all point to a common mechanism of induction. The inducer displaces Asp156 from the magnesium ion in the binding pocket, leading to a short cascade of rearrangements of salt bridges that results in the allosteric change. The increased flexibility of the induced form of the protein is suggested to contribute to the decrease in binding affinity to DNA on induction.

The binding motif (pharmacophore) for induction and the changes in the structure of the binding site that accompany induction have been determined from molecular-dynamics simulations on the tetracycline-repressor signal-transduction protein. The changes and the induction mechanism are discussed and compared with conclusions drawn from earlier X-ray structures. The differences in inducer strength of tetracycline and 5a,6-anhydrotetracycline are discussed with respect to their interaction in the MD simulations.

We present a computational model study designed to simulate the results of time-resolved fluorescence spectra of tryptophan in proteins. In such measurements, the occurrence of more than one fluorescence lifetime is generally attributed to the existence of several tryptophan rotamers and/or structural conformations of the protein structure. The protein system we chose for this initial study is the tetracycline repressor (TetR), an interesting model system for the investigation of the mechanisms of transcriptional regulation. Fluorescence resonance energy transfer (FRET) from tryptophan to tetracycline is frequently observed in complexes of the TetR with the antibiotic tetracycline. We use a combined classical/quantum mechanical approach to model the structure and the spectroscopic properties of the TetR-tetracycline complex. A classical molecular dynamics simulation provides input geometries for semiempirical quantum mechanical/molecular mechanical (QM/MM) single-point configuration interaction (CI) calculations, which are used to calculate tryptophan vertical absorption and fluorescence energies and intensities as well as relative FRET rate constants. These rate constants together with the Einstein coefficients for spontaneous emission and an assumed rate for nonradiative deactivation allow us to simulate fluorescence decay curves with and without FRET and for the entire ensemble as well as for individual rotamers. Our results indicate that the classical "rotamer model", used to explain the multiexponential fluorescence-decay curves of time-resolved tryptophan emission spectra, can be extended to systems with FRET acceptors present in the protein matrix but that the interpretation of the fitted lifetimes is different to that usually used.

We report a consistent set of AMBER force-field parameters for the most common phosphorylated amino acids, phosphoserine, phosphothreonine, phosphotyrosine, and phosphohistidine in different protonation states. The calculation of atomic charges followed the original restrained electrostatic potential fitting procedure used to determine the charges for the parm94/99 parameter set, taking alpha-helical and beta-strand conformations of the corresponding ACE-/NME-capped model peptide backbone into account. Missing force-field parameters were taken directly from the general AMBER force field (gaff) and the parm99 data set with minor modifications, or were newly generated based on ab initio calculations for model systems. Final parameters were validated by geometry optimizations and molecular-dynamics simulations. Template libraries for the phosphorylated amino acids in Leap format and corresponding frcmod parameter files are made available. [Figure: see text].

Focusing on the similarity and divergence of GPCR subtypes and their ligand interactions, we generated dopamine D2, D3, and D4 receptor models based on the rhodopsin crystal structure and refined these with an extensive MM/MD protocol. After validation by diagnostic experimental data, subtype-specific relative positions of TM1, 2, 6, and 7 and bending angles of TM7 were found. To sample the conformational space of the complex, we performed simulated-annealing runs of the receptor protein with the sub-nanomolar antagonist spiperone. Docking a representative set of ligands, we were able to identify one superior model for each subtype when excellent correlations between predicted energies of binding and experimental affinities (r2 = 0.72 for D2, 0.91 for D3 and 0.77 for D4) could be observed. Further analysis revealed general ligand interactions with ASP3.32 and aromatic residues in TM6/7 and individual key interactions with TM1 and TM2 residues of the D3 and D4 receptor models, respectively.

Piritrexim (PTX) (1), a lipophilic inhibitor of the human dihydrofolate reductase, has been evaluated as an anticancer agent. The synthesis of four structural variations (2-5) of PTX is reported. The PTX analogues 2-5 were obtained by reaction of suitable C3-building blocks with pyrimidine-2,4,6-triamine (14) or with cyanacetamide (7) and guanidine (10). The evaluation of 2-4 for antitumor activity against a panel of 60 human cancer cell lines showed inhibitory effects on the growth of the cell lines. These data are supported by molecular modeling and docking studies, which show that compounds 2-4 share the same binding mode within the DHFR active site. Moreover, the estimated ligand binding energies are in good agreement with the experimental activity data.

Inhibitory glycine receptors mediate rapid synaptic inhibition in mammalian spinal cord and brainstem. The previously identified hyperekplexia mutation GLRA1(P250T), located within the intracellular TM1-2 loop of the GlyR alpha1 subunit, results in altered receptor activation and desensitization. Here, elementary steps of ion channel function of alpha1(250) mutants were resolved and shown to correlate with hydropathy and molar volume of residue alpha1(250). Single-channel recordings and rapid activation kinetic studies using laser pulse photolysis showed reduced conductance but similar open probability of alpha1(P250T) mutant channels. Molecular dynamics simulation of a helix-turn-helix motif representing the intracellular TM1-2 domain revealed alterations in backbone conformation, indicating an increased flexibility in these mutants that paralleled changes in elementary steps of channel function. Thus, the architecture of the TM1-2 loop is a critical determinant of ion channel conductance and receptor desensitization.