Conformational Adaptation Research Articles

Characterizing the Binding Interaction between Erlotinib and Calf Thymus DNA In Vitro Using Multi‐Spectroscopic Methodologies and Viscosity Measurement Combined with Molecular Docking and DFT Calculation

AbstractThe binding interaction between erlotinib (ELTN) and calf thymus DNA (ct‐DNA) was characterized with the help of multi‐spectroscopic approaches, viscosity measurement and molecular docking as well as density functional theory (DFT) calculation to get critical information regarding ELTN binding to DNA. The findings confirmed that ELTN acted with ct‐DNA and formed the ELTN‐DNA complex with the binding constant of 2.37×103 M−1 (298 K) and the binding rate of ELTN was larger than 99% when C(ELTN)=8 μM and C(DNA) > 50.6 μM at 298 K, ELTN bound to the rich A−T minor groove of ct‐DNA, and the helical configuration of ct‐DNA slightly changes after binding ELTN but still kept B‐form while the conformation, atomic charge distribution, dipole moment, and frontier molecular orbitals of ELTN in the DNA complex obviously altered to satisfy with a larger global Hardness and the conformational adaptation. During the binding process, the ELTN interacting with ct‐DNA is endothermic, spontaneous, and entropy‐driven because of ΔH0 > 0, ΔG0 < 0, and |ΔH0|<|TΔS0|, and the dominated driving force was hydrophobic interaction.

The Subtle Trade-Off between Evolutionary and Energetic Constraints in Protein-Protein Interactions.

Life machinery, although overwhelmingly complex, is rooted on a rather limited number of molecular processes. One of the most important is protein-protein interaction. Metabolic regulation, protein folding control, and cellular motility are examples of processes based on the fine-tuned interaction of several protein partners. The region on the protein surface devoted to the recognition of a specific partner is essential for the function of the protein and is, therefore, likely to be conserved during evolution. On the other hand, the physical chemistry of amino acids underlies the mechanism of interactions. Both evolutionary and energetic constraints can then be used to build scoring functions capable of recognizing interaction sites. Our working hypothesis is that residues within the interaction interface tend at the same time to be evolutionarily conserved (to preserve their function) and to provide little contribution to the internal stabilization of the structure of their cognate protein, to facilitate conformational adaptation to the partner. Here, we show that for some classes of protein partners (for example, those involved in signal transduction and in enzymes) evolutionary constraints play the key role in defining the interaction surface. In contrast, energetic constraints emerge as more important in protein partners involved in immune response, in inhibitor proteins, and in structural proteins. Our results indicate that a general-purpose scoring function for protein-protein interaction should not be agnostic of the biological function of the partners.

Understanding molecular features of aggregation-resistant tau conformer using oxidized monomer

BackgroundAggregation of tau into paired helical filament (PHF) is a hallmark of Alzheimer's disease (AD), and Cys-mediated disulfide bond formation plays a vital role in tau fibrillation. While intermolecular disulfide bond between Cys residues in microtubule-binding repeat (MTBR) region facilitates tau aggregation, intramolecular disulfide bond attenuates the same, though the molecular basis for such phenomenon remains obscure. Thus intramolecular disulfide-bonded tau monomer might be an excellent model to understand the unique features of aggregation-resistant tau conformer. MethodsWe synthesized the Cys cross-linked tau40 monomer by oxidation and characterized the altered conformational dynamics in the molecule by Hydrogen-deuterium exchange, limited proteolysis and fluorescence quenching. ResultsDeuterium exchange study showed that rigidity was imparted in the core PHF region of oxidized tau40 in MTBR segment, consisting of the fundamental PHF6 motif. Conformational rigidity was prominent in C-terminal tail region also. Limited proteolysis supported reduced accessibility of MTBR region in the molecule. ConclusionsPHF formation of oxidized tau40 might be attenuated either by induction of intramolecular H-bonding between the regions of high β-structure propensity in second and third MTBR (R2, R3), thus preventing intermolecular interaction between them, or by imparted rigidity in R2–R3, preventing the formation of extended β-structure preceding fibrillation. Data indicated plausible effect of conformational adaptation on the nucleation process of oxidized tau40 assembly. General significanceOur findings unravel the essential molecular features of aggregation-resistant tau conformer. Therapeutics stabilizing such conformers in vivo might be of high benefit in arresting tau assembly during AD and other tauopathies.

Deciphering evolution of immune recognition in antibodies

BackgroundAntibody, the primary effector molecule of the immune system, evolves after initial encounter with the antigen from a precursor form to a mature one to effectively deal with the antigen. Antibodies of a lineage diverge through antigen-directed isolated pathways of maturation to exhibit distinct recognition potential. In the context of evolution in immune recognition, diversity of antigen cannot be ignored. While there are reports on antibody lineage, structural perspective with respect to diverse recognition potential in a lineage has never been studied. Hence, it is crucial to evaluate how maturation leads to topological tailoring within a lineage enabling them to interact with significantly distinct antigens.ResultsA data-driven approach was undertaken for the study. Global experimental mouse and human antibody-antigen complex structures from PDB were compiled into a coherent database of germline-linked antibodies bound with distinct antigens. Structural analysis of all lineages showed variations in CDRs of both H and L chains. Observations of conformational adaptation made from analysis of static structures were further evaluated by characterizing dynamics of interaction in two lineages, mouse VH1–84 and human VH5–51. Sequence and structure analysis of the lineages explained that somatic mutations altered the geometries of individual antibodies with common structural constraints in some CDRs. Additionally, conformational landscape obtained from molecular dynamics simulations revealed that incoming pathogen led to further conformational divergence in the paratope (as observed across datasets) even while maintaining similar overall backbone topology. MM-GB/SA analysis showed binding energies to be in physiological range. Results of the study are coherent with experimental observations.ConclusionsThe findings of this study highlight basic structural principles shaping the molecular evolution of a lineage for significantly diverse antigens. Antibodies of a lineage follow different developmental pathways while preserving the imprint of the germline. From the study, it can be generalized that structural diversification of the paratope is an outcome of natural selection of a conformation from an available ensemble, which is further optimized for antigen interaction. The study establishes that starting from a common lineage, antibodies can mature to recognize a wide range of antigens. This hypothesis can be further tested and validated experimentally.

Torsional flexibility of undecorated catechol diether compound as potent NNRTI targeting HIV-1 reverse transcriptase

Conformational adaptation of non-nucleoside reverse transcriptase inhibitor (NNRTI) via torsional flexibility is found to be very significant for targeting human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) mutants. Catechol diether derivative including flexible torsions is new potent NNRTI with picomolar activity. Moreover, this derivative also reveals the good solubility, low toxicity and potent inhibition for HIV-1 mutants. In this study, torsional flexibility of an undecorated catechol diether compound in the binding pocket of wild type and mutants (Y181C and K103N/Y181C) HIV-1 RT is investigated by using QM/MM calculations. From the results, the uracil ring is found to exhibit more flexibility in the NNIBP. On the contrary, potential energy surfaces show that high energy is encountered by changing of the corresponding torsion of the cyanovinyl aryl ring indicating the limitation for torsional flexibility. For pointing out the key interaction for the binding, the residual interaction energies are performed by means of QM calculations. Important attractive interactions through hydrogen bonds between the inhibitor and K102, K/N103, V106, and Y188 are observed. The catechol ring is proposed to be modified in order to strengthen interactions with surrounding amino acids. The results may help for the designing of new potent NNRTIs.

Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions.

Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been suggested among the risk factors in those pathologies. Its main effect is the N-homocysteinylation of proteins due to the interaction between the highly reactive homocysteine thiolactone (HTL) and lysine residues. The aim of this study was to evaluate the effect of N-homocysteinylation on the erythropoietic and antiapoptotic abilities of EPO, which can be a consequence of structural changes in the modified protein. We found that both cellular functions were altered in the presence of HTL-EPO. A decreased net positive charge of HTL-EPO was detected by capillary zone electrophoresis, while analysis of polyacrylamide gel electropherograms suggested formation of aggregates. Far-UV spectra, obtained by Circular Dichroism Spectroscopy, indicated a switch of the protein's secondary structure from α-helix to β-sheet structures. Results of Congo red and Thioflavin T assays confirm the formation of repetitive β-sheet structures, which may account for aggregates. Accordingly, Dynamic Light Scattering analysis showed a markedly larger radius of the HTL-EPO structures, supporting the formation of soluble oligomers. These structural changes might interfere with the conformational adaptations necessary for efficient ligand-receptor interaction, thus affecting the proliferative and antiapoptotic functions of EPO. The present findings may contribute to explain the resistance exhibited by patients with cardio-renal syndrome to treatment with rhuEPO, as a consequence of structural modifications due to protein N-homocysteinylation.

Hybrasurfs-A New Class of Hyperbranched Surfactants.

A hyperbranched (HB) polyester carrying peripheral allyl groups was prepared by melt-condensation of a suitably designed AB2 monomer bearing two allyl ester groups and one hydroxyl group. The periphery of the hyperbranched polymer was co-clicked with two different organic thiols, namely, hexadecane thiol and 3-mercaptopropionic acid, using the thiol-ene reaction. Three different samples with varying mole fractions of the hydrophilic carboxylic acid groups were prepared; the conformational adaptability of the hyperbranched polymer backbone permitted these amphiphilic systems to form Janus structures that exhibit surfactant-like properties and, therefore, we have termed them hybrasurfs. These polymers behave like clusters of surfactants that have been stitched at the waist by the HB polymer backbone; the Langmuir isotherms revealed the formation of a monolayer, and in two of the samples having higher mole fractions of hexadecyl segments a weak inflection in the isotherm is seen. This suggests a densification, typically implying the crystallization of the alkyl segment at the air-water interface. The monolayers were transferred onto a substrate, and their heights were estimated using atomic force microscopy; the values thus obtained were in reasonable agreement with the expected value. The water contact angles of the substrates bearing the transferred monolayers of the three different samples (transferred at two different points along the isotherm) were measured; it was seen that the sample carrying the highest mole fraction of hexadecyl chains exhibited a significantly larger contact angle when compared to that of the other two samples. Interestingly, these hybrasurfs also formed vesicles in water and were shown to encapsulate water-soluble dyes, such as Eosin Y. Thus, this class of readily accessible amphiphilic HB polymers that behave as a cluster of surfactants opens some interesting possibilities for further exploration.

A molecular simulation analysis of vitamin D targets interleukin 13 (IL13) as an alternative to mometasone in asthma.

Asthma, a chronic lung disease characterized by obstruction of airway passage is characterized by inflammation and hyperresponsiveness with increase in the number of eosinophils. Interleukin-13, plays a significant role in causing inflammation during an asthmatic attack by bronchial constriction. Mometasone, a glucocorticoid has been used as the first line of administration for people affected with asthma for almost a decade. However, in several cases, people treated with mometasone have faced systemic and local side effects. To reduce these side effects, we hypothesized vitamin D that can be used as a substitute to mometasone. For this purpose, we employed the use of molecular docking and simulation studies for comparative study. The docking studies revealed the binding residues of interleukin-13 which are bound to the active site. Among all, we noticed three binding residue Leu83, His84 and Arg86 common for both mometasone and vitamin D. Also, the binding energies share a significant similarity between them. The docked complexes of mometasone and vitamin D with interleukin-13 were evaluated with molecular dynamics simulation. Consistently, the MD analysis uncovered the interesting note on conformational adaptation between the complexes as well as that vitamin D has the complementary binding efficiency to interleukin-13 as compared to mometasone. The substitution of vitamin D might provide a promising gateway to reduce the side effects caused by mometasone and also reduce the cost for treatment of asthma patients.

Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics.

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1–4, 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+, helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg2+, the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg2+. NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+. We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated interhelical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.

Inhibition of Heat-Induced Flocculation of Myosin-Based Emulsions through Steric Repulsion by Conformational Adaptation-Enhanced Interfacial Protein with an Alkaline pH-Shifting-Driven Method.

Protein conformational rearrangement triggered by adsorption to the hydrophobic interface of oil droplets has long been considered as a key factor in emulsification. In this study, an alkaline pH-shifting-driven conformational adaptation enhanced interfacial proteins was used to improve their stability against heat-induced flocculation of myosin emulsions. We used the unfolded myosin at pH 12 to emulsify soy oil and then readjusted the pH of the emulsion to neutral. The corresponding myosin emulsion (0.5% w/v protein, 10% v/v soy oil, and 0.6 M NaCl) almost not flocculated when heated at 75 °C for 30 min. Moreover, after thermal treatment, the particle size of the emulsion was not significantly increased ( P > 0.05) and the emulsion did not exhibit a creaming phenomenon after a week. Based on the circular dichroism and Fourier transform infrared analysis, we speculated the superiority of the emulsion is closely related to the alkaline pH-shifting-driven conformational adaptation enhanced interfacial protein. Additionally, the resulting steric stabilization in overcoming the attractive hydrophobic forces between denatured protein molecules coated droplets might be the main factor for the inhibition of heat-induced flocculation of the emulsion. Our research may have important implications for the formulation of protein-stabilized oil-in-water emulsions.

The cytokine secretion profile of mesenchymal stromal cells is determined by surface structure of the microenvironment

Mesenchymal stromal cells (MSC) secrete factors that contribute to organ homeostasis and repair in a tissue specific manner. For instance, kidney perivascular mesenchymal stromal cells (kPSCs) can facilitate renal epithelial repair through secretion of hepatocyte growth factor (HGF) while the secretome of bone marrow MSCs gives rise to immunosuppression. Stromal cells function in a complex 3-dimensional (3D) connective tissue architecture that induces conformational adaptation. Here we tested the hypothesis that surface topography and associated cell adaptations dictate stromal cell function through tuning of the cytokines released. To this end, we cultured human bone marrow and kidney perivascular stromal cells in the TopoWell plate, a custom-fabricated multi-well plate containing 76 unique bioactive surface topographies. Using fluorescent imaging, we observed profound changes in cell shape, accompanied by major quantitative changes in the secretory capacity of the MSCs. The cytokine secretion profile was closely related to cell morphology and was stromal cell type specific. Our data demonstrate that stromal cell function is determined by microenvironment structure and can be manipulated in an engineered setting. Our data also have implications for the clinical manufacturing of mesenchymal stromal cell therapy, where surface topography during bioreactor expansion should be taken into account to preserve therapeutic properties.

Effect of altered solution conditions on tau conformational dynamics: Plausible implication on order propensity and aggregation

Intrinsically disordered protein tau plays a central role in maintaining neuronal network by stabilizing microtubules in axon. Tau reportedly possesses random coil architecture, which is largely inert to alteration in solution conditions. However, the presence of transient compact conformers and residual structure has been evident from previous reports. Also, during Alzheimer's disease, misfolded tau detaches from microtubule and forms ordered filaments, which is the hallmark of the disease. Despite its fundamental role in neuronal physiology and in pathological cascade of several fatal neurodegenerative diseases, tau conformational dynamics remains poorly understood. In the present study, we have explored the effect of ionic strength, temperature and solvent polarity on tau40 conformational preferences using ion mobility mass spectrometry. Investigation of collision cross section revealed that while low ionic strength, elevated temperature and reduced solvent polarity mostly induced partial collapse in tau40 conformers, higher ionic strength led to an expansion of the molecule. Limited proteolysis identified segments of tau40 projection domain and proline-rich region having high order propensity and a C-terminal region having vulnerability for further expansion at altered solution conditions. The high susceptibility for disorder-to-order transition in the above region of the protein might have crucial implication on its role as microtubule spacers, and in cellular signaling cascade. The conformational adaptation of tau40 did not enhance the heparin-induced aggregation proclivity of the protein. Nevertheless, the observed correlation of electrostatic interaction with fibrillation propensity of tau40 might indicate plausible link between hyperphosphorylation at diseased state with tau conformation and self-assembly.

Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

The hepatitis B virus (HBV) icosahedral nucleocapsid is assembled from 240 chemically identical core protein molecules and, structurally, comprises four groups of symmetrically nonequivalent subunits. We show here that this asymmetry is reflected in solid-state NMR spectra of the capsids, in which peak splitting is observed for a subset of residues. We compare this information to dihedral angle variations from available 3D structures and also to computational predictions of "dynamic" domains and molecular hinges. We find that although, at the given resolution, dihedral angles variations directly obtained from the X-ray structures are not precise enough to be interpreted, the chemical-shift information from NMR correlates, and interestingly goes beyond, information from bioinformatics approaches. Our study reveals the high sensitivity with which NMR can detect the residues allowing the subtle conformational adaptations needed in lattice formation. Our findings are important for understanding the formation and modulation of protein assemblies in general.

Cryo-EM structure of a herpesvirus capsid at 3.1 Å.

Structurally and genetically, human herpesviruses are among the largest and most complex of viruses. Using cryo-electron microscopy (cryo-EM) with an optimized image reconstruction strategy, we report the herpes simplex virus type 2 (HSV-2) capsid structure at 3.1 angstroms, which is built up of about 3000 proteins organized into three types of hexons (central, peripentonal, and edge), pentons, and triplexes. Both hexons and pentons contain the major capsid protein, VP5; hexons also contain a small capsid protein, VP26; and triplexes comprise VP23 and VP19C. Acting as core organizers, VP5 proteins form extensive intermolecular networks, involving multiple disulfide bonds (about 1500 in total) and noncovalent interactions, with VP26 proteins and triplexes that underpin capsid stability and assembly. Conformational adaptations of these proteins induced by their microenvironments lead to 46 different conformers that assemble into a massive quasisymmetric shell, exemplifying the structural and functional complexity of HSV.

RNA and RNP Structures that Contribute to Viral Pathogenesis

RNA viruses persist to pose serious threats to human health and global economies. Disease progression mediated by viral pathogenesis requires numerous intersections between host proteins and viral RNA (vRNA) elements. Host‐vRNA complexes drive essential processes in the replication cycles of viruses; as such, they represent novel targets for therapeutic intervention. Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family are cellular proteins often usurped by RNA viruses. My lab studies the molecular mechanisms by which HIV and Enterovirus 71 redirects hnRNPs to control viral gene expression. To that end, we have designed an integrated biophysical framework to elucidate the structures of vRNAs and protein‐vRNA complexes. Here, we describe 3D structures of conserved HIV and EV71 RNA elements that regulate splicing and translation. We further show that conformational adaptation and slow protein motions are fundamental properties by which hnRNP A1 and hnRNP H achieve cognate RNA recognition. To globally identify determinants of specificity, we used High Throughput Sequencing Equilibrium (HTS‐EQ) binding experiments to characterize the complete affinity distribution of human hnRNP A1 bound to the HIV ESS3 stem loop. HTS‐EQ involves competitive binding of hnRNP A1 to a completely randomized pool of ESS3 apical loop variants (n=16,384). Quantitative analysis of the resulting affinity distribution reveals RNA sequence; motif copy number; motif spacing; and secondary structure determine specificity by modulating the rates of productive hnRNP A1‐RNA encounters. Collectively, our work is offering mechanistic insights into the strategies by which viruses coopt cellular RNA binding proteins.Support or Funding InformationNIH R01 GM101979NIH U54 GM103297This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Dynamics of Gangliosides.

Computational methodologies have immense potential to delineate the dynamic conformations of glycoconjugates including gangliosides, thereby characterizing the conformational adaptability of their glycans upon interacting with various target proteins. Replica-exchange molecular dynamics simulations have been employed to effectively explore the vast conformational spaces of large, branched carbohydrate moieties. When experimentally validated using NMR, molecular simulations can provide dynamical views of molecular recognition events involving the ganglioside glycans.

Mutational Analysis of a Conserved Glutamate Reveals Unique Mechanistic and Structural Features of the Phosphatase PRL-3.

Phosphatase of regenerating liver (PRL)-3 (PTP4A3) has gained much attention in cancer research due to its involvement in tumor promoting and metastatic processes. It belongs to the protein tyrosine phosphatase (PTP) superfamily and is thought to follow the catalytic mechanism shared by this family, which aside from the conserved active-site amino acids includes a conserved glutamic acid residue that is usually required for the integrity of the active site in PTPs. We noted that in structures of PRL-3, PRL-1, and PTEN these residues do not clearly align and therefore we sought to investigate if the glutamic acid residue fulfills its usual function in these proteins. Although this residue was essential for PTEN’s catalytic activity, it was nonessential for PRL-1 and PRL-3. Surprisingly, the mutation E50R increased PRL-3 activity against all tested in vitro substrates and also enhanced PRL-3-promoted cell adhesion and migration. We show that the introduction of Arg50 leads to an enhancement of substrate turnover for both PRL-3 and, to a lesser extent, PRL-1, and that the stronger gain in activity correlates with a higher structural flexibility of PRL-3, likely allowing for conformational adaptation during catalysis. Thus, in contrast to its crucial functions in other PTPs, this conserved glutamic acid can be replaced in PRL-3 without impairing the structural integrity. The variant with enhanced activity might serve as a tool to study PRL-3 in the future.

Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: A combined spectroscopic and docking simulation approach

A comparative biophysical study on the individual conformational adaptation embraced by two homologous serum albumins (SA) (bovine and human) towards a potential anticancer bioorganic compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)- dione (CBIQD) is apparent from the discrimination in binding behavior and the ensuing consequences accomplished by combined in vitro optical spectroscopy, in silico molecular docking and molecular dynamics (MD) simulation. The Sudlow site I of HSA although anion receptive, harbors neutral CBIQD in Sudlow site I (subdomain IIA, close to Trp) of HSA, while in BSA its prefers to snugly fit into Sudlow site II (subdomain IIIA, close to Tyr). Based on discernable diminution of HSA mean fluorescence lifetime as a function of biluminophore concentration, facile occurrence of fluorescence resonance energy transfer (FRET) is substantiated as the probable quenching mechanism accompanied by structural deformations in the protein ensemble. CBIQD establishes itself within HSA close to Trp214, and consequently reduces the micropolarity of the cybotactic environment that is predominantly constituted by hydrophobic amino acid residues. The stronger association of CBIQD with HSA encourages an allosteric modulation leading to slight deformation in its secondary structure whereas for BSA the association is comparatively weaker. Sudlow site I of HSA is capable to embrace a favorable conformation like malleable gold to provide room for incoming CBIQD, whereas for BSA it behaves more like rigid cast-iron which does not admit any change thus forcing CBIQD to occupy an altogether different binding location i.e. the Sudlow site II. The anticancer CBIQD is found to be stable within the HSA scaffold as vindicated by root mean square deviation (RMSD) and root mean square fluctuation (RMSF) obtained by MD simulation. A competitively inhibited esterase-like activity of HSA upon CBIQD binding to Lys199 and Arg257 residues, plausibly envisions that similar naphthalimide based prodrugs, bearing ester functionality, can be particularly activated by Sudlow site I of HSA. The consolidated spectroscopic research described herein may encourage design of naphthalimide based pro-drugs for effective in vivo biodistribution using HSA-based drug delivery systems.

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitamin B12 (57 Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN)18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.

Restricted amide rotation with steric hindrance induced multiple conformations

The CN bond character is dependent directly upon the resonance-contributor structure population driven by the delocalized nitrogen lone-pair of electrons. In the case of N, N-dibenzyl-ortho-toluamide (o-DBET), the molecule adopts subpopulations of conformers with distinct NMR spectral features, particularly at low temperatures. This conformational adaptation is unique to o-DBET, while the corresponding meta- and para- forms do not show such behavior. Variable-temperature (VT) NMR, two-dimensional exchange spectroscopy (EXSY), and qualitative molecular modeling studies are used to demonstrate how multiple competing interactions such as restricted amide rotation and steric hindrance effects can lead to versatile molecular adaptations in the solution state.