Atomistic simulations of 2D bicomponent self-assembly: from molecular recognition to self-healing

Supramolecular two-dimensional engineering epitomizes the design of complex molecular architectures through recognition events in multicomponent self-assembly. Despite being the subject of in-depth experimental studies, such articulated phenomena have not been yet elucidated in time and space with atomic precision. Here we use atomistic molecular dynamics to simulate the recognition of complementary hydrogen-bonding modules forming 2D porous networks on graphite. We describe the transition path from the melt to the crystalline hexagonal phase and show that self-assembly proceeds through a series of intermediate states featuring a plethora of polygonal types. Finally, we design a novel bicomponent system possessing kinetically improved self-healing ability in silico, thus demonstrating that a priori engineering of 2D self-assembly is possible.

The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition

Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the characterization of Ig domains of intracellular origin--in particular, modular components of the titin myofilament. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization.

Tuning Molecular Recognition of RNA and DNA via Sugar Modification

This minireview highlights three aspects of our recent work in the area of sugar modified oligonucleotide analogues. It provides an overview over recent results on the conformationally constrained analogue tricyclo-DNA with special emphasis of its antisense properties, it summarizes results on triple-helix forming oligodeoxynucleotides containing pyrrolidino-nucleosides with respect to DNA recognition via the dual recognition mode, and it highlights the advantageous application of the orthogonal oligonucleotidic pairing system homo-DNA in molecular beacons for DNA diagnostics

Supramolecular Organization of Heptapyrenotide Oligomers—An in Depth Investigation by Molecular Dynamics Simulations

We present a molecular modeling study based on ab initio and classical molecular dynamics calculations, for the investigation of the tridimensional structure and supramolecular assembly formation of heptapyrenotide oligomers in water solution. Our calculations show that free oligomers self-assemble in helical structures characterized by an inner core formed by π- stacked pyrene units, and external grooves formed by the linker moieties. The coiling of the linkers has high ordering, dominated by hydrogen-bond interactions among the phosphate and amide groups. Our models support a mechanism of longitudinal supramolecular oligomerization based on interstrand pyrene intercalation. Only a minimal number of pyrene units intercalate at one end, favoring formation of very extended longitudinal chains, as also detected by AFM experiment. Our results provide a structural explanation of the mechanism of chirality amplification in 1:1 mixtures of standard heptapyrenotides and modified oligomers with covalently linked deoxycytidine, based on selective molecular recognition and binding of the nucleotide to the groove of the left-wound helix.

Role of electrostatic interactions for ligand recognition and specificity of peptide transporters.

BACKGROUND Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.

Phenotypic and genetic characterization of Pasteurella multocida and related isolates from rabbits in Switzerland

Several bacteria belonging to the family Pasteurellaceae are potential pathogens in rabbits. In particular, Pasteurella multocida is considered to be important, and outbreaks caused by this species result in considerable economic losses in rabbitries. However, Pasteurellaceae spp. isolated from rabbits are poorly characterized, and thus, proper identification of P. multocida isolates from these animals is problematic and often unsatisfactory, thereby hampering epidemiological investigations. Therefore, 228 isolates from rabbit populations originating from a breeding and fattening organization with group management and postmortem cases with pasteurellosis from individual owners were phenotypically and genotypically analyzed using biochemical tests and repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Furthermore, 41 samples representing observed phenotypes were selected for phylogenetic analysis using 16S ribosomal RNA and rpoB genes. The REP-PCR typing and phylogenetic analyses correlated well and appeared to be distinct molecular methods for characterization of rabbit isolates. Phenotyping, however, diverged from molecular recognition, reflecting the problematic conventional diagnosis of these strains. The fermentation of sorbitol appeared to be an imprecise indicator for P. multocida subspecies classification. According to REP-PCR and sequencing results, 82% of the isolates were characterized as P. multocida subsp. multocida, 3% as P. multocida subsp. septica, and 5% as P. multocida. Further, 5% were identified as Pasteurella canis. The other 5% represented a homogeneous group of unknown species belonging to the Pasteurellaceae. Samples obtained from individual postmortem cases demonstrated a higher phenotypic and genetic heterogeneity than samples from group management rabbits.

Polar Nature of Biomimetic Fluorapatite/Gelatin Composites: A Comparison of Bipolar Objects and the Polar State of Natural Tissue

The correspondence of the state of alignment of macromolecules in biomimetic materials and natural tissues is demonstrated by investigating a mechanism of electrical polarity formation: An in vitro grown biomimetic FAp/gelatin composite is investigated for its polar properties by second harmonic (SHGM) and scanning pyroelectric microscopy (SPEM). Hexagonal prismatic seed crystals formed in gelatin gels represent a monodomain polar state, due to aligned mineralized gelatin molecules. Later growth stages, showing dumbbell morphologies, develop into a bipolar state because of surface recognition by gelatin functionality: A reversal of the polar alignment of macromolecules, thus, takes place close to that basal plane of the seed. In natural hard tissues (teeth and bone investigated by SPEM) and the biomimetic FAp/gelatin composite, we find a surprising analogy in view of growth-induced states of polarity: The development of polarity in vivo and in vitro can be explained by a Markov-type mechanism of molecular recognition during the attachment of macromolecules.

[Molecular analyses for (early) recognition of hematologic diseases - sense and sensibility for molecular analysis: the art of intelligent decision-making]

During the last 10 years several molecular markers have been established as useful tools among the armamentarium of a hematologist. As a consequence, the number of performed hematologic molecular analyses has immensely increased. Often, such tests replace or complement other laboratory methods. Molecular markers can be useful in many ways: they can serve for diagnostics, describe the prognostic profile, predict which types of drugs are indicated, and can be used for the therapeutic monitoring of the patient to indicate an adequate response or predict resistance or relapse of the disease. Many markers fulfill more than one of these aspects. Most important, however, is the right choice of analyses at the right time-points!

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.