“Setting paint” analogy for the hydrophobic self‐association of tropoelastin into elastin‐like hydrogel

Alkaline tropoelastin solutions (pH 11) were optically clear at low temperatures, but a firm gel formed when the temperature was raised to 37°C. Reversion to a clear solution took place if the temperature was lowered to below 20°C within less than 2 h, but not if 37°C was maintained for several hours. The precipitated elastin‐like hydrogel thus formed did not visually redissolve at low temperatures. Tropoelastin hydrogel was stable to subsequent washings with alkaline solution at 37°C, but at 4°C some hydrogel redissolved showing that association is at least partly reversible. Washing the hydrogel with neutral 8M urea solution at 4°C dissolved less than 10% of tropoelastin in 24 h. We characterized this phenomenon by combining temperature‐controlled light microscopy analysis, ¹H NMR spectroscopy (temperature, diffusion, and relaxation time studies), and UV‐absorption‐based concentration measurements. The self‐association of tropoelastin at pH 11 is due to hydrophobic interactions in an emulsion‐like system in which the spherules coalesce in a manner like a water‐based latex paint that forms a durable hydrophobic sheet as water and the organic solvent evaporate. In the present case, the sedimentation and entanglement of the tropoelastin porous sheets means that reverse dissolution is a kinetically slow process. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 321–330, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Thermodynamically controlled supramolecular polymerization of cytochrome b562

A hemoprotein‐based supramolecular polymer that has a covalently linked heme moiety on the protein surface has been constructed based on interprotein heme–heme pocket interactions of the chemically modified apocytochrome b₅₆₂ ( 1 ‐H63C). The thermodynamic properties of the polymer have been investigated by means of size exclusion chromatography, UV–vis spectroscopy, and circular dichroism spectroscopy. The results indicate that, as with other synthetic systems reported so far, the 1 ‐H63C hemoprotein assembly is thermodynamically controlled in aqueous solution: the degree of polymerization is dependent on the 1 ‐H63C concentration and is modulated by the addition of the end‐capping units, native heme, and/or apocytochrome b₅₆₂ mutant (apoH63C). These properties suggest a potential use for the hemoprotein self‐assembly in preparation of stimuli‐responsive functional nanobiomaterials. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 194–200, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti‐inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin^50–59 sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well‐defined conformation in methanol, its structure was distinct from the corresponding 50–59 fragment in the native ubiquitin molecule. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 423–431, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Recent research has implicated the C‐terminus of G‐protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C‐tail has proven a formidable task. Here, a peptide corresponding to the full‐length C‐tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α‐helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self‐association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (¹⁵N‐ and ¹³C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐helical domains. The first domain, S401‐F412, corresponds to the helix 8 common to G protein‐coupled receptors while the second domain, A440‐M461, is a newly identified structural motif in the distal region of the carboxyl‐terminus of the receptor. Molecular modeling of the C‐tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 565–573, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Human tropoelastin sequence: Dynamics of polypeptide coded by exon 6 in solution

Calorimetric studies were performed on exon 6 in powdered form and in solution [water and 2,2,2‐trifluoroethanol (TFE), a structure‐inducing solvent or cosolvent]. Dynamic dielectric spectroscopy (DDS) analyses were realized in water and 20% TFE. The major role of solvent–peptide organization is evidenced with these techniques. Calorimetric measurements reveal the structural water organization around the polypeptide as well as the presence of hydrophobic interactions in TFE solution. Dielectric measurements showed for exon 6/water a decrease of relaxations times of bulk solvent implying a faster dynamics with a slight increase of the activation entropy, suggesting that exon 6 probably creates disorder within the solvent. For TFE/water mixtures, an influence of exon 6 on its environment was seen with a relaxation associated with the exon 6/solvent interactions reinforced by storage of 72 h. Finally, exon 6/solvent interactions were clearly observed with additionof TFE. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 943–952, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Specific DNA G‐quadruplexes bind to ethanolamines

A significant number of G‐quadruplex‐forming sequences have been revealed in human genome by bioinformatic searches, implying that G‐quadruplexes may be involved in important biological processes and may be new chemotherapeutic targets. Therefore, it is important to discover the potential interactions of G‐quadruplexes with other molecules or groups. Here we describe a class of G‐quadruplexes, which can bind to ethanolamine groups that widely exist in biomolecules and drug molecules. The specific interaction of these G‐quadruplexes with ethanolamine groups was identified by high performance affinity chromatography (HPAC) using immobilized ethanolamine and diethanolamine as stationary phase reagents. The circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE) show that these ethanolamine binding quadruplexes adopt an intramolecularly parallel structure. The relationship of ethanolamine binding and G‐quadruplexe structure provides new clues for the G‐quadruplex‐related studies as well as for the molecular designs of therapeutic reagents. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 874–883, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Glycerol‐induced folding of unstructured disulfide‐deficient lysozyme into a native‐like conformation

2SS[6‐127,64‐80] variant of lysozyme which has two disulfide bridges, Cys6‐Cys127 and Cys64‐Cys80, and lacks the other two disulfide bridges, Cys30‐Cys115 and Cys76‐Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native‐like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4°C. Then, ¹H‐¹⁵N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D₂O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A‐, B‐, and C‐helices and β3‐strand, and especially centered on Ile 55 and Leu 56. In 2SS[6‐127,64‐80], long‐range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 665–675, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Changes in the quaternary structure of amelogenin when adsorbed onto surfaces

Amelogenin is a unique protein that self‐assembles into spherical aggregates called “nanospheres” and is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin onto substrates is of great interest because protein‐surface interactions are critical to its function. We report studies of the adsorption of amelogenin onto self‐assembled monolayers containing COOH end group functionality as well as single crystal fluoroapatite, a biologically relevant surface. We found that although our solutions contained only nanospheres of narrow size distribution, smaller structures such as dimers or trimers were observed on the hydrophilic surfaces. This suggests that amelogenin can adsorb onto surfaces as small structures that “shed” or disassemble from the nanospheres that are present in solution. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 103–107, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effect of pH on molecular constitution and distribution of hemoglobin in living erythrocyte

The molecular constitution of in situ hemoglobin (Hb) and their distribution in living erythrocyte were investigated versus pH using the technique of confocal Raman microscopy. Both Raman point spectra and line mapping measurements were performed on living erythrocytes in suspensions with pH values from 4.82 to 9.70. It was found that the Hb inside a living erythrocyte would dissociate into monomer/dimer when the cells are in low and high pH environments. In contrast to the homogeneous distribution of the Hbs in the cells in neutral suspension, there are more Hbs distributing around the cell membrane or binding to the membrane as pH increases. While in low pH, as the cell become spherical, most of the Hbs distribute to the central part of the cell. In summary, our investigation suggests that the variation of the external pH not only brings changes in the morphology and membrane structure of an erythrocyte, but also affects the constitution and distribution of its intracellular Hbs, thereby the flexibility of the cell membrane and the oxygenation ability of the Hb. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 348–354, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Gel formation and low-temperature intramolecular conformation transition of a triple-helical polysaccharide lentinan in water

The gelation behavior of the triple-helical polysaccharide lentinan fractions having different molecular weights in water at 25 degrees C were studied by using a rheometer. The analysis of concentration and molecular weight dependence of shear stress and shear viscosity showed that aqueous lentinan is a typical shear-thinning fluid, possessing potential as a viscosity control agent, and that a weak gel with entangled network structure formed. The dynamic oscillatory behavior of lentinan in the temperature range of 1-15 degrees C was also investigated by rheologic method. The storage modulus G' and complex viscosity eta* increased first with decreasing temperature, and underwent a maximum centered at 7-9 degrees C, and then decreased with further decreasing temperature. This abnormal phenomenon was ascribed to formation of rigid structure in the gel state, which was confirmed by the experimental results from micro-DSC. The micro-DSC curves showed that an endothermic peak appeared at 7-9 degrees C for lentinan in water upon heating, which was attributable to the intramolecular order-disorder structure transition similar to triple-helical polysaccharide schizophyllan. Namely, at lower temperature, the side glucose residues of lentinan (triplix II) formed a well-organized triple-helical structure (triplix I) through hydrogen-bonding with the surrounding water molecules. Moreover, this conformation transition was proved to be thermally reversible. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 852-861, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Influence of chondroitin sulfate on collagen gel structure and mechanical properties at physiologically relevant levels

The ability to alter collagen organization could lead to more physiologically relevant scaffolds for tissue engineering. This study examined collagen organization in the presence of polysaccharide and the resulting effects on viscoelastic properties. Fibrillogenesis in the presence of chondroitin sulfate (CS) resulted in changes in the collagen network organization with an increase in void space present. The increased void space caused by CS addition correlated with a decreased stiffness of the collagen gel. These changes occurred with physiologically relevant ratios of collagen to CS, at physiological pH and ionic strength, and without a decrease in the amount of collagen incorporated into fibrils. The addition of dextran, an uncharged polysaccharide, yielded no change in network void space or mechanical properties. Changes in fibril diameter caused by CS or dextran were not correlated with mechanical properties. The results of this study demonstrate that collagen organization can be modified by the addition of GAG, leading to altered matrix mechanical properties. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 841-851, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

FtsZ condensates: An in vitro electron microscopy study

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto‐filaments. Although FtsZ assembles into single proto‐filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z‐ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 340–350, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Kinetics of the self‐aggregation and film formation of poly‐L‐proline at high temperatures explored by circular dichroism spectroscopy

Poly‐L ‐proline has been used as a model system for various purposes over a period of more than 60 years. Its relevance among the protein/peptide community stems from its use as a reference system for determining the conformational distributions of unfolded peptides and proteins, its use as a molecular ruler, and from the pivotal role of proline residues in conformational transitions and protein–protein interactions. While several studies indicate that polyproline can aggregate and precipitate in aqueous solution, a systematic study of the aggregation process is still outstanding. We found, by means of UV‐circular dichroism and IR measurements, that polyproline is predominately monomeric at room temperature at millimolar concentrations. Upon heating, the polypeptide stays in its monomeric state until the temperature reaches a threshold of ca. 60°C. At higher temperatures, the peptide aggregates as a film on the inside surface of the employed cuvette. The process proceeds on a time scale of 10³ s and can best be described by a bi‐exponential relaxation function. The respective CD and IR spectra are qualitatively different from the canonical spectra of polyproline in aqueous solution, and are indicative of a highly packed state. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 451–457, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Interaction of α‐gliadin with polyanions: Design considerations for sequestrants used in supportive treatment of celiac disease

Copolymers of sodium 4‐styrene sulfonate (SS) and hydroxyethyl methacrylate (HEMA) were investigated as sequestrants of α‐gliadin, a gluten protein, for the treatment of gluten intolerance. The interactions of α‐gliadin with poly(SS) and poly(HEMA‐co‐SS) with 9 and 26 mol% SS content were studied at gastric (1.2) and intestinal (6.8) pH using circular dichroism and measurements of turbidity, dynamic light scattering and zeta potential. The interactions and their influence on α‐gliadin secondary and aggregated structures depended mainly on the ratio of polymer negative and protein positive charges at pH 1.2, and on polymer SS content at polymer concentrations providing in excess of negative charges at either pH. Poly(SS) could not form complex particles with α‐gliadin in a sufficient excess of negative charges. Copolymerization with HEMA enhanced the formation of complex particles. Poly(HEMA‐co‐SS) with intermediate SS content was found to be the most effective sequestrant for α‐gliadin. This study provides insight into design considerations for polymer sequestrants used in the supportive treatment of celiac disease. © 2009 Wiley Periodicals, Inc. Biopolymers 93:418–428, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Chaperone‐like activities of different molecular forms of β‐casein. Importance of polarity of N‐terminal hydrophilic domain

As a member of intrinsically unstructured protein family, β‐casein (β‐CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone‐like activity in vitro. Like many chaperones, native β‐CN does not contain cysteinyl residues and exhibits strong tendencies for self‐association. The chaperone‐like activities of three recombinant β‐CNs wild type (WT) β‐CN, C4 β‐CN (with cysteinyl residue in position 4) and C208 β‐CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native β‐CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β‐CND) of C4‐β‐CN and C208 β‐CN were also studied and their chaperone‐like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β‐CN, C208 β‐CN, C4 β‐CN and C4 β‐CND exhibited significantly lower chaperone‐like activities than native β‐CN. Dimerization of C208 β‐CN with two distal hydrophilic domains considerably improved its chaperone‐like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N‐terminal hydrophilic domain as important functional elements in enhancing the chaperone‐like activity of native β‐CN. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 623–632, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Physicochemical studies on the biopolymer inulin: A critical evaluation of its self‐aggregation,aggregate‐morphology,interaction with water,and thermal stability

Physicochemical properties viz., aggregation, molar mass, shape, and size of chicory inulin in solution were determined by fluorimetry, DLS, SLS, TEM, and viscometry methods. The thermal stability of the biopolymer was examined by TGA, DTA, and DSC measurements. The water vapor adsorption of desiccated inulin was also studied by the isopiestic method, and the data were analyzed in the light of the BET equation. On the basis of the obstruction to ion conductance by the inulin aggregates in solution and analysis of the data, the extent of hydration of inulin in solution was estimated. The result was coupled with the intrinsic viscosity, [η], of inulin to ascertain the shape of the biopolymer aggregates in aqueous solution. The critical aggregation concentration (cac) of inulin in aqueous as well as in salt solution was assessed by fluorimetry. The weight average molar mass, , of inulin monomer and its aggregate was found to be 4468 and 1.03 × 10⁶ g/mol, respectively, in aqueous solution. This aggregated mass was 2.4 × 10⁶ g/mol in 0.5M NH₄SCN solution. The [η] values of the soft supramolecular aggregates in solution (without and with salt) were small and comparable with globular proteins evidencing spherical geometry of the biopolymer aggregates as supported by the TEM results. In DMSO, rod‐like aggregates of inulin was found by the TEM study. The [η] of the biopolymer in the DMSO medium was therefore, higher than that in the aqueous medium. Unlike aqueous medium, the aggregation in DMSO was not associated with a cac. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 687–699, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structure and conformational stability of a tetrameric thermostable N‐succinylamino acid racemase

The N‐succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co²⁺ by using hydrodynamic and spectroscopic techniques. The Co²⁺, among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by α‐helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten‐globule‐like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co²⁺ did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 757–772, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Synthesis of quadruplex‐forming tetra‐end‐linked oligonucleotides: Effects of the linker size on quadruplex topology and stability

G‐quadruplexes are characteristic structural arrangements of guanine‐rich DNA sequences that abound in regions with relevant biological significance. These structures are highly polymorphic differing in the number and polarity of the strands, loop composition, and conformation. Furthermore, the cation species present in solution strongly influence the topology of the G‐quadruplexes. Recently, we reported the synthesis and structural studies of new G‐quadruplex forming oligodeoxynucleotides (ODNs) in which the 3′‐ and/or the 5′‐ends of four ODN strands are linked together by a non‐nucleotidic tetra‐end‐linker (TEL). These TEL‐ODN analogs having the sequence TGGGGT are able to form parallel G‐quadruplexes characterized by a remarkable high thermal stability. We report here an investigation about the influence of the reduction of the TEL size on the molecularity, topology, and stability of the resulting TEL‐G‐quadruplexes using a combination of circular dichroism (CD), CD melting, ¹H NMR spectroscopy, gel electrophoresis, and molecular modeling data. We found that all TEL‐(TGGGGT)₄ analogs, regardless the TEL size and the structural orientation of the ODN branches, formed parallel TEL‐G‐quadruplexes. The molecular modeling studies appear to be consistent with the experimental CD and NMR data revealing that the G‐quadruplexes formed by TEL‐ODNs having the longer TEL (L 1 ‐ 4 ) are more stable than the corresponding G‐quadruplexes having the shorter TEL (S 1 ‐ 4 ). The relative stability of S 1 ‐ 4 was also reported. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 466–477, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com