Insight into the stability of cross‐β amyloid fibril from molecular dynamics simulation

Amyloid fibrils are considered to play causal roles in the pathogenesis of amyloid‐related degenerative diseases such as Alzheimer's disease, type II diabetes mellitus, the transmissible spongiform encephalopathies, and prion disease. The mechanism of fibril formation is still hotly debated and remains an important open question. In this study, we utilized molecular dynamics (MD) simulation to analyze the stability of hexamer for eight class peptides. The MD results suggest that VEALYL and MVGGVV‐1 are the most stable ones, then SNQNNY, followed by LYQLEN, MVGGVV‐2, VQIVYK, SSTSAA, and GGVVIA. The statistics result indicates that hydrophobic residues play a key role in stabilizing the zipper interface. Single point and two linkage mutants of MVGGVV‐1 confirmed that both Met1 and Val2 are key hydrophobic residues. This is consistent with the statistics analysis. The stability results of oligomer for MVGGVV‐1 suggest that the intermediate state should be trimer (3‐0) and tetramer (2‐2). These methods can be used in stabilization study of other amyloid fibril. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 578–586, 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     

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     

Study on the inhibitory mechanism and binding mode of the hydroxycoumarin compound NSC158393 to HIV‐1 integrase by molecular modeling

Human immunodeficiency virus type 1 integrase (IN) is an essential enzyme in the life cycle of this virus and also an important target for the study of anti‐HIV drugs. In this work, the binding modes of the wild type IN core domain and the two mutants, that is, W132G and C130S, with the 4‐hydroxycoumarin compound NSC158393 were evaluated by using the “relaxed complex” molecular docking approach combined with molecular dynamics (MD) simulations. Based on the monomer MD simulations, both of the two substitutions affect not only the stability of the 128–136 peptides, but also the flexibility of the functional 140s loop. In principle, NSC158393 binds the 128–136 peptides of IN; however, the specific binding modes for the three systems are various. According to the binding mode of NSC158393 with WT, NSC158393 can effectively interfere with the stability of the IN dimer by causing a steric hindrance around the monomer interface. Additionally, through the comparative analysis of the MD trajectories of the wild type IN and the IN‐NSC158393 complex, we found that NSC15893 may also exert its inhibitory function by diminishing the mobility of the function loop of IN. Three key binding residues, that is, W131, K136, and G134, were discovered by energy decomposition calculated with the Molecular Mechanics Generalized Born Surface Area method. Characterized by the largest binding affinity, W131 is likely to be indispensable for the ligand binding. All the above results are consistent with experiment data, providing us some helpful information for understanding the mechanism of the coumarin‐based inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 700–709, 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 role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 373–383, 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 studies of SNARE complex and its interaction with complexin by molecular dynamics simulation

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four‐helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q‐SNARE complex instead of synaptobrevin to decrease the Q‐SNARE flexibility. The complexin alpha‐accessory helix and the C‐terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha‐accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha‐accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q‐SNARE complex. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 560–570, 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     

NMR structure of duplex DNA containing the α‐OH‐PdG·dA base pair: A mutagenic intermediate of acrolein

Acrolein, a cell metabolic product and main component of cigarette smoke, reacts with DNA generating α‐OH‐PdG lesions, which have the ability to pair with dATP during replication thereby causing G to T transversions. We describe the solution structure of an 11‐mer DNA duplex containing the mutagenic α‐OH‐PdG·dA base pair intermediate, as determined by solution nuclear magnetic resonance (NMR) spectroscopy and retrained molecular dynamics (MD) simulations. The NMR data support a mostly regular right‐handed helix that is only perturbed at its center by the presence of the lesion. Undamaged residues of the duplex are in anti orientation, forming standard Watson‐Crick base pairs alignments. Duplication of proton signals at and near the damaged base pair reveals the presence of two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. The α‐OH‐PdG adduct assumes a syn conformation pairing to its partner dA base that is protonated at pH 6.6. The three‐dimensional structure obtained by restrained molecular dynamics simulations show hydrogen bond interactions that stabilize α‐OH‐PdG in a syn conformation and across the lesion containing base pair. We discuss the implications of the structures for the mutagenic bypass of acrolein lesions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 391–401, 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     

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     

Structural and electrostatic characterization of Pariacoto virus: Implications for viral assembly

We present the first all‐atom model for the structure of a T = 3 virus, pariacoto virus (PaV), which is a nonenveloped, icosahedral RNA virus and a member of the Nodaviridae family. The model is an extension of the crystal structure, which reveals about 88% of the protein structure but only about 35% of the RNA structure. New modeling methods, combining coarse‐grained and all‐atom approaches, were required for developing the model. Evaluation of alternative models confirms our earlier observation that the polycationic N‐ and C‐terminal tails of the capsid proteins must penetrate deeply into the core of the virus, where they stabilize the structure by neutralizing a substantial fraction of the RNA charge. This leads us to propose a model for the assembly of small icosahedral RNA viruses: nonspecific binding of the protein tails to the RNA leads to a collapse of the complex, in a fashion reminiscent of DNA condensation. The globular protein domains are excluded from the condensed phase but are tethered to it, so they accumulate in a shell around the condensed phase, where their concentration is high enough to trigger oligomerization and formation of the mature virus. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 530–538, 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     

Potential allosteric modulators of the proteasome activity

Proteasome, consisting of a tube‐shaped proteolytic core particle and attached to it regulatory modules, is a multifunctional enzymatic complex essential for the ubiquitin‐proteasome metabolic pathway. Due to its immense involvement in regulation of cellular physiology, the proteasome is an acknowledged anticancer drug target and potential target to treat inflammatory or degenerative diseases. So far, competitive inhibitors of the core particle gain most consideration as drugs. We postulate that noncompetitively‐acting small‐molecule compounds would provide excellent means to precisely regulate actions of the proteasome. In this study, we evaluated five short peptides based on sequences of two proteins known to interact with the core proteasome: HIV‐1 Tat and PA28/REG activator. We performed Circular Dichroism (CD), Fourier Transformed Infrared Spectroscopy (FTIR), and Nuclear Magnetic Resonance (NMR) analysis, supplemented by MD simulations, and tested influence of the peptides on performance of the core particle active sites and functioning of regulatory modules. We found that PP2‐containing Tat peptides are noncompetitive inhibitors of the core, interfering with the actions of PA28αβ activator. In addition, at low concentrations the turn‐prone Tat2 is able to activate the latent core. The random coil‐structured PA28‐derived peptides display only weak or nondetectable direct effects on the core activities, exhibiting, however, a positive cooperation with activity‐enhancing actions of PA28αβ. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 481–495, 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     

Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage,a phosphodiester isostere

Modified internucleotide linkage featuring the C3′‐O‐P‐CH₂‐O‐C4″ phosphonate grouping as an isosteric alternative to the phosphodiester C3′‐O‐P‐O‐CH₂‐C4″ bond was studied in order to learn more on its stereochemical arrangement, which we showed earlier to be of prime importance for the properties of the respective oligonucleotide analogues. Two approaches were pursued: First, the attempt to prepare the model dinucleoside phosphonate with ¹³C‐labeled CH₂ group present in the modified internucleotide linkage that would allow for a more detailed evaluation of the linkage conformation by NMR spectroscopy. Second, the use of ab initio calculations along with molecular dynamics (MD) simulations in order to observe the most populated conformations and specify main structural elements governing the conformational preferences. To deal with the former aim, a novel synthesis of key labeled reagent (CH₃O)₂P(O)¹³CH₂OH for dimer preparation had to be elaborated using aqueous ¹³C‐formaldehyde. The results from both approaches were compared and found consistent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 514–529, 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     

Conformations and properties of the L‐tryptophyl‐containing peptides in solution,depending on the pH—Theoretical study vs. experiments

The conformational preference and electronic properties of three L ‐tryptophyl‐containing dipeptides, i.e., glycyl‐L ‐tryptophane (H‐Gly‐Trp‐OH), L ‐alanyl‐L ‐tryptophane (H‐Ala‐Trp‐OH), and L ‐methionyl‐L ‐tryptophane (L ‐Met‐Trp‐OH) in solution depending on the pH of the media are studied both theoretically and experimentally. The effect of the protonation of the COO^? and deprotonation of the NH as well as the alkaline hydrolysis of the amide fragment in a strong basic media on the electronic spectra are discussed. Ab initio and density functional theory (DFT) methods as well as the time‐dependent DFT (TD‐DFT) method as a function of the basis set are performed with a view to obtain the geometry and electronic properties of all of the species as well as the intermediate, obtained in the alkaline hydrolysis mechanism. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 727–734, 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     

Hsp70 molecular chaperones and Parkinson's disease

Because over expression of Hsp70 molecular chaperones suppresses the toxicity of aberrantly folded proteins that occur in Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and various polyQ‐diseases (Huntington's disease and ataxias), Hsp70 is garnering attention as a possible therapeutic agent for these various diseases. Here, I review progress in this fascinating field of molecular chaperones and neurodegeneration and describe our current understanding of the mechanisms by which Hsp70 protects cells from the PD‐related protein called alpha‐synuclein (α‐syn). © 2009 Wiley Periodicals, Inc. Biopolymers 93: 218–228, 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     

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     

Comparison of the internalization of targeted dendrimers and dendrimer‐entrapped gold nanoparticles into cancer cells

Dendrimer‐based nanotechnology significantly advances the area of targeted cancer imaging and therapy. Herein, we compared the difference of surface acetylated fluorescein isocyanate (FI) and folic acid (FA) modified generation 5 (G5) poly(amidoamine) dendrimers (G5.NHAc‐FI‐FA), and dendrimer‐entrapped gold nanoparticles with similar modifications ([(Au⁰)_51.2‐G5.NHAc‐FI‐FA]) in terms of their specific internalization to FA receptor (FAR)‐overexpressing cancer cells. Confocal microscopic studies show that both G5.NHAc‐FI‐FA and [(Au⁰)_51.2‐G5.NHAc‐FI‐FA] exhibit similar internalization kinetics regardless of the existence of Au nanoparticles (NPs). Molecular dynamics simulation of the two different nanostructures reveals that the surface area and the FA moiety distribution from the center of the geometry are slightly different. This slight difference may not be recognized by the FARs on the cell membrane, consequently leading to similar internalization kinetics. This study underlines the fact that metal or inorganic NPs entrapped within dendrimers interact with cells in a similar way to that of dendrimers lacking host NPs. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 936–942, 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     

Molecular dynamics simulations of xDNA

xDNA is a modified DNA, which contains natural as well as expanded bases. Expanded bases are generated by the addition of a benzene spacer to the natural bases. A set of AMBER force‐field parameters were derived for the expanded bases and the structural dynamics of the xDNA decamer ( xT5 ′ G xT A xC xG C xA xG T3′ ) · ( xA5′ C T xG C G xT A xC A3′) was explored using a 22 ns molecular dynamics simulation in explicit solvent. During the simulation, the duplex retained its Watson‐Crick base‐pairing and double helical structure, with deviations from the starting B‐form geometry towards A‐form; the deviations are mainly in the backbone torsion angles and in the helical parameters. The sugar pucker of the residues were distributed among a variety of modes; C2′ endo, C1′ exo, O4′ endo, C4′ exo, C2′ exo, and C3′ endo. The enhanced stacking interactions on account of the modification in the bases could help to retain the duplex nature of the helix with minor deviations from the ideal geometry. In our simulation, the xDNA showed a reduced minor groove width and an enlarged major groove width in comparison with the NMR structure. Both the grooves are larger than that of standard B‐DNA, but major groove width is larger than that of A‐DNA with almost equal minor groove width. The enlarged groove widths and the possibility of additional hydration in the grooves makes xDNA a potential molecule for various applications. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 351–360, 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     

Small‐molecule reductants inhibit multicatalytic activity of AA‐NADase from Agkistrodon acutus venom by reducing the disulfide‐bonds and Cu(II) of enzyme

AA‐NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA‐NADase contains Cu(II) and has disulfide‐bond linkages between two peptide chains. The effects of the reduction of the disulfide‐bonds and Cu(II) in AA‐NADase by small‐molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA‐NADase has six disulfide‐bonds and fifteen free cysteine residues. L‐ascorbate inhibits AA‐NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA‐NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2‐carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide‐bonds in AA‐NADase. Apo‐AA‐NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo‐AA‐NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide‐bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA‐NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L‐ascorbate are endogenous inhibitors to the multicatalytic activity of AA‐NADase. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 141–149, 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