Formation Of Α-helical Structure Research Articles

Research on the secondary structure and hydration water around human serum albumin induced by ethanol with infrared and near-infrared spectroscopy

Given the role of ethanol played in the precipitation of various proteins, it is of great significance for the refined purification of proteins to understand the mechanism of protein secondary structure and hydration effect under the ethanol perturbation. Infrared (IR) spectroscopy and near-infrared (NIR) spectroscopy combined with chemometrics and aquaphotomics were utilized to get a generalized picture of the second structure of human serum albumin (HSA) and the surrounding hydrogen bond network of water after perturbation with different concentrations of ethanol ranging from 0% to 45% (v/v). The two-dimensional correlation (2DCOS) and moving window two-dimensional correlation analysis (MW-2DCOS) of IR spectroscopy provide evidence that the native structure of HSA was stabilized by the formation of α-helical structure at a low concentration of ethanol. However, the formation of intermolecular β-sheet structure by increasing ethanol is an important reason for the aggregation and precipitation of HSA. The analysis of NIR spectroscopy with the McCabe-Fisher method and aquaphotomics showed that free hydrogen bonded water was dynamic and fluctuating, 0%-20% ethanol could enhance the hydrogen bonded water cluster while the increasement of ethanol from 20% to 45% would lead to the formation of weak hydrogen bonded water cluster. These measurements provide a novel perspective on the relationship between ethanol and HSA, which is useful for maintaining activity and improving purity during the purification of proteins or other biological macromolecules.

Conserved Glycines Control Disorder and Function in the Cold-Regulated Protein, COR15A.

Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil–helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze–thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of α-helical structure and the increased α-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil–helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of α-helical structure during freezing induced dehydration.

Physicochemical and Structural Studies on Shaping of β-hairpin in Proteins as a First Stage of Amyloid Formation.

The aggregation of proteins or their digested fragments through β-sheet structures has a great significance because it leads to neurodegenerative diseases, which are a problem of the aging societies of the developed countries. Short peptides are typically used as models to study the formation of specific structures. However, while the formation of α-helical structure was investigated thoroughly, until recently, there have been much fewer studies on the formation of β-structure. In this review, recent experimental and theoretical studies of β-hairpin-forming peptides, both model alaninebased systems, and those based on the fragments of real proteins, are summarized with regard to the role of hydrophobic, local, and Coulombic interactions. It is demonstrated that the presence of charged residues can induce a bent structure not only owing to the formation of salt bridges if oppositely- charged residues present at the ends of a sequence but also through shielding the hydrophobic interior by like-charged residues at the end of the sequence.

Structure of RNA Stem Loop B from the Picornavirus Replication Platform.

The presumptive RNA cloverleaf at the start of the 5'-untranslated region of the picornavirus genome is an essential element in replication. Stem loop B (SLB) of the cloverleaf is a recognition site for the host polyC-binding protein, which initiates a switch from translation to replication. Here we present the solution structure of human rhinovirus isotype 14 SLB using nuclear magnetic resonance spectroscopy. SLB adopts a predominantly A-form helical structure. The stem contains five Watson-Crick base pairs and one wobble base pair and is capped by an eight-nucleotide loop. The wobble base pair introduces perturbations into the helical parameters but does not appear to introduce flexibility. However, the helix major groove appears to be accessible. Flexibility is seen throughout the loop and in the terminal nucleotides. The pyrimidine-rich region of the loop, the apparent recognition site for the polyC-binding protein, is the most disordered region of the structure.

Nucleotide contributions to the structural integrity and DNA replication initiation activity of noncoding y RNA.

Noncoding Y RNAs are small stem-loop RNAs that are involved in different cellular processes, including the regulation of DNA replication. An evolutionarily conserved small domain in the upper stem of vertebrate Y RNAs has an essential function for the initiation of chromosomal DNA replication. Here we provide a structure-function analysis of this essential RNA domain under physiological conditions. Solution state nuclear magnetic resonance and far-ultraviolet circular dichroism spectroscopy show that the upper stem domain of human Y1 RNA adopts a locally destabilized A-form helical structure involving eight Watson-Crick base pairs. Within this helix, two G:C base pairs are highly stable even at elevated temperatures and therefore may serve as clamps to maintain the local structure of the helix. These two stable G:C base pairs frame three unstable base pairs, which are located centrally between them. Systematic substitution mutagenesis results in a disruption of the ordered A-form helical structure and in the loss of DNA replication initiation activity, establishing a positive correlation between folding stability and function. Our data thus provide a structural basis for the evolutionary conservation of key nucleotides in this RNA domain that are essential for the functionality of noncoding Y RNAs during the initiation of DNA replication.

Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10.

A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [(13)C, (15)N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80% of the resonances of the (13)C-(1)H moieties at the 1', 2', 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY.

An In Vitro Selection Protocol for Threose Nucleic Acid (TNA) Using DNA Display.

Threose nucleic acid (TNA) is an unnatural genetic polymer composed of repeating threofuranosyl sugars linked by 2' and 3' phosphodiester bonds. TNA is capable of forming antiparallel Watson-Crick duplex structures in a self-pairing mode, and can also cross-pair opposite complementary strands of DNA and RNA. The solution NMR structure of a self-complementary TNA duplex reveals that TNA adopts an A-form helical structure, which explains its ability to exchange genetic information with natural genetic polymers. In a recent advance, a TNA aptamer was evolved from a pool of random sequences using an engineered polymerase that can copy DNA sequences into TNA. This unit details the steps required to evolve functional TNA molecules in the laboratory using a method called DNA display. Using this approach, TNA molecules are physically linked to their encoding double-stranded DNA template. By linking TNA phenotype with DNA genotype, one can select for TNA molecules with a desired function and recover their encoding genetic information by PCR amplification. Each round of selection requires ∼3 days to complete and multiple rounds of selection and amplification are required to generate functional TNA molecules.

Controlled Synthesis of Amino Acid-Based pH-Responsive Chiral Polymers and Self-Assembly of Their Block Copolymers

Leucine/isoleucine side chain polymers are of interest due to their hydrophobicity and reported role in the formation of α-helical structures. The synthesis and reversible addition-fragmentation chain transfer (RAFT) polymerization of amino acid-based chiral monomers, namely Boc-L-leucine methacryloyloxyethyl ester (Boc-L-Leu-HEMA, 1a), Boc-L-leucine acryloyloxyethyl ester (Boc-L-Leu-HEA, 1b), Boc-L-isoleucine methacryloyloxyethyl ester (Boc-L-Ile-HEMA, 1c), and Boc-L-isoleucine acryloyloxyethyl ester (Boc-L-Ile-HEA, 1d), are reported. The controlled nature of the polymerization of the said chiral monomers in N, N-dimethylformamide (DMF) at 70 °C is evident from the formation of narrow polydisperse polymers, the molecular weight controlled by the monomer/chain transfer agent (CTA) molar ratio and the linear relationship between molecular weight and monomer conversion. The resulting well-defined polymers were used as macro-CTAs to prepare corresponding diblock copolymers by RAFT polymerization of methyl (meth)acrylate monomers. Deprotection of Boc groups in the homopolymers and block copolymers under acidic conditions produced cationic, pH-responsive polymers with primary amine moieties at the side chains. The optical activity of the homopolymers and block copolymers were studied using circular dichroism (CD) spectroscopy and specific rotation measurements. The self-assembling nature of the block copolymers to produce highly ordered structures was illustrated through dynamic light scattering (DLS) and atomic force microscopy (AFM) studies. The side chain amine functionality instills pH-responsive behavior, which makes these cationic polymers attractive candidates for drug delivery applications, as well as for conjugation of biomolecules.

Steric Restrictions of RISC in RNA Interference Identified with Size-Expanded RNA Nucleobases

Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

Gene Silencing Activity of siRNA Molecules Containing Phosphorodithioate Substitutions

Chemically synthesized small interfering RNAs (siRNAs) have been widely used to identify gene function and hold great potential in providing a new class of therapeutics. Chemical modifications are desired for therapeutic applications to improve siRNA efficacy. Appropriately protected ribonucleoside-3'-yl S-[β-(benzoylmercapto)ethyl]pyrrolidino-thiophosphoramidite monomers were prepared for the synthesis of siRNA containing phosphorodithioate (PS2) substitutions in which the two non-bridging oxygen atoms are replaced by sulfur atoms. A series of siRNAs containing PS2 substitutions have been strategically designed, synthesized, and evaluated for their gene silencing activities. These PS2-siRNA duplexes exhibit an A-form helical structure similar to unmodified siRNA. The effect of PS2 substitutions on gene silencing activity is position-dependent, with certain PS2-siRNAs showing activity significantly higher than that of unmodified siRNA. The relative gene silencing activities of siRNAs containing either PS2 or phosphoromonothioate (PS) linkages at identical positions are variable and depend on the sites of modification. 5'-Phosphorylation of PS2-siRNAs has little or no effect on gene silencing activity. Incorporation of PS2 substitutions into siRNA duplexes increases their serum stability. These results offer preliminary evidence of the potential value of PS2-modified siRNAs.

Physicochemical Mechanism for the Enhanced Ability of Lipid Membrane Penetration of Polyarginine

Arginine-rich, cell-penetrating peptides (e.g., Tat-peptide, penetratin, and polyarginine) are used to carry therapeutic molecules such as oligonucleotides, DNA, peptides, and proteins across cell membranes. Two types of processes are being considered to cross the cell membranes: one is an endocytic pathway, and another is an energy-independent, nonendocytic pathway. However, the latter is still not known in detail. Here, we studied the effects of the chain length of polyarginine on its interaction with an anionic phospholipid large unilamellar vesicle (LUV) or a giant vesicle using poly-l-arginine composed of 69 (PLA69), 293 (PLA293), or 554 (PLA554) arginine residues, together with octaarginine (R8). ζ-potential measurements confirmed that polyarginine binds to LUV via electrostatic interactions. Circular dichroism analysis demonstrated that the transition from the random coil to the α-helix structure upon binding to LUV occurred for PLA293 and PLA554, whereas no structural change was observed for PLA69 and R8. Fluorescence studies using membrane probes revealed that the binding of polyarginine to LUV affects the hydration and packing of the membrane interface region, in which the degree of membrane insertion is greater for the longer polyarginine. Isothermal titration calorimetry measurements demonstrated that although the binding affinity (i.e., the Gibbs free energy of binding) per arginine residue is similar among all polyarginines the contribution of enthalpy to the energetics of binding of polyarginine increases with increasing polymer chain length. In addition, confocal laser scanning microscopy showed that all polyarginines penetrate across giant vesicle membranes, and the order of the amount of membrane penetration is R8 ≈ PLA69 < PLA293 ≈ PLA554. These results suggest that the formation of α-helical structure upon lipid binding drives the insertion of polyarginine into the membrane interior, which appears to enhance the membrane penetration of polyarginine.

A Coarse Grain RNA Model for Exploration of RNA Conformational Space

Several recent studies have suggested that RNA three-dimensional structure and dynamics are highly restricted to a small set of allowed conformations by topological constraints that are encoded at the secondary structure level. We have developed a coarse-grained model of RNA implemented within the CHARMM molecular dynamics package that allows us to further characterize the nature of RNA topological constraints. In this coarse grain model, each residue is represented using three pseudo-atoms for the phosphate, sugar, and base moieties respectively. Secondary structure is specified by modeling bonds between paired bases and parameterizing these regions to adopt A-form helical structure. All non-base paired residues are modeled without torsional potentials or attractive non-bonded forces, preserving only connectivity and repulsive steric terms. Thus, the energy landscape between different helical orientations is effectively flat, allowing efficient exploration of topologically allowed conformations. We benchmark our simulations using results from prior NMR and bioinformatics studies of two-way helix junctions. Moreover, simulations starting from a linear chain of the 76 residue tRNA-Phe molecule show that our model is able to sample the native conformation with minimal computational effort. We also show that the size of the conformational ensemble is reduced by over an order of magnitude when a limited set of three non-crystallographically determined tertiary contacts are used as restraints. In fact, the mean all phosphate RMSD over an ensemble of 100,000 structures has a value of 10 Å. We also present preliminary results of simulations done on RNAs with greater than 200 residues. These results suggest topological constraints alone, coupled with a few important tertiary contacts for larger RNAs, are enough to significantly constrain the available conformational ensemble and suggest a new approach to RNA structure prediction that is applicable to very large RNAs.

Raman and Raman optical activity (ROA) analysis of RNA structural motifs in Domain I of the EMCV IRES.

Raman and Raman optical activity (ROA) spectra were collected for four RNA oligonucleotides based on the EMCV IRES Domain I to assess the contributions of helix, GNRA tetraloop, U·C mismatch base pair and pyrimidine-rich bulge structures to each. Both Raman and ROA spectra show overall similarities for all oligonucleotides, reflecting the presence of the same base paired helical regions and GNRA tetraloop in each. Specific bands are sensitive to the effect of the mismatch and asymmetric bulge on the structure of the RNA. Raman band changes are observed that reflect the structural contexts of adenine residues, disruption of A-form helical structure, and incorporation of pyrimidine bases in non-helical regions. The ROA spectra are also sensitive to conformational mobility of ribose sugars, and verify a decrease in A-type helix content upon introduction of the pyrimidine-rich bulge. Several Raman and ROA bands also clearly show cooperative effects between the mismatch and pyrimidine-rich bulge motifs on the structure of the RNA. The complementary nature of Raman and ROA spectra provides detailed and highly sensitive information about the local environments of bases, and secondary and tertiary structures, and has the potential to yield spectral signatures for a wide range of RNA structural motifs.

Probing the Self-Assembly and the Accompanying Structural Changes of Hydrophobin SC3 on a Hydrophobic Surface by Mass Spectrometry

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate α-helical state to the stable end form called the β-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the α-helical state. The β-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39–S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of α-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the β-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme

Background: Phosphoramidate oligonucleotide analogs containing N3′–P5′ linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N–P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. Results: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3′–P5′ bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5′ splicing reaction. Conclusions: Ribo-N3′–P5′ phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein–RNA interactions.

Alcohol-induced versus anion-induced states of α-chymotrypsinogen A at low pH

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K 3FeCN 6, Na 2SO 4, KClO 4 and KCl) on the acid-induced state of α-chymotrypsinogen A, a predominantly β-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of α-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K 3FeCN 6>Na 2SO 4>KClO 4>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.

Equilibrium unfolding (folding) pathway of a model H-type pseudoknotted RNA: the role of magnesium ions in stability.

In T2 and related T-even bacteriophages, the upstream autoregulatory mRNA leader sequence of gene 32 folds into a simple tertiary structural motif, a hairpin (H)-type pseudoknot. This pseudoknot is derived from 32 contiguous nucleotides which form two coaxially stacked helical stems that adopt a pseudocontinuous A-form helical structure. These stems are connected by two nonequivalent single-stranded loops. The equilibrium unfolding pathway of a 36-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T2 gene 32 mRNA pseudoknot has been probed as a function of [Mg2+] by analysis of dual optical wavelength, equilibrium thermal melting profiles. A van't Hoff model based on multiple sequential, two-state unfolding transitions has been applied to the resultant data. Compensatory base pair substitutions incorporated into the helical stems have been used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is minimally described by three sequential unfolding transitions. The helix-helix junction region melts first in a low-enthalpy transition, followed by the unfolding of the remainder of helical stem 2, and then, all of stem 1. The total enthalpy of unfolding (folding) at [Mg2+] >/= 1 mM is accounted for by the secondary structure alone, suggesting that, if any non-Watson-Crick or tertiary structure exists in this conformation, it makes little or no enthalpic contribution to the stability of the molecule. Consistent with this, the [Mg2+] dependence of individual unfolding transitions within the pseudoknot is well-described by differential extents of delocalized binding of Mg2+ to folded and unfolded regions of the molecule. At a fixed [Mg2+], the helix junction region and stem 2 sequester more Mg2+ ions than the stem 1 hairpin; a larger fraction of these ions are then released upon unfolding. Two base or base pair substitution mutant RNAs which destabilize the helical junction and/or the base of stem 2 appear to sequester fewer ions, with a correspondingly smaller number of these ions released upon unfolding.

Binding properties of newly identified Xenopus proteins containing dsRNA-binding motifs

Although most RNA-binding proteins recognize a complex set of structural motifs in their RNA target, the double-stranded (ds) RNA-binding proteins are limited to interactions with double helices. Recently, it has been discovered that some dsRNA-binding proteins share regions of amino-acid similarity known as dsRNA-binding motifs. A Xenopus ovary cDNA expression library was screened with radiolabeled dsRNA to identify previously uncharacterized dsRNA-binding proteins. The analysis of an incomplete cDNA identified during the screen led to the discovery of two longer cDNAs of related sequence. The proteins encoded by these cDNAs each contained two dsRNA-binding motifs, in glycine. The nucleic-acid-binding properties of a fusion protein containing the two dsRNA-binding motifs and the auxiliary domain were analyzed using a gel mobility shift assay. The fusion protein bound dsRNA of a variety of different sequences, and exhibited a preference for binding to dsRNA and RNA-DNA hybrids over other nucleic acids. Appropriate mRNAs, corresponding to each cDNA, were detected in polyadenylated RNA isolated from Xenopus stage VI oocytes, but translation of one of the mRNAs appeared to be masked until meiotic maturation. dsRNA-binding motifs are often found in proteins that bind dsRNA, and our results show that they can be associated with auxiliary domains rich in arginine and glycine. These motifs can confer very tight binding to dsRNA. Binding can also occur to RNA-DNA hybrids, suggesting recognition of some aspect of the A-form helical structure that is adopted by both dsRNA and RNA-DNA hybrids.

Formation of α-Helix 88-98 is Essential in the Establishment of Higher-order Structure from Reduced Lysozyme

Lys96 and Lys97 in lysozyme are located at the C terminus of α-helix 88-98. The positive charges of these residues are supposed to stabilize the helical structure, and these residues are conserved as the basic amino acids among c-type lysozymes. The renaturation rate of reduced mutant lysozyme, where both Lys96 and Lys97 were mutated together to Ala, was slower than that of native lysozyme at pH 8·0 and 37°C by SH-SS interchange reactions. In order to investigate the reason, the peptide fragment 36-105 (where we can obtain information of the interaction between helix 88-98 and Trp62 and Trp63 residues) was prepared. CD spectra were compared between peptide fragment 36-105 and its acetylated form, where the positive charges of Lys96 and Lys97 were eliminated, and it was elucidated that the displacement of positive charges at the C terminus of the helix caused the shift of the advantageous structure of the fragment from α-helix to coil. Moreover, we obtained evidence that there was interaction of the helix with Trp62 and/or Tro63, which maintained a thermodynamically stable higher-order structure. Therefore, these results suggest that the formation of α-helical structure in 88-98 is a significant factor in the establishment of native structure from reduced lysozyme.

Ribonucleotide-induced helical alteration in DNA prevents nucleosome formation.

Several polynucleotides that assume an A-form helical structure in solution are unable to form nucleosomes. We attempted to establish a relationship between the ease of the A-form----B-form helix transition and ease of nucleosome formation by reconstituting nucleosomes using ribosubstituted DNA containing various levels of ribonucleotides. Instead we discovered that, when riboadenosine is substituted for deoxyriboadenosine, even one ribonucleotide per 125 base pairs of DNA reduces nucleosome formation and that DNA containing greater than 5% ribonucleotide is completely unable to form nucleosomes. Ribosubstituted DNA restriction fragments exhibited altered mobility on native 6% polyacrylamide gels, indicating an altered helical structure (probably bending). The effects on both nucleosome formation and gel mobility are nucleotide specific and are correlated, being greatest for riboadenosine and decreasing in the order riboadenosine greater than riboguanosine greater than ribocytosine. The results are consistent with the hypothesis that the rate of nucleosome formation can be drastically reduced by isolated local perturbations, such as kinking or bending, in the helical structure of DNA.