Molar Ellipticity Research Articles

Influence of functionalized nanoparticles on conformational stability of type I collagen for possible biomedical applications

Collagen–nanoparticle interactions are vital for many biomedical applications including drug delivery and tissue engineering applications. Iron oxide nanoparticles synthesized using starch template according to our earlier reported procedures were functionalized by treating them with Gum Arabic (GA), a biocompatible polysaccharide, so as to enhance the interaction between nanoparticle surfaces and collagen. Viscosity, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) techniques have been used to study the collagen–nanoparticle interactions. The relative viscosity for collagen–nanoparticle conjugate was found to increase with increase in concentration of the nanoparticle within the concentration range investigated, which is due to the aggregation of protein onto the surface of nanoparticle. The CD spectra for the collagen–nanoparticle at different concentration ratios do not have much variation in the Rpn values (ratio of positive peak intensity over negative peak intensity) after functionalization with GA. The variation of molar ellipticity values for collagen–nanoparticle is due to the glycoprotein present in GA. The collagen triple helical structure is maintained after interaction with nanoparticles. The FTIR spectra of native collagen, Coll–Fs (nanoparticle without functionalization) and Coll–FsG (nanoparticle functionalized with GA) show clearly the amide I, II, III bands, with respect to collagen. The ability of polysaccharide stabilized/functionalized nanoparticles to maintain the collagen properties would help in its biomedical applications.

Abstract B44: Mithramycin exhibits dual binding mode and acts as an epigenetic switch

Abstract The recognition of epigenetic defects in all types of cancer has led to a marked achievement in cancer research in recent years. Histone acetylation, an important post translational epigenetic modification is altered in various diseases and the balance between acetylation and deacetylation is hampered. Reversibility of such epigenetic events provides a target for chemotherapeutic intervention. The major hurdle for drugs used for brain targeting is to cross the blood brain barrier. Mithramycin (MTR), a clinically approved DNA-binding antitumor antibiotic has been found to cross the blood-brain barrier and is in its preclinical trials in Huntington's disease (HD) [Stack et al., Hum Mol Genet, 2007]. It improved altered nucleosome homeostasis in HD mice, normalizing the chromatin pattern. It has the ability to rebalance epigenetic histone modifications. The binding properties of MTR with histone proteins have been examined from the perspective of the current proposition from our laboratory to classify small DNA binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode) [Ghosh et al., Biochim Biophys Acta, 2010; Dasgupta et al., J Biosciences, 2012]. Steady state fluorescence spectroscopy, circular dichroism (CD) spectroscopy and in vitro Histone Acetyl Transferase (HAT) assay have been employed as experimental tools. Spectrofluorimetric measurements and CD studies show that MTR binds with micromolar dissociation constant to core histone assembly and its individual histone components. A single isoelliptic point in the CD spectra indicates formation of one type of complex between MTR and histones. Complex formation leads to decrease in molar ellipticity in the far UV CD spectra of core histones. In contrast to MTR-DNA interaction, association of MTR with histones does not require the presence of bivalent metal ions. MTR thus exhibits dual binding mode since it binds to histone proteins present in chromatin besides DNA. These results led us to investigate the role of MTR as a potential epigenetic modulator. In vitro HAT assays using CREB-binding protein (CBP) HAT domain, a transcriptional coactivator, showed that MTR inhibits H3 K18 acetylation (specifically mediated mark by CBP/p300 in vivo) in H3.3 recombinant histone whereas it induces H3 K18 hyperacetylation in H3.1/H4 tetramer as well as core histones. H3 K18 acetylation is correlated with poor prognosis in patients with prostrate, pancreatic, lung, breast and kidney cancers and loss of this modification is an important event in tumor progression [Seligson et al., Nature 2005; Elsheikh et al., Cancer Res, 2009]. The interaction of MTR with histones hints at a different scenario in MTR-chromatin interaction. Experiments are in progress to address the effect of MTR on other epigenetic marks. The significance of these in vitro experiments would also be examined by performing cell line based experiments. These data would be useful in understanding the mechanism of action of anticancer drugs as well as in designing new-generation therapeutics. Citation Format: Amrita Banerjee, Chandrima Das, Dipak Dasgupta. Mithramycin exhibits dual binding mode and acts as an epigenetic switch. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B44.

Mithramycin interacts with core histones and modulates epigenetic modifications

BackgroundMithramycin (MTR), a clinically approved DNA-bindingantitumor antibiotic has been found to cross the blood-brain barrier and is in its preclinical trials in Huntington sdisease (HD) [1]. It improved altered nucleosome homeos-tasis in HD mice, normalizing the chromatin pattern. Ithas the ability to rebalance epigenetic histone modifica-tions. The binding properties of MTR with histone pro-teins have been examined from the perspective of thecurrent proposition from our laboratory to classify smallDNA binding molecules in terms of their ability to bindchromosomal DNA alone (single binding mode) or bothhistones and chromosomal DNA (dual binding mode)[2,3].Materials and methodsChemicals were from Sigma, histones from New Eng-land Biolabs, antibodies from Abacm, Active Motif.Spectrofluorimetry, circular dichroism (CD) studies, invitro histone acetyl transferase (HAT) assay and westernblot analysis were employed as experimental tools.ResultsSpectrofluorimetric measurements and CD studies showthat MTR binds with micromolar dissociation constant tocore histone assembly and its individual histone compo-nents. A single isoelliptic point in the CD spectra indicatesformation of one type of complex between MTR and his-tones. Complex formation leads to decrease in molar ellip-ticity in the far UV CD spectra of core histones. Theseresults led us to investigate the role of MTR as a potentialepigenetic modulator. In vitro HAT assays using CREB-binding protein (CBP) HAT domain, a transcriptionalcoactivator, showed that MTR inhibits H3 K18 acetylation(specifically mediated mark by CBP/p300 in vivo ) in H3.3recombinant histone whereas it induces H3 K18 hyperace-tylation in H3.1/H4 tetramer as well as core histones.ConclusionsMTR exhibits dual binding mode since it binds to histoneproteins present in chromatin besides DNA [4]. It altersCBP induced H3 K18 acetylation. In contrast to MTR-DNA interaction, association of MTR with histones doesnot require obligatory presence of bivalent metal ions. Theinteraction of MTR with histones hints at a different sce-nario in MTR-chromatin interaction. Experiments are inprogress to address the effect of MTR on other epigeneticmarks.Thesignificanceofthesein vitro experimentswould also be examined by performing cell line basedexperiments. These data would be useful in understandingthe mechanism of action of anticancer drugs as well as indesigning new-generation therapeutics.

Circular dichroism of anthocyanidin 3-glucoside self-aggregates

Self-association constants for the flavylium cations of the six most common anthocyanidin 3-glucosides were determined by circular dichroism (CD) and UV-Vis spectroscopy. Along with previous (1)H NMR results, all measurements were consistent with a monomer-dimer model. The CD spectra of the anthocyanidin 3-glucosides were similar to the analogues 3,5-diglucosides. All dimers of the anthocyanidin 3-glucosides exhibited left-handed CD signals, with petunidin-3-glucoside and myrtillin having the most intense signals. In addition, the magnitude of the molar ellipticity, [θ], was generally higher for the 3-glucosides than for the 3,5-diglucosides. For all six anthocyanins studied, the CD absorption spectra of their dimers showed evidence of the splitting of the monomer absorption into lower (J aggregates) and higher (H aggregates) energy bands. The angle and the distance between the dipolar moments of the two monomers comprising the dimer were obtained from the lower energy absorption band. While the angle was more or less similar in all six dimers, the separation distance between the monomer dipole moments differed dramatically. The intensity of the CD signal displayed a linear dependence with the inverse square of the dipole moment distances.

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Dual pH and temperature responsive helical copolymer libraries with pendant chiral leucine moieties

Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to afford two different chiral copolymer series having opposite chiroptical properties based on 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and Boc-L/D-leucine methacryloyloxyethyl ester (Boc-L/D-Leu-HEMA) with varying co-monomer proportions. The random nature of the copolymers was indicated by the reactivity ratios of the two monomers for the two systems (MEO2MA with Boc-L-Leu-HEMA and Boc-D-Leu-HEMA), and further supported by smooth variation of the single glass transition temperature (Tg) determined from differential scanning calorimetry (DSC). With an increasing amino acid content in the copolymer, the specific rotation and molar ellipticity values increased, as examined by polarimeter and circular dichroism (CD) spectroscopy, respectively. After Boc-group cleavage under acidic conditions, all the copolymers exhibited cationic nature, as confirmed by dynamic light scattering (DLS), as well as the dual thermo and pH-dependent solubility behaviour in aqueous solution. The temperature induced phase transition of these copolymers was investigated as a function of the copolymer compositions over the pH range from 5.5 to 6.5 using a UV-Vis spectrophotometer. Furthermore, the chiral recognition ability of the synthesized monomers and homopolymers towards 1,1′-bi-2-naphthol (rac-BINOL) was studied by 1H NMR spectroscopy.

Designing of nitrile hydratase from alkaline protease using quanidine hydrochloride and cobalt metal ion

Alkaline protease (22.5kDa) from Bacillus licheniformis was denatured using 3–6M Guanidine hydrochloride followed by renaturation with cobalt metal ion in buffer solution (pH 7.5) in order to design cobalt dependent nitrile hydratase AP-xMGdn-Co, where x=3, 4, 5 and 6. The native alkaline protease showed a 20% residual molar ellipticity at 212nm while on treatment with Gdn-HCl, it got reduced to 12% due to nearly complete collapse of the globular structure of the protein. The observed circular dichroism spectra of the renatured proteases with Co metal ion are different from the native enzyme, because those molecules contain many metal chelating sites through its coordinating amino acid residues. The introduction of cobalt centered new active site was evidenced by the fluorescence spectroscopy (appearance of a new peak at 360nm) and ICP analysis (0.0476–0.0553mg/L). The specific activities of the modified enzymes were measured using the selective catalytic hydrolysis of 3-cyanopyridine to nicotinamide. The highest specific activity of renatured enzymes (AP-xMGdn-Co) were obtained at optimum pH of 7.5 and temperature of 50°C, due to the proper co-ordination of amino acid residue with cobalt metal ion and the appropriate arrangement of α-helix, β-strand, turn and unordered segment in the globular structure.

The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t1/2) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.

Structural and Functional Relationships between the Lectin and Arm Domains of Calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.

Antibody Recognition of a Human Chorionic Gonadotropin Epitope (hCGβ66–80) Depends on Local Structure Retained in the Free Peptide

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical β3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGβ(66-80) and the closely related luteinizing hormone (LH) fragment LHβ(86-100), which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.

Mapping the IκB Kinase β (IKKβ)-binding Interface of the B14 Protein, a Vaccinia Virus Inhibitor of IKKβ-mediated Activation of Nuclear Factor κB

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKβ subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKβ, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKβ and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKβ. In contrast, a second monomeric mutant (F130K) neither bound IKKβ nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKβ interaction. Thus, the dimerization and IKKβ-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.

Conformational Folding and Stability of the HET-C2 Glycolipid Transfer Protein Fold: Does a Molten Globule-like State Regulate Activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 Å) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.

Designed Coiled Coils Promote Folding of a Recombinant Bacterial Collagen

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

A 68 residue N-terminal fragment of pro-atrial natriuretic peptide is a monomeric intrinsically unstructured protein

The mature pro forms of the cardiac natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are proteolytically processed to their active hormone forms (28 and 32 residues, respectively) and N-terminal (NT)-pro fragments (68 and 76 residues, respectively). Far-ultraviolet circular dichroism (UV CD), 1D and 2D-homonuclear nuclear magnetic resonance (NMR), size exclusion-high performance liquid chromatography (SE-HPLC) and analytical ultracentrifuge sedimentation equilibrium (AUCSE) data are obtained for NT-proANP. CD data showed a large negative molar ellipticity for NT-proANP of -14,800° cm(2)/dmol at 199-200 nm. The intensity of the 1D-(1)H NMR spectra in the amide region for NT-proANP was very low and confined to ~8-8.6 ppm. Furthermore, cross-correlation resonance peaks were absent in the corresponding 2D-(1)H NOE spectra for NT-proANP in this region. The elution peak for this fragment from a G2000SW size-exclusion column was 20.4'; myoglobin (~17 K) was also eluted at 20.4'. No higher molecular weight oligomers were evident in the AUCSE experiments for NT-proANP. Collectively, the physical data demonstrate that NT-proANP, like NT-proBNP, is primarily a disordered, monomeric protein. Lastly, we compare the predictions from two in silico metaserver disorder algorithms, MeDor and MetaPrDOS, to the experimental data.

Oxidative Modifications of the C-terminal Domain of Tropoelastin Prevent Cell Binding

Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin α(v)β(3) and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO(-)) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO(-) treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO(-) treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO(-) prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.

Sequence-independent Control of Peptide Conformation in Liposomal Vaccines for Targeting Protein Misfolding Diseases

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule β-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing β-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized β-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

Synthesis and Spectroscopic Studies of the Aminoglycoside (Neomycin)−Perylene Conjugate Binding to Human Telomeric DNA

Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

Evolution of Metamorphism in Thymidylate Synthases Within the Primate Lineages

Crystal structures of human thymidylate synthase (hTS) revealed that the protein exists in active and inactive conformations, defined by the position of a loop containing the active site nucleophile. TS is highly homologous among diverse species; however, the residue at position 163 (hTS) differs among species. Arginine at this position is predicted by structural modeling to enable conformational switching. Arginine or lysine is reported at this position in all mammals in the GenBank and Ensembl databases, with arginine reported in only primates. Sequence analysis of the TS gene of representative primates revealed that arginine occurs at this relative position in all primates except a representative of prosimians. Mutant human proteins were created with residues at position 163 that occur in TSs from prokaryotes and eukaryotes. Catalytic constants (k(cat)) of mutant enzymes were 45-149% of hTS, with the lysine mutant (R163K) exhibiting the highest k(cat). The effect of lysine substitution on solution structure and on ligand binding was investigated. R163K exhibited higher intrinsic fluorescence, a more negative molar ellipticity, and higher dissociation constants (K(d)) for ligands that modulate protein conformation than hTS. Temperature effects on intrinsic fluorescence and catalytic activity of hTS and R163K are consistent with proteins populating different conformational states. The data indicate that the enzyme with arginine at the position corresponding to 163 (hTS) evolved after the divergence of prosimians and simians and that substitution of lysine by arginine confers unique structural and functional properties to the enzyme expressed in simian primates.