Molecular Recognition

Turning molecular recognition into an effective mechanical response is critical for many applications ranging from molecular motors and responsive materials to sensors. Herein, we demonstrate how the energy of the molecular recognition between a supramolecular host and small alkylammonium salts can be harnessed to perform a nanomechanical task in a univocal way. Nanomechanical Si microcantilevers (MCs) functionalized by a film of tetra-phosphonate cavitands were employed to screen as guests the compounds of the butylammonium chloride series 1-4, which comprises a range of low molecular weight (LMW) molecules (molecular mass < 150 Da) that differ from each other by one or a few N-methyl groups (molecular mass 15 Da). The cavitand surface recognition of each individual guest drove a specific MC bending (from a few to several tens of nanometer), disclosing a direct, label-free, and real-time mean to sort them. The complexation preferences of tetraphosphonate cavitands toward ammonium chloride guests 1-4 were independently assessed by isothermal titration calorimetry. Both direct and displacement binding experiments concurred to define the following binding order in the alkylammonium series: 2 > 3 ≈ 1 ≫ 4. This trend is consistent with the number of interactions established by each guest with the host. The complementary ITC experiments showed that the host-guest complexation affinity in solution is transferred to the MC bending. These findings were benchmarked by implementing cavitand-functionalized MCs to discriminate sarcosine from glycine in water.

Molecular imprinting is a process that allows for the synthesis of artificial receptors for a given target molecule based on synthetic polymers. The target molecule acts as a template around which interacting and cross-linking monomers are arranged and co-polymerized to form a cast-like shell. In essence, a molecular memory is imprinted in the polymer, which is now capable of selectively binding the target. Molecularly imprinted polymers (MIPs) thus possess the most important feature of biological antibodies -specific molecular recognition. They can thus be used in applications where selective binding events are of importance, such as immunoassays, affinity separation, biosensors, and directed synthesis and catalysis. Since its beginnings in the 1970s, the technique of molecular imprinting has greatly diversified during the last decade both from a materials point of view and from an application point of view. Still, there is much room for further improvement. The key challenges, in particular the binding site homogeneity and water compatibility of MIPs, and the possibility of synthesizing MIPs specific for proteins, are actively addressed by research groups over the World. Other important points are the conception of composite materials based on MIPs, in order to include additional interesting properties into the material, and the synthesis of very small and quasisoluble MIPs, close in size to proteins.

Background: Eps15 homology (EH) domains are protein interaction modules binding to peptides containing Asn-Pro-Phe (NPF) motifs and mediating critical events during endocytosis and signal transduction. The EH domain of POB1 associates with Eps15, a protein characterized by a striking string of DPF triplets, 15 in human and 13 in mouse Eps15, at the C-terminus and lacking the typical EH-binding NPF motif. Results: By screening a multivalent nonapeptide phage display library we have demonstrated that the EH domain of POB1 has a different recognition specificity since it binds to both NPF and DPF motifs. The region of mouse Eps15 responsible for the interaction with the EH domain of POB1 maps within a 18 amino acid peptide (residues 623-640) that includes three DPF repeats. Finally, mutational analysis in the EH domain of POB1, revealed that several solvent exposed residues, while distal to the binding pocket, mediate specific recognition of binding partners through both hydrophobic and electrostatic contacts. In the present study we have analysed the binding specificity of the POB1 EH domain. We show that it differs from other EH domains since it interacts with both NPF-and DPFcontaining sequences. These unusual binding properties could be attributed to a different conformation of the binding pocket that allows to accommodate negative charges; moreover, we identified a cluster of solvent exposed Lys residues, which are only found in the EH domain of POB1, and influence binding to both NPF and DPF motifs. The characterization of structures of the DPF ligands described in this study and the POB1 EH domain will clearly determine the involvement of the positive patch and the rationalization of our findings.

Helix-loop-helix (HLH) and helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that mediate selective pairing among members of a large transcription factor family involved in cell fate determination. To investigate the molecular rules underlying recognition specificity and to isolate molecules interfering with cell proliferation and differentiation control, we assembled two molecular repertoires obtained by directed randomization of the binding surface in these two domains. For this strategy we selected the Heb HLH and Max Zip regions as molecular scaffolds for the randomization process and displayed the two resulting molecular repertoires on phage capsids. By affinity selection, many domains were isolated that bound to the proteins Mad, Rox, MyoD, and Id2 with different levels of affinity. Although several residues along an extended surface within each domain appeared to contribute to dimerization, some key residues critically involved in molecular recognition could be identified. Furthermore, a number of charged residues appeared to act as switch points facilitating partner exchange. By successfully selecting ligands for four of four HLH or HLHZip proteins, we have shown that the repertoires assembled are rather general and possibly contain elements that bind with sufficient affinity to any natural HLH or HLHZip molecule. Thus they represent a valuable source of ligands that could be used as reagents for molecular dissection of functional regulatory pathways. Phage, Plasmids, and GST Fusion Proteins-DNA sequences encoding Max bHLHZip (Ala 22 to Leu 102 ) and repertoires of HLH and bHL-* This work was funded by a grant from the Associazione Italiana Ricerca sul Cancro.

Two receptors, a diaminotriazine derivative (DAT) and diamidopyridine (DAP), are complementary to the electroactive naphthalimide (N) through three-point hydrogen bonding. The association constants of the two receptors were evaluated for both the fully oxidized and the radical anion forms of N. In the oxidized state, the two receptors displayed identical binding constants. Diamidopyridine, however, lowers the reduction potential of naphthalimide to a far greater extent than does diaminotriazine, indicating a greater affinity for diamidopyridine by naphthalimide in the radical anion form. This behavior was mirrored by EPR experiments that showed small deviations from the hyperfine coupling pattern of N red in the presence of DAT, with greater effects seen for the Nred‚DAP complex. Computational simulations using the UB3LYP/6-311+G-(d,p)//UHF/6-31G(d) hybrid gave theoretical hyperfine constants in good quantitative agreement with the experimental results. Using this correlation, we determined that electrostatics and hydrogen bond polarizability play key roles in controlling redox-modulated molecular recognition.

Cassia senna, famous for its medicinal qualities, has been crucial in traditional medicine, providing remedies for many ailments. Its seeds, which are abundant in bioactive compounds such as lectins, have significant pharmaceutical potential. In this study, we investigated the
biochemical intricacies and therapeutic potential of CsSL, a lectin found in Cassia senna seeds.
Hemagglutination assays revealed robust CsSL activity, reaching its peak with 50 mM Tris-HCl buffer at pH 7.5. Ammonium sulfate fractionation was used to purify CsSL, with Fr40 showing the highest activity, confirming successful isolation. GlcNAc-agarose affinity chromatography
further purified CsSL, showing its affinity for galactose residues. CsSL is stable, retains its full activity for 45 minutes at 30-40°C, and exhibits pH dependence, with peak hemagglutinating activity occurring between pH 3.5 and 4.5 and sharply declining beyond pH 7. Nbromosuccinimide
(NBS) modification showed that half of CsSL's tryptophan residues is crucial for its activity, as evidenced by decreased lectin activity. Therapeutically, CsSL demonstrated notable analgesic effects in acetic acid-induced writhing and thermal sensitivity tests. The 12 mg/kg dose particularly showed a remarkable antinociceptive effect (p<0.05), completely reducing the writhing reflex by 100% compared to that of the negative control Moreover, CsSL had potent gastroprotective effects, reducing ethanol-induced gastric lesions in a dose-dependent
manner. Notably, doses of 0.25 mg/kg and 0.5 mg/kg provided substantial stomach lining protection (p<0.05), outperforming the standard ulcer medication pantoprazole. CsSL exhibits promise for pain relief and ulcer prevention, with significant analgesic and gastroprotective effects. However, further research is needed to fully understand its underlying mechanisms and clinical applications, as this research has exciting potential in biomedicine and biotechnology.

A molecularly imprinted polymer (MIP) designed to enable the selective extraction of carbamazepine (CBZ) from effluent wastewater and urine samples has been synthesised using a non-covalent molecular imprinting approach. The MIP was evaluated chromatographically in the first instance and its affinity for CBZ also confirmed by solid-phase extraction (SPE). The optimal conditions for SPE consisted of conditioning of the cartridge using acidified water purified from a Milli-Q system, loading of the sample under basic aqueous conditions, clean-up using acetonitrile and elution with methanol. The attractive molecular recognition properties of the MIP gave rise to good CBZ recoveries (80%) when 100 mL of effluent water spiked with 1 g L -1 was percolated through the polymer. For urine samples, 2 mL samples spiked with 2.5 g L -1 CBZ were extracted with a recovery of 65%. For urine, the linear range was 0.05-24 mg L -1 , the limit of detection was 25 g L -1 and precision, expressed as relative standard deviation at 0.5 mg L -1 (n = 3), was 3.1% and 12.6% for repeatability and reproducibility between days, respectively.

We discuss several concepts of handling molecule-adsorbent and hetero-molecular structures on an individual molecular basis. Molecular recognition using scanning tunneling microscopy underpins the fundamental progress made. "Beyond imaging" implies repositioning, patterning, and exploring the functionality of individual molecules. Using porphyrin-and fullerene-based systems, we discuss issues such as conformational analysis, supramolecular systems, patterning, and the fabrication of a molecular adding machine. These examples form the beginnings of a bottom-up approach to fabrication and "sciengineering" from a molecule-bymolecule perspective.

The terpenoid chiral selectors dehydroabietic acid, 12,14-dinitrodehydroabietic acid and friedelin have been covalently linked to silica gel yielding three chiral stationary phases CSP 1, CSP 2 and CSP 3, respectively. The enantiodiscriminating capability of each one of these phases was evaluated by HPLC with four families of chiral aromatic compounds composed of alcohols, amines, phenylalanine and tryptophan amino acid derivatives and b-lactams. The CSP 3 phase, containing a selector with a large friedelane backbone is particularly suitable for resolving free alcohols and their derivatives bearing fluorine substituents, while CSP 2 with a dehydroabietic architecture is the only phase that efficiently discriminates 1,1 0 -binaphthol atropisomers. CSP 3 also gives efficient resolution of the free amines. All three phases resolve well the racemates of N-trifluoracetyl and N-3,5-dinitrobenzoyl phenylalanine amino acid ester derivatives. Good enantioseparation of b-lactams and N-benzoyl tryptophan amino acid derivatives was achieved on CSP 1. In order to understand the structural factors that govern the chiral molecular recognition ability of these phases, molecular dynamics simulations were carried out in the gas phase with binary diastereomeric complexes formed by the selectors of CSP 1 and CSP 2 and several amino acid derivatives. Decomposition of molecular mechanics energies shows that van der Waals interactions dominate the formation of the diastereomeric transient complexes while the electrostatic binding interactions are primarily responsible for the enantioselective binding of the (R)-and (S)-analytes. Analysis of the hydrogen bonds shows that electrostatic interactions are mainly associated with the formation of N-HÁ Á ÁO@C enantioselective hydrogen bonds between the amide binding sites from the selectors and the carbonyl groups of the analytes. The role of mobile phase polarity, a mixture of n-hexane and propan-2-ol in different ratios, was also evaluated through molecular dynamics simulations in explicit solvent.

The terpenoid chiral selectors dehydroabietic acid, 12,14-dinitrodehydroabietic acid and friedelin have been covalently linked to silica gel yielding three chiral stationary phases CSP 1, CSP 2 and CSP 3, respectively. The enantiodiscriminating capability of each one of these phases was evaluated by HPLC with four families of chiral aromatic compounds composed of alcohols, amines, phenylalanine and tryptophan amino acid derivatives and b-lactams. The CSP 3 phase, containing a selector with a large friedelane backbone is particularly suitable for resolving free alcohols and their derivatives bearing fluorine substituents, while CSP 2 with a dehydroabietic architecture is the only phase that efficiently discriminates 1,1 0 -binaphthol atropisomers. CSP 3 also gives efficient resolution of the free amines. All three phases resolve well the racemates of N-trifluoracetyl and N-3,5-dinitrobenzoyl phenylalanine amino acid ester derivatives. Good enantioseparation of b-lactams and N-benzoyl tryptophan amino acid derivatives was achieved on CSP 1. In order to understand the structural factors that govern the chiral molecular recognition ability of these phases, molecular dynamics simulations were carried out in the gas phase with binary diastereomeric complexes formed by the selectors of CSP 1 and CSP 2 and several amino acid derivatives. Decomposition of molecular mechanics energies shows that van der Waals interactions dominate the formation of the diastereomeric transient complexes while the electrostatic binding interactions are primarily responsible for the enantioselective binding of the (R)-and (S)-analytes. Analysis of the hydrogen bonds shows that electrostatic interactions are mainly associated with the formation of N-HÁ Á ÁO@C enantioselective hydrogen bonds between the amide binding sites from the selectors and the carbonyl groups of the analytes. The role of mobile phase polarity, a mixture of n-hexane and propan-2-ol in different ratios, was also evaluated through molecular dynamics simulations in explicit solvent.

A 1:1 mixture of a tris(Zn IIcyclen) (1: [Zn 3 L 1 ], L 1 1,3,5-tris-(1,4,7,10-tetraazacyclododecan-1-ylmethyl)benzene) and trithiocyanuric acid (TCA) yielded a 4:4 self-assembly complex [(Zn 3 L 1 ) 4 -(TCA 3À ) 4 ] (6) through the formation of Zn II ÀS À coordination bonds and hydrogen bonds between 1,3,5-triazine N and cyclen NH (cyclen 1,4,7,10-tetraazacyclododecane); the supramolecular capsule structure was revealed by X-ray crystal structure analysis. The capsule exterior represents a twisted cuboctahedral framework containing a nanoscale truncated tetrahedral cavity. The crystal data: formula C 144 H 308 N 72 O 58 S 12 Zn 12 (6[NO 3 ] 12 ¥ 22 H 2 O), M r 5145.75, cubic, space group F432 (No. 209), a 39.182(1) ä, V 60153(3) ä 3 , Z 8, R 0.100, R w 0.259. Lipophilic organic molecules with the matching sizes, for example, ([D 4 ]-2,2,3,3)-3-(trimethylsilyl)propionic acid (TSP), 1-adamantanecarboxylic acid, 2,4-dinitrophenol (2,4-DNP), adaman-tane (ADM), or the tetra-n-propylammonium (TPA) cation, are encapsulated in the inner cavity, as revealed by remarkable upfield shifts of the 1 H NMR signals of these guest molecules. The encapsulation of ADM was confirmed by X-ray crystal structure analysis. Crystal data of the ADMencapsulating complex: formula C 154 H 334 N 72 O 63 S 12 Zn 12 (6-ADM[NO 3 ] 12 ¥ 27 H 2 O), M r 5372.06, cubic, space group F432 (No. 209), a 39.061( ) ä, V 59599(3) ä 3 , Z 8, R 0.103, R w 0.263. The 4:4 self-assembly was stabilized by incorporation of one of these guest molecules. The apparent 4:4 self-assembly constants for 6 in the presence of an excess amount of a guest TPA, log K app (K app [6-TPA]/ [1] 4 [TCA] 4 ) (m À7 )), were determined to be 34.0 AE 2.0 and 35.5 AE 3.0 by potentiometric pH and UV spectrophotometric titrations, respectively. An apparent encapsulation constant for 2,4-DNP, log K enc (K enc [6-2,4-DNP]/[6][2,4-DNP] (m À1 )), was 6.0 AE 0.1 at pH 7.0 (50 mm HEPES with I 0.1 (NaNO 3 )), as determined by UV titrations. The lipophilicity of the inner cavity was close to that of 2-propanol, as a quantum yield (F) of 0.24 AE 0.1 for the fluorescent emission of 7-diethylaminocoumarin-1carboxylic acid (20 mm) in the capsule was close to the F of 0.22 found for 2-propanol. Encapsulation properties of the present Zn II -containing cage have been compared with those of cyclodextrins and Fujita×s Pd II -containing supramolecular cage. The exterior chirality of the 4:4 complex was controlled from within by an encapsulated chiral guest molecule, 2,10-camphorsultam, as indicated by Cotton effects in the circular dichroism spectra.

Nuclear import of proteins is determined by specific signals that allow them to bind to receptors that mediate their energy-dependent transport through the nuclear pore. These signals are termed nuclear localization signals and do not constitute a specific consensus sequence. Among them, the most characterized correspond to monopartite and bipartite nuclear localization signals, which interact with the importin α/β heterodimer. We previously described a cytotoxic variant of human pancreatic-ribonuclease that is actively transported into the nucleus. Here, we show that this protein interacts with importin α through different basic residues, including Lys1 and the arginine clusters 31-33 and 89-91. Although these residues are scattered along the sequence, they are close in the three-dimensional structure of the protein and their topological disposition strongly resembles that of a classical bipartite nuclear localization signal.

This study aimed to elucidate the structural nature of the polydisperse, nanoscopic components in the solution and the solid states of partially reduced polyoxomolybdate derived from the {Mo132} keplerate, {(Mo)Mo5}12‐{Mo2 acetate}30. Designer tripodal hexamine‐tris‐crown ethers and nanoscopic molybdate coprecipitated from aqueous solution. These microcrystalline solids distributed particle radii between 2–30 nm as assayed by transmission electron microscopy (TEM). The solid materials and their particle size distributions were snap shots of the solution phase. The mother liquor of the preparation of the {Mo132} keplerate after three days revealed large species (r=20–30 nm) in the coprecipitate, whereas {Mo132} keplerate redissolved in water revealed small species (3–7 nm) in the coprecipitate. Nanoparticles of coprecipitate were more stable than solids derived solely from partially reduced molybdate. The TEM features of all material analyzed lacked facets on the nanometer length sca...

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody. Aptamers are a novel type of ligand composed of nucleic acids. DNA aptamers in particular have many advantages over antibodies. Therefore, we tried to isolate the DNA aptamer for mouse PrP. We developed a competitive selection method and tried to screen the DNA aptamer with it. In the fourth round of selection, several clones of the aptamer with an affinity to PrP were enriched, and clone 4-9 showed the highest affinity of all. The investigation by aptamer blotting and Western blotting showed that clone 4-9 was specifically able to recognize both α-PrP and β-PrP. Moreover, it was indicated that clone 4-9 could recognize the flexible region of the N-terminal domain of PrP. These characteristics suggest that clone 4-9 might be a useful tool in prion-disease diagnosis and research.

Turning molecular recognition into an effective mechanical response is critical for many applications ranging from molecular motors and responsive materials to sensors. Herein, we demonstrate how the energy of the molecular recognition between a supramolecular host and small alkylammonium salts can be harnessed to perform a nanomechanical task in a univocal way. Nanomechanical Si microcantilevers (MCs) functionalized by a film of tetra-phosphonate cavitands were employed to screen as guests the compounds of the butylammonium chloride series 1-4, which comprises a range of low molecular weight (LMW) molecules (molecular mass < 150 Da) that differ from each other by one or a few N-methyl groups (molecular mass 15 Da). The cavitand surface recognition of each individual guest drove a specific MC bending (from a few to several tens of nanometer), disclosing a direct, label-free, and real-time mean to sort them. The complexation preferences of tetraphosphonate cavitands toward ammonium chloride guests 1-4 were independently assessed by isothermal titration calorimetry. Both direct and displacement binding experiments concurred to define the following binding order in the alkylammonium series: 2 > 3 ≈ 1 ≫ 4. This trend is consistent with the number of interactions established by each guest with the host. The complementary ITC experiments showed that the host-guest complexation affinity in solution is transferred to the MC bending. These findings were benchmarked by implementing cavitand-functionalized MCs to discriminate sarcosine from glycine in water.

La Broncopneumopatia Cronica Ostruttiva (BPCO) è un’infiammazione polmonare progressiva caratterizzata da limitazione di flusso espiratorio. Ad oggi a livello mondiale si stima che 328 milioni di persone siano affette da BPCO e si prevede che diventi la quarta causa di morte entro il 2030 con più di 65 milioni di casi l’anno. Il successo dei trattamenti medici mediante acidi nucleici, come DNA e silencing interference RNA è fortemente legato al design di efficienti tecnologie di rilascio. I dendrimeri cationici rappresentano un’interessante possibilità come vettori grazie a noti vantaggi di efficienza, di basso costo, di produzione e di versatilità d’applicazione nel trattamento di molteplici malattie e disturbi. Il presente lavoro di tesi, svolto presso l’Istituto Dalle Molle di Studi sull’Intelligenza Artificiale di Lugano (CH), ha avuto lo scopo di investigare a livello computazionale su scala atomica il meccanismo di legame di diversi dendrimeri con un modello di siRNA, per l’ot...

Aerolysin is the paradigmatic member of a large family of toxins that convert from a water-soluble monomer/dimer into a membrane-spanning oligomeric pore. While there is x-ray crystallographic data of its water-soluble conformation, the most recent structural model of the membrane-inserted pore is based primarily on data of water-soluble tetradecamers of mutant protein, together with computational modeling ultimately performed in vacuum. Here we examine this pore model with atomic force microscopy (AFM) of membrane-associated wild-type complexes and all-atom molecular dynamics (MD) simulations in water. In striking contrast to a disc-shaped cap region predicted by the present model, the AFM images reveal a star-shaped complex, with a central ring surrounded by seven radial projections. Further, the MD simulations suggest that the locations of the receptor-binding (D1) domains in the present model are not correct. However, a modified model in which the D1 domains, rather than localiz...

Cantilever-type all-organic microelectromechanical systems based on molecularly imprinted polymers for specific analyte recognition are used as chemical sensors. They are produced by a simple spray-coating-shadow-masking process. Analyte binding to the cantilever generates a measurable change in its resonance frequency. This allows label-free detection by direct mass sensing of low-molecular-weight analytes at nanomolar concentrations.

The application of the SOM network in drug design and molecular diversity is discussed. In particular, examples of the applications of the Comparative Molecular Surface Analysis (CoMSA) are reviewed. Molecular surface is a fuzzy category, inspired by the macroscopic world, which has no unique equivalent in the molecular scale. However, it is somewhere near the area where the molecular recognition processes are taking place. Consequently, the methods that analyze this region promise better efficiency than procedures that are based on uniform grids. An important advantage of the CoMSA method is the possibility for the generation of fuzzy molecular representations together with its ability to discover such aspects of molecular similarity that can be easily overlooked by a chemist. The ability for data compression is a further advantage. It has also been shown that the fast processing of the comparative Kohonen mapping enables one to implement this method in the field of molecular diversity.

Background: Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. Methodology/Principal Findings: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at .1,000-fold faster rate, and reduce costs ,250fold for generating 10 6 ligand sequences. Conclusions/Significance: Our analyses demonstrates that whereas this approach correlates with the traditional colonycounting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.

The increasing prevalence of antibiotic-resistant bacteria is creating a real challenge for health care systems worldwide, making the development of novel antibiotics a necessity. In addition to the development of new antibiotics, there is an urgent need for in-depth characterization of the mechanisms of bacterial resistance toward new drugs. Here, we used essential oils extracted in our laboratory from Piper cubeba against methicillin-resistant Staphylococcus aureus ATCC 43300, one of the most prominent antibiotic-resistant bacteria. Effects of the essential oils extracted from P cubeba on bacteria were mainly evaluated using 2 powerful microscopy techniques: atomic force microscopy and transmission electron microscopy. High-resolution atomic force microscopy images of the cells were obtained close to their native environment by immobilizing the cells on porous Polyether sulfone membranes, which were prepared in our laboratory with a wide range and distribution of pore sizes and de...

Standard procedure: Solutions of D-glucose in D 2 O/S mixtures (S = MeOH-d 4 , DMSO-d 6 , or MeCN-d 3 ) or D 2 O/KSCN were prepared and allowed to equilibrate overnight. 1 H NMR analysis indicated that the anomeric ratio (α/β) was ca. 40:60 in all cases. Aliquots were added to 700 μL of a solution of receptor 1b in the same solvent system (0.7 μmol, [1b] initial = 1.0 mM) in a NMR sample tube. The sample tube was shaken carefully after each addition and 1 H-NMR spectra were recorded at 296 K. Signals A, B and C (see Fig. S1) moved downfield during the titration and were used to calculate binding constants. A representative set of spectra for a titration in water are shown in ref. [11b]. The data were analysed using a specifically written non-linear least squares curve-fitting program implemented within Excel. Binding curves are shown in Figs. S2af (D 2 O/MeOH-d 4 ), S3a-d (D 2 O/DMSO-d 6 ) and S4a-d (D 2 O/MeCN-d 3 ). Peak B moved the furthest during the titrations and appeared to give the best-fitting curves. The binding constants derived from this peak were therefore used in Figure 2a.

A number of new cationic ligands have been designed and synthesized for the selective resolution and purification of the trypszin-like proteases. A series of ligands based on 4-[2'-methyl-4'-(2",4"-dichloro-l",~,S-triazin-6ylamino) phenylazo]benzamidine were able to bind to trypsin and the trypsin-like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4aminophenyltrimethylammonium or 4-aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis-cationic ligands bound pancreatic kallikrein in solution and following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis-cationic ligands was used to purify pancreatic kallikrein 9.5-fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.

A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de nouo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.

Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.

Infective metacyclic promastigote forms of Leishmania mexicana are introduced by the bite of sandfly vectors into their human hosts where they transform into the amastigote form. The kinetics of this process was examined in vitro in response to different combinations of temperature (26 mC or 32 mC), pH (7n2 or 5n5), and exposure to human serum. Little transformation occurred at 26 mC\pH 7n2, intermediate levels at 26 mC\pH 5n5 and 32 mC\ pH 7n2, and the greatest response at 32 mC\pH 5n5. Transformation was stimulated by exposure to normal human serum, but was markedly reduced when serum previously incubated at 56 mC for 1 h was used (complement heat-inactivated). This stimulatory effect was reproduced by exposure to a single purified component of human serum, C-reactive protein (CRP). Binding of CRP to the whole surface of L. mexicana metacyclic promastigotes, including the flagella, was demonstrated by an indirect fluorescent antibody test. The effect of purified CRP was dose dependent and occurred using normal serum concentrations. The stimulatory effect of whole serum was oblated by CRP depletion and restored by addition of purified CRP. The effects of cAMP analogues indicated that transformation could be mediated via an adenylate cyclase cascade.

Herein we demonstrate the electrochemical activation and detection of intermolecular halogen bonding (XB) in solution by generating the cation radical of iodo-trimethyltetrathiafulvalene (I-Me 3 TTF * + ) in the presence of various Lewis-basic anions. This work is leading to the endorsement and generalization of the concept of electrostimulating XB for molecular recognition by adjusting the properties of the XB donor via the electrochemical reversible generation of charged species. The formation of the interaction between I-Me 3 TTF * + and Lewis bases has been investigated by cyclic voltammetry allowing to quantify the strength of the interaction (K Ox = 100 to 400 M À 1 and ΔG = À 12 to À 15 kJ mol À 1 ). Moreover spectroelectrochemical measurements along with TD-DFT calculations have confirmed XB formation upon generation of the radical cation. The present work opens broad perspectives for the development of redox switchable XB donors in the field of molecular recognition in chemical and biological systems where noncovalent interactions are usually key governing parameters.

The polymerization of norepinephrine, and the properties of the related polymer polynorepinephrine, started to be investigated barely 9 years ago and only few works were produced so far, mainly in materials science and medicine. An unexpectedly low relevance, especially if compared with the interest toward dopamine and polydopamine, differing from norepinephrine only for a hydroxyl group and whose properties were deeply investigated and applied to an impressive number of subject areas. We show here that in some cases, norepinephrine and dopamine monomers can be exchanged without virtually affecting the experimental results. But even more interesting, the choice of norepinephrine can positively influence the properties of the final polymer. In particular, the smoother and more hydrophilic surface of polynorepinephrine may enhance cell adhesion and proliferation, increase the activity of conjugated biomolecules, and induce higher cellular uptake of nanodrugs. Moreover, polynorepinephrine presents an additional anchoring point that can be exploited for further functionalization. Nevertheless, despite its potential for bioconjugation and molecular recognition, polynorepinephrine has not yet been considered in biosensing. Here we report our feelings in terms of perspective use of polynorepinephrine as new functional monomer for biomimetic receptor development by molecular imprinting, with application in affinity biosensing.

The engineering of complex architectures from functional molecules on surfaces provides new pathways to control matter at the nanoscale. In this article, we present a combined study addressing the self-assembly of the amino acid l -methionine on Ag(111). Scanning tunneling microscopy data reveal spontaneous ordering in extended molecular chains oriented along high-symmetry substrate directions. At intermediate coverages, regular biomolecular gratings evolve whose periodicity can be tuned at the nanometer scale by varying the methionine surface concentration. Their characteristics and stability were confirmed by helium atomic scattering. X-ray photoemission spectroscopy and high-resolution scanning tunneling microscopy data reveal that the l -methionine chaining is mediated by zwitterionic coupling, accounting for both lateral links and molecular dimerization. This methionine molecular recognition scheme is reminiscent of sheet structures in amino acid crystals and was corroborated b...

The present study involves molecular docking, molecular dynamics (MD) simulation studies, and Caco-2 cell monolayer permeability assay to investigate the effect of structural modifications on PepT1-mediated transport of thyrotropin releasing hormone (TRH) analogs. Molecular docking of four TRH analogs was performed using a homology model of human PepT1 followed by subsequent MD simulation studies. Caco-2 cell monolayer permeability studies of four TRH analogs were performed at apical to basolateral and basolateral to apical directions. Inhibition experiments were carried out using Gly-Sar, a typical PepT1 substrate, to confirm the PepT1-mediated transport mechanism of TRH analogs. P app of the four analogs follows the order: NP-1894 < NP-2378 < NP-1896 < NP-1895. Higher absorptive transport was observed in the case of TRH analogs, indicating the possibility of a carrier-mediated transport mechanism. Further, the significant inhibition of the uptake of Gly-Sar by TRH analogs confirmed the PepT1-mediated transport mechanism. Glide docking scores of all the four analogues were in good agreement with their transport rates, suggesting the role of substrate binding affinity in the PepT1-mediated transport of TRH analogs. MD simulation studies revealed that the polar interactions with amino acid residues present in the active site are primarily responsible for substrate binding, and a downward trend was observed with the increase in bulkiness at the N-histidyl moiety of TRH analogs. Copyright

Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core composition, illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural analysis and molecular dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core volumes, which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding...

The application of principals of molecular recognition to the design of organic and inorganic solids is leading to remarkable progress in the preparation of porous and chiral materials. A new area of supramolecular complexes is metallamacrocycles, large molecules that are sewn together using metal linkages. Metallacrowns are a member of this class of inorganic complexes. In this article we develop a strategy for the preparation of solids that are chiral using metallacrowns as the centerpiece. First, we introduce the available structure types for these molecules and then proceed to describe some of the physical properties associated with this unique molecular assembly. We demonstrate that chirality can be incorporated simply and inexpensively into these molecules and then we show how the metallacrowns can be used as building blocks to make layered solids with chiral channels. Finally, we assess more general strategies that could be used to engineer truly porous and chiral materials with high metal densities.

The application of principals of molecular recognition to the design of organic and inorganic solids is leading to remarkable progress in the preparation of porous and chiral materials. A new area of supramolecular complexes is metallamacrocycles, large molecules that are sewn together using metal linkages. Metallacrowns are a member of this class of inorganic complexes. In this article we develop a strategy for the preparation of solids that are chiral using metallacrowns as the centerpiece. First, we introduce the available structure types for these molecules and then proceed to describe some of the physical properties associated with this unique molecular assembly. We demonstrate that chirality can be incorporated simply and inexpensively into these molecules and then we show how the metallacrowns can be used as building blocks to make layered solids with chiral channels. Finally, we assess more general strategies that could be used to engineer truly porous and chiral materials with high metal densities.

A novel protocol for use of molecularly imprinted polymer (MIP) in analysis of melamine is presented. Design of polymer for melamine has been achieved using a combination of computational techniques and laboratory trials, the former greatly reducing the duration of the latter. The compatibility and concerted effect of monomers and solvents were also investigated and discussed. Two novel open source tools were presented which are: the online polymer calculator from mipdatabase.com and the application of the Gromacs modelling suite to determine the ideal stoichiometric ratio between template and functional monomer. The MIP binding was characterised for several structural analogues at 1-100 μM concentrations. The use of DVB as cross-linking polymer and itaconic acid as functional monomer allowed synthesis of MIP with imprint factor for melamine IF=2.25. This polymer was used in HPLC for the rapid detection of melamine in spiked milk samples with an experimental run taking 7-8 minutes. This approach demonstrated the power of virtual tools in accelerated design of MIPs for practical applications.

Phage display screening of a surface-immobilized adenine derivative led to the identification of a heptameric peptide with selectivity for adenine as demonstrated through quartz crystal microbalance (QCM) studies. The peptide demonstrated a concentration dependent affinity for an adeninyl moiety decorated surface (K D of 968 ± 53.3 μM), which highlights the power of piezoelectric sensing in the study of weak interactions.

We have determined the solution structure of the complex between an arginine-glycine-rich RGG peptide from the human fragile X mental retardation protein (FMRP) and an in vitro-selected guanine-rich (G-rich) sc1 RNA. The bound RNA forms a newly discovered G-quadruplex separated from the flanking duplex stem by a mixed junctional tetrad. The RGG peptide is positioned along the major groove of the RNA duplex, with the G-quadruplex forcing a sharp turn of R(10)GGGGR(15) at the duplex-quadruplex junction. Arg10 and Arg15 form cross-strand specificity-determining intermolecular hydrogen bonds with the major-groove edges of guanines of adjacent Watson-Crick G•C pairs. Filter-binding assays on RNA and peptide mutations identify and validate contributions of peptide-RNA intermolecular contacts and shape complementarity to molecular recognition. These findings on FMRP RGG domain recognition by a combination of G-quadruplex and surrounding RNA sequences have implications for the recognition o...

Erythrocytes (RBCs) constitute a very interesting class of cells both for their physiological function and for a variety of peculiarities. Due to their exceptionally strong relationship with the environment, the morphology and nanoscale characteristics of these cells can reveal their biochemical status and structural integrity. Among the possible subjects of investigations, the RBCs' ageing is of the utmost importance. This is a fundamental phenomenon that, in physiological conditions, triggers the cell turnover and ensures the blood homeostasis. With these premises, in recent years, we have presented an atomic force microscopy-based methodology to characterize the patterns of RBC ageing from the morphological point of view. In the present work, we used an ageing protocol more similar to the physiological conditions and we used differential scanning calorimetry and atomic force microscopy to probe the cross correlation between important structural and functional proteins. We also assessed the role played by fundamental structural and membrane proteins in the development of the most relevant morphological intermediates observed along the ageing. Furthermore, we coupled the morphological ageing patterns to the (bio)chemical alterations detected by Raman spectroscopy. This allowed identifying the chronology of the ageing morphologies and the metabolic pathways most involved in their development. As a whole, the present study provides the base to correlate specific molecular alterations to the development of structural anomalies, and these latter to the functional status of blood cells.

The aim of this study is to prepare an ion-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(l)cysteine methyl ester) [PHEMAC] cryogel which can be used to remove Fe 3+ ions from beta thalassemia patient plasma in vitro. Fe 3+ , as a template, was initially complexed with N-methacryloyl-(l)-cysteine methyl ester (MAC) to form the complex monomer MAC-Fe 3+ polymerized to constitute a Fe 3+ ionimprinted PHEMAC-Fe 3+ cryogel by cryo-polymerization. The template (Fe 3+ ) was removed from the polymer by using 0.1 M EDTA solution. The specific surface area of PHEMAC-Fe 3+ cryogel was found to be 32.7 m 2 /g. A maximum Fe 3+ adsorption capacity of 75 g/g was observed with PHEMAC-Fe 3+ cryogel. It was determined that PHEMAC-Fe 3+ cryogel possesses high selectivity coefficients for Fe 3+ /Cd 2+ and Fe 3+ /Ni 2+ , which are 10.5 and 2.3, respectively, times greater than PHEMAC (non-imprinted cryogel produced in the absence of Fe 3+ ).

Imprinting chondroitin sulfate (CS)/silica composites with Pb(II) and Cu(II) cations was explored with CS of bovine and different fish species origin. The process was based on the assumption that particular arrangements of the linear CS chains in aqueous solution, induced so as to accommodate cross complexation with the cations, would be embodied into a tridimensional matrix created through an organoalkoxysilane sol-gel scheme. The presence of Cu(II) in the synthesis of the composites did not result in the production of significantly stronger Cu(II)-oriented binding arrangements, and therefore, the imprinting was not successful. Inversely, for Pb(II), the materials obtained exhibited a &quot;memory&quot; effect for the Pb(II) ions, expressed in the observation of stronger (13%-44%) binding as compared to the nonimprinted counterparts, and increased selectivity (1.5-2 folds) against Cd(II). The imprinting features observed were dependent on the CS source. However, it was not possible...