Biophysical characterization and unfolding of LEF4 factor of RNA polymerase from AcNPV

Late expression factor 4 (LEF4) is one of the four subunits of Autographa californica nuclear polyhedrosis virus (AcNPV) RNA polymerase. LEF4 was overexpressed in Escherichia coli and recombinant protein was subjected to structural characterization. Chemical induced unfolding of LEF4 was investigated using intrinsic fluorescence, hydrophobic dye binding, fluorescence quenching, and circular dichroism (CD) techniques. The unfolding of LEF4 was found to be a non‐two state, biphasic transition. Intermediate states of LEF4 at 2M GnHCl and 4M urea shared some common structural features and hence may lie on the same pathway of protein folding. Steady‐state fluorescence and far‐UV CD showed that while there was considerable shift in the wavelength of emission maximum (λ_max), the secondary structure of LEF4 intermediates at 2M GnHCl and 4M urea remained intact. Further, temperature induced denaturation of LEF4 was monitored using far‐UV CD. This study points to the structural stability of LEF4 under the influence of denaturants like urea and temperature. Although LEF4 is an interesting model protein to study protein folding intermediates, in terms of functional significance the robust nature of this protein might reflect one of the several strategies adapted by the virus to survive under very adverse environmental and physiological conditions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 574–582, 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     

Hairpin ribozyme catalysis: A surface‐enhanced Raman spectroscopy study

The existence of an “RNA world” as an early step in the history of life increases the interest for the characterization of these biomolecules. The hairpin ribozyme studied here is a self‐cleaving/ligating motif found in the minus strand of the satellite RNA associated with Tobacco ringspot virus. Surface‐enhanced Raman spectroscopy (SERS) is a powerful tool to study trace amounts of RNA. In controlled conditions, a SERS signal is proportional to the amount of free residues adsorbed on the metal surface. On RNA cleavage, residues are unpaired and free to interact with metal. SERS procedures are used to monitor and quantify the catalysis of ribozyme cleavage at biological concentrations in real time; thus, they propose an interesting alternative to electrophoretic methods. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 384–390, 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     

Viral structural transition mechanisms revealed by multiscale molecular dynamics/order parameter extrapolation simulation

On the basis of an all‐atom multiscale analysis theory of nanosystem dynamics, a multiscale molecular dynamics/order parameter extrapolation (MD/OPX) approach has recently been developed. It accelerates MD for long‐time simulation of large bionanosystems and addresses rapid atomistic fluctuations and slowly varying coherent dynamics simultaneously. In this study, MD/OPX is optimized and implemented to simulate viral capsid structural transitions. Specifically, 200 ns MD/OPX simulation of the swollen state of cowpea chlorotic mottle virus capsid reveals that it undergoes significant energy‐driven shrinkage in vacuum, which is a symmetry‐breaking process involving local initiation and front propagation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 61–73, 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     

On the mechanism of SDS‐induced protein denaturation

To understand the mechanism of ionic detergent‐induced protein denaturation, this study examines the action of sodium dodecyl sulfate on ferrocytochrome c conformation under neutral and strongly alkaline conditions. Equilibrium and stopped‐flow kinetic results consistently suggest that tertiary structure unfolding in the submicellar and chain expansion in the micellar range of SDS concentrations are the two major and discrete events in the perturbation of protein structure. The nature of interaction between the detergent and the protein is predominantly hydrophobic in the submicellar and exclusively hydrophobic at micellar levels of SDS concentration. The observation that SDS also interacts with a highly denatured and negatively charged form of ferrocytochrome c suggests that the interaction is independent of structure, conformation, and ionization state of the protein. The expansion of the protein chain at micellar concentration of SDS is driven by coulombic repulsion between the protein‐bound micelles, and the micelles and anionic amino acid side chains. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 186–199, 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     

Probing nanostructures of bacterial extracellular polymeric substances versus culture time by Raman microspectroscopy and atomic force microscopy

The structure of a bacterial cell wall may alter during bacterial reproduction. Moreover, these cell wall variations, on a nanoscale resolution, have not yet fully been elucidated. In this work, Raman spectroscopy and atomic force microscopy (AFM) technique are applied to evaluate the culture time‐dependent cell wall structure variations of Pseudomonas putida KT2440 at a quorum and single cell level. The Raman spectra indicate that the appearance of DNA/RNA, protein, lipid, and carbohydrates occurs till 6 h of cultivation time under our experimental conditions. AFM characterization reveals the changes of the cellular surface ultrastructures over the culture time period, which is a gradual increase in surface roughness during the time between the first two and eight hours cultivation time. This work demonstrates the feasibility of utilizing a combined Raman spectroscopy and AFM technique to investigate the cultivation time dependence of bacterial cellular surface biopolymers at single cell level. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 171–177, 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     

Conformational stability of the full‐atom hexameric model of the ClpB chaperone from Escherichia coli

The Escherichia coli heat shock protein ClpB, a member of the Hsp100 family, plays a crucial role in cellular thermotolerance. In co‐operation with the Hsp70 chaperone system, it is able to solubilize proteins aggregated by heat shock conditions and refold them into the native state in an ATP‐dependent way. It was established that the mechanism of ClpB action depends on the formation of a ring‐shaped hexameric structure and the translocation of a protein substrate through an axial channel. The structural aspects of this process are not fully known. By means of homology modeling and protein–protein docking, we obtained a model of the hexameric arrangement of the full‐length ClpB protein complexed with ATP. A molecular dynamics simulation of this model was performed to assess its flexibility and conformational stability. The high mobility of the “linker” M‐domain, essential for the renaturing activity of ClpB, was demonstrated, and the size and shape of central channel were analyzed. In this model, we propose the coordinates for a loop between b4 and B6 structural elements, not defined in previous structural research, which faces the inside of the channel and may therefore play a role in substrate translocation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 47–60, 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     

Peptidic modulators of protein‐protein interactions: Progress and challenges in computational design

With the decline in productivity of drug‐development efforts, novel approaches to rational drug design are being introduced and developed. Naturally occurring and synthetic peptides are emerging as novel promising compounds that can specifically and efficiently modulate signaling pathways in vitro and in vivo. We describe sequence‐based approaches that use peptides to mimic proteins in order to inhibit the interaction of the mimicked protein with its partners. We then discuss a structure‐based approach, in which protein‐peptide complex structures are used to rationally design and optimize peptidic inhibitors. We survey flexible peptide docking techniques and discuss current challenges and future directions in the rational design of peptidic inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 505–513, 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     

Heating‐induced conformational change of a novel β‐(1→3)‐D‐glucan from Pleurotus geestanus

Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical–chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel β‐(1→3)‐D ‐glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical β‐(1→3)‐D ‐glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8–12.5°C and 25–60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro‐differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating‐induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating‐induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121–131, 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     

Sequence‐dependent folding and unfolding of ligand‐bound purine riboswitches

Riboswitch regulation of gene expression requires ligand‐mediated RNA folding. From the fluorescence lifetime distribution of bound 2‐aminopurine ligand, we resolve three RNA conformers (C_o, C_i, C_c) of the liganded G‐ and A‐sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg²⁺, together with results from mutagenesis, suggest that C_o and C_i are partially unfolded species compromised in key loop‐loop interactions present in the fully folded C_c. These data verify that the ligand‐bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand‐mediated folding of the G‐sensing riboswitch is more effective, less dependent on Mg²⁺, and less debilitated by mutation, than the A‐sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence‐dependent RNA dynamics, which adjust the balance of ligand‐mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine‐tuning of gene regulatory mechanisms. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 953–965, 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     

Uncover the conserved property underlying sequence‐distant and structure‐similar proteins

It is widely accepted that a protein's sequence determines its structure. The surprising finding that proteins of distant sequence can adopt similar 3D structures has raised interesting questions regarding underlying conserved properties that are essential for protein folding and stability. Uncovering the conserved properties may shed light on the folding mechanism of proteins and help with the development of computational tools for protein structure prediction. We compiled and analyzed a structure pair dataset of 66 high‐resolution and low sequence identity (16–38%) soluble proteins. Structure deviation for each pair was confirmed by calculating its C_α SiMax value and comparing its potential energy per residue. Analysis of favorable inter‐residue interactions for each structure pair indicated that the average number of inter‐residue interactions within each structure represents a conserved feature of homologous structures of distant sequence. Detailed comparison of individual types of interactions showed that the average number of either hydrophobic or hydrogen bonding interactions remains unchanged for each structure pair. These findings should be of help to improving the quality of homology models based on templates of low sequence identity, thus broadening the application of homology modeling techniques for protein studies. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 340–347, 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     

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     

Thermal stability and conformational structure of salmon calcitonin in the solid and liquid states

Salmon calcitonin (sCT) was selected as a model protein drug for investigating its intrinsic thermal stability and conformational structure in the solid and liquid states by using a Fourier transform infrared (FT‐IR) microspectroscopy with or without utilizing thermal analyzer. The spectral correlation coefficient (r) analysis between two second‐derivative IR spectra was applied to quantitatively estimate the structural similarity of sCT in the solid state before and after different treatments. The thermal FT‐IR microspectroscopic data clearly evidenced that sCT in the solid state was not effected by temperature and had a thermal reversible property during heating–cooling process. Moreover, the high r value of 0.973 or 0.988 also evidenced the structural similarity of solid‐state sCT samples before and after treatments. However, sCT in H₂O exhibited protein instability and thermal irreversibility after incubation at 40°C. The temperature‐induced conformational changes of sCT in H₂O was occurred to transform the α‐helix/random coil structures to β‐sheet structure and also resulted in the formation of intramolecular and intermolecular β‐sheet structures. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 200–207, 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     

Electrostatic interactions with histone tails may bend linker DNA in chromatin

Is linker DNA bent in the 30‐nm chromatin fiber at physiological conditions? We show here that electrostatic interactions between linker DNA and histone tails including salt condensation and release may bend linker DNA, thus affecting the higher order organization of chromatin. © 2005 Wiley Periodicals, Inc. Biopolymers 81: 20–28, 2006 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     

Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants

Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant‐robust broad‐specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS‐PAGE and N‐terminal sequencing. We observe well‐defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for α‐lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 221–231, 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     

Oligomerization of adenosin‐5′‐O‐ylmethylphosphonate,an isopolar AMP analogue: Evaluation of the route to short oligoadenylates

In an attempt to prepare a library of short oligoadenylate analogues featuring both the enzyme‐stable internucleotide linkage and the 5′‐O‐methylphosphonate moiety and thus obtain a pool of potential RNase L agonists/antagonists, we studied the spontaneous polycondensation of the adenosin‐5′‐O‐ylmethylphosphonic acid (p_cA), an isopolar AMP analogue, and its imidazolide derivatives employing N,N′‐dicyclohexylcarbodiimide under nonaqueous conditions and uranyl ions under aqueous conditions, respectively. The RP LC–MS analyses of the reaction mixtures per se, and those obtained after the periodate treatment, along with analyses and separations by capillary zone electrophoresis, allowed us to characterize major linear and cyclic oligoadenylates obtained. The structure of selected compounds was supported, after their isolation, by NMR spectroscopy. Ab initio calculation of the model structures simulating the AMP‐imidazolide and p_cA‐imidazolide offered the explanation why the latter compound exerted, in contrast to AMP‐imidazolide, a very low stability in aqueous solutions. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 277–289, 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     

Bioencapsulation of apomyoglobin in nanoporous organosilica sol–gel glasses: Influence of the siloxane network on the conformation and stability of a model protein

Nanoporous sol–gel glasses were used as host materials for the encapsulation of apomyoglobin, a model protein employed to probe in a rational manner the important factors that influence the protein conformation and stability in silica‐based materials. The transparent glasses were prepared from tetramethoxysilane (TMOS) and modified with a series of mono‐, di‐ and tri‐substituted alkoxysilanes, R_nSi(OCH₃)_4?n (R = methyl‐, n = 1; 2; 3) of different molar content (5, 10, 15%) to obtain the decrease of the siloxane linkage (? Si? O? Si? ). The conformation and thermal stability of apomyoglobin characterized by circular dichroism spectroscopy (CD) was related to the structure of the silica host matrix characterized by ²⁹Si MAS NMR and N₂ adsorption. We observed that the protein transits from an unfolded state in unmodified glass (TMOS) to a native‐like helical state in the organically modified glasses, but also that the secondary structure of the protein was enhanced by the decrease of the siloxane network with the methyl modification (n = 0 < n = 1 < n = 2 < n = 3; 0 < 5 < 10 < 15 mol %). In 15% trimethyl‐modified glass, the protein even reached a maximum molar helicity (?24,000 deg. cm² mol^?1) comparable to the stable folded heme‐bound holoprotein in solution. The protein conformation and stability induced by the change of its microlocal environment (surface hydration, crowding effects, microstructure of the host matrix) were discussed owing to this trend dependency. These results can have an important impact for the design of new efficient biomaterials (sensors or implanted devices) in which properly folded protein is necessary. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 895–906, 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     

A proposed model for dragline spider silk self‐assembly: Insights from the effect of the repetitive domain size on fiber properties

Dragline spider silk has been intensively studied for its superior qualities as a biomaterial. In previous studies, we made use of the baculovirus mediated expression system for the production of a recombinant Araneus diadematus spider silk dragline ADF4 protein and its self‐assembly into intricate fibers in host insect cells. In this study, our aim was to explore the function of the major repetitive domain of the dragline spider silk. Thus, we generated an array of synthetic proteins, each containing a different number of identical repeats up to the largest recombinantly expressed spider silk to date. Study of the self‐assembly properties of these proteins showed that depending on the increasing number of repeats they give rise to different assembly phenotypes, from a fully soluble protein to bona fide fibers with superior qualities. The different assembly forms, the corresponding chemical resistance properties obtained as well as ultrastructural studies, revealed novel insights concerning the structure and intermolecular interactions of the repetitive and nonrepetitive domains. Based on these observations and current knowledge in the field, we hereby present a comprehensive hypothetical model for the mechanism of dragline silk self‐assembly and fiber formation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 458–468, 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