Purification and properties of the cellular and scrapie hamster prion proteins

During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.     

The role of electrostatic interaction in triggering the unraveling of stable helix 1 in normal prion protein. A molecular dynamics simulation investigation

The conversion of normal prion protein (PrPC) into scrapie isoform (PrPSc) is a key event in the pathogenesis of prion diseases. However, the conversion mechanism has given rise to much controversy. For instance, there is much debate on the behavior of helix 1 (H1) in the conversion. A series of experiments demonstrated that H1 in isolated state was very stable under a variety of conditions. But, other experiments indicated that helices 2 and 3 rather than H1 were retained in PrPSc. In this paper, molecular dynamics (MD) simulation is employed to investigate the dynamic behavior of H1. It is revealed that although the helix 1 of Human PrPC (HuPrPC) is very stable in the isolated state, it becomes unstable when incorporated into native HuPrPC, which likely results from the long-range electrostatic interaction between Asp147 and Arg208 located in the helices 1 and 3, respectively. This explanation is supported by experimental evaluation and MD simulation on D147N mutant of HuPrPC that the mutant becomes a little more stable than the wild type HuPrPC. This finding not only help to reconcile the existing debate on the role of helix 1 in the PrPC-->PrPSc transition, but also reveals a possible mechanism for triggering the PrPC-->PrPSc conversion.     

Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 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 the cysteine‐rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF‐like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor‐ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand‐binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine‐rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28‐Ala70) by heteronuclear NMR, with a ¹³C‐/¹⁵N‐labeled sample. The tertiary structure of the CRD comprises a β‐sheet with two strands, followed by a 3₁₀ helix and a C‐terminal α‐helix, and is stabilized by three disulfide bonds connecting Cys36‐Cys49, Cys52‐Cys67, and Cys55‐Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1‐C2 module type), but not to the CRD of B‐cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1‐D2 module type). This is the first structural report about the A1‐C2 type CRD that could bind to the known target.     

Synthesis and conformational analysis of salivary proline‐rich peptide P‐B

The 57‐amino acid human salivary polypeptide P‐B has been synthesized by the solid‐phase method using 9‐fluorenylmethoxycarbonyl (Fmoc) strategy. The circular dichroism (CD) spectroscopy, Fourier‐transform infrared spectroscopy (FTIR) and molecular modeling methods have been used for conformational studies of P‐B. Examination of the CD spectra of P‐B showed the content of the secondary structure to be independent of temperature over the range 0–60 °C at pH = 7 as well as over the pH range of 2–12 at 37 °C. P‐B adopts predominantly unordered structure with locally appearing β‐turns. The cumulative results obtained using the CD and FTIR spectroscopic techniques indicate the percentage of the polyproline type‐II (PPII) helix being as low as about 10%. Similarly, the molecular dynamics (MD) simulations reveal only a short PPII helix in the C‐terminal fragment of the peptide (Pro⁵¹–Pro⁵⁴), which constitutes 7%. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring the free energy landscape of a model β‐hairpin peptide and its isoform

Secondary structural transitions from α‐helix to β‐sheet conformations are observed in several misfolding diseases including Alzheimer's and Parkinson's. Determining factors contributing favorably to the formation of each of these secondary structures is therefore essential to better understand these disease states. β‐hairpin peptides form basic components of anti‐parallel β‐sheets and are suitable model systems for characterizing the fundamental forces stabilizing β‐sheets in fibrillar structures. In this study, we explore the free energy landscape of the model β‐hairpin peptide GB1 and its E2 isoform that preferentially adopts α‐helical conformations at ambient conditions. Umbrella sampling simulations using all‐atom models and explicit solvent are performed over a large range of end‐to‐end distances. Our results show the strong preference of GB1 and the E2 isoform for β‐hairpin and α‐helical conformations, respectively, consistent with previous studies. We show that the unfolded states of GB1 are largely populated by misfolded β‐hairpin structures which differ from each other in the position of the β‐turn. We discuss the energetic factors contributing favorably to the formation of α‐helix and β‐hairpin conformations in these peptides and highlight the energetic role of hydrogen bonds and non‐bonded interactions. Proteins 2014; 82:2394–2402. © 2014 Wiley Periodicals, Inc.     

Early autoimmune destruction of islet grafts is associated with a restricted repertoire of IGRP-specific CD8+ T cells in diabetic nonobese diabetic mice

beta cell replacement via islet or pancreas transplantation is currently the only approach to cure type 1 diabetic patients. Recurrent beta cell autoimmunity is a critical factor contributing to graft rejection along with alloreactivity. However, the specificity and dynamics of recurrent beta cell autoimmunity remain largely undefined. Accordingly, we compared the repertoire of CD8+ T cells infiltrating grafted and endogenous islets in diabetic nonobese diabetic mice. In endogenous islets, CD8+ T cells specific for an islet-specific glucose-6-phosphatase catalytic subunit-related protein derived peptide (IGRP206-214) were the most prevalent T cells. Similar CD8+ T cells dominated the early graft infiltrate but were expanded 6-fold relative to endogenous islets. Single-cell analysis of the TCR alpha and beta chains showed restricted variable gene usage by IGRP206-214-specific CD8+ T cells that was shared between the graft and endogenous islets of individual mice. However, as islet graft infiltration progressed, the number of IGRP206-214-specific CD8+ T cells decreased despite stable numbers of CD8+ T cells. These results demonstrate that recurrent beta cell autoimmunity is characterized by recruitment to the grafts and expansion of already prevalent autoimmune T cell clonotypes residing in the endogenous islets. Furthermore, depletion of IGRP206-214-specific CD8+ T cells by peptide administration delayed islet graft survival, suggesting IGRP206-214-specific CD8+ T cells play a role early in islet graft rejection but are displaced with time by other specificities, perhaps by epitope spread.     

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

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

Temperature-jump kinetics of the dC-G-T-G-A-A-T-T-C-G-C-G double helix containing a G . T base pair and the dC-G-C-A-G-A-A-T-T-C-G-C-G double helix containing an extra adenine

The kinetics of helix formation were investigated using the temperature-jump technique for the following two molecules: dC-G-T-G-A-A-T-T-C-G-C-G, which forms a double helix containing a G·T base pair(the G·T 12-mer), and dC-G-C-A-G-A-A-T-T-C-G-C-G, which forms a double helix containing an extra adenine (the 13-mer). When data were analyzed in an all-or-none model, the activation energy for the helix association process was 22 ± 4 kcal/mol for the G·T 12-mer and 16 ± 7 kcal/mol for the 13-mer. The activation energy for the helix-dissociation process was 68 ± 2 kcal/mol for the G·T 12-mer and 74 ± 3 kcal/mol for the 13-mer. Rate constants for recombination were near 10⁵s^?1M^?1 in the temperature range from 32 to 47°C; for the dissociation process, the rate constants varied from 1s^?1 near 32°C to 130s^?1 near 47°C. Possible effects of hairpin loops and fraying ends on the above data are discussed.     

CD and NMR studies of prion protein (PrP) helix 1. Novel implications for its role in the PrPC-->PrPSc conversion process

The conversion of prion helix 1 from an alpha-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC-->PrPSc conversion.     

Slow spontaneous ��-to-�� structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences

The solid‐state conformations of two αγ hybrid peptides Boc‐[Aib‐γ⁴(R)Ile]₄‐OMe 1 and Boc‐[Aib‐γ⁴(R)Ile]₅‐OMe 2 are described. Peptides 1 and 2 adopt C₁₂‐helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C₁₂ motifs. The structure of peptide 2 reveals the formation of eight successive C₁₂ hydrogen‐bonded turns. Average backbone dihedral angles for αγ C₁₂ helices are peptide 1 , Aib; φ (°) = ?57.2 ± 0.8, ψ (°) = ?44.5 ± 4.7; γ⁴(R)Ile; φ (°) = ?127.3 ± 7.3, θ₁ (°) = 58.5 ± 12.1, θ₂ (°) = 67.6 ± 10.1, ψ (°) = ?126.2 ± 16.1; peptide 2 , Aib; φ (°) = ?58.8 ± 5.1, ψ (°) = ?40.3 ± 5.5; ψ⁴(R)Ile; φ (°) = ?123.9 ± 2.7, θ₁ (°) = 53.3 θ 4.9, θ ₂ (°) = 61.2 ± 1.6, ψ (°) = ?121.8 ± 5.1. The tendency of γ⁴‐substituted residues to adopt gauche–gauche conformations about the C^α–C^β and C^β–C^γ bonds facilitates helical folding. The αγ C₁₂ helix is a backbone expanded analog of α peptide 3₁₀ helix. The hydrogen bond parameters for α peptide 3₁₀ and α‐helices are compared with those for αγ hybrid C₁₂ helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.     

Propensities of peptides containing the Asn‐Gly segment to form β‐turn and β‐hairpin structures

The propensities of peptides that contain the Asn‐Gly segment to form β‐turn and β‐hairpin structures were explored using the density functional methods and the implicit solvation model in CH₂Cl₂ and water. The populations of preferred β‐turn structures varied depending on the sequence and solvent polarity. In solution, β‐hairpin structures with βI′ turn motifs were most preferred for the heptapeptides containing the Asn‐Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X‐ray structures. The sequence, H‐bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β‐hairpin structure of each heptapeptide, although the β‐turn segments played a role in promoting the formation of β‐hairpin structures and the β‐hairpin propensity varied. In the heptapeptides containing the Asn‐Gly segment, the β‐hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β‐turns and β‐hairpins containing the Asn‐Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β‐hairpin mimics and the design of binding epitopes for protein–protein and protein–nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653–664, 2016.     

Clustered negative charges on the lipid membrane surface induce beta-sheet formation of prion protein fragment 106-126

The conformational conversion of prion protein (PrP) from an alpha-helix-rich normal cellular isoform (PrPC) to a beta-sheet-rich pathogenic isoform (PrPSc) is a key event in the development of prion diseases, and it takes place in caveolae, cavelike invaginations of the plasma membrane. A peptide homologous to residues 106-126 of human PrP (PrP106-126) is known to share several properties with PrPSc, e.g., the capability to form a beta-sheet and toxicity against PrPC-expressing cells. PrP106-126 is thus expected to represent a segment of PrP that is involved in the formation of PrPSc. We have examined the effect of lipid membranes containing negatively charged ganglioside, an important component of caveolae, on the secondary structure of PrP106-126 by circular dichroism. The peptide forms an alpha-helical or a beta-sheet structure on the ganglioside-containing membranes. The beta-sheet content increases with an increase of the peptide:lipid ratio, indicating that the beta-sheet formation is linked with self-association of the positively charged peptide on the negatively charged membrane surface. Analogous beta-sheet formation is also induced by membranes composed of negatively charged and neutral glycerophospholipids with high and low melting temperatures, respectively, in which lateral phase separation and clustering of negatively charged lipids occur as shown by Raman spectroscopy. Since ganglioside-containing membranes also exhibit lateral phase separation, clustered negative charges are concluded to be responsible for the beta-sheet formation of PrP106-126. In caveolae, clustered ganglioside molecules are likely to interact with the residue 106-126 region of PrPC to promote the PrPC-to-PrPSc conversion.     

Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.     

Thermodynamic and kinetic parameters of an oligonucleotide hairpin helix

The thermodynamics of the hairpin helix-single strand transition of A₆C₆U₆ has been analyzed by a staggering zipper model with consideration of single strand stacking. This analysis yields an enthalpy change of +11 kcal/mole for the formation of a first, isolated base pair. The stability constant of a first (intramolecular) base pair in A₆C₆U₆ is around 2 × 1O^?5 at 25°C, whereas a first (intermoleciilar) base pair in an A₆ · U₆ helix is characterised by a stability constant of about 4 × 10^?3M^?1 (25°C, extrapolated from A_n · V_n oligomer measurements). These data indicate a destabilizing effect of the C₆ loop.The rate constant of hairpin helix formation is 2 to 3 × 10⁴ sec^?1 associated with an activation enthalpy of +2.5 kcal/mote. The rate of helix dissociation of the A₆C₆U₆ hairpin is in the range of 10³ to lO⁵ sec^?1 with an activation enthalpy of 21 $\text{kcal}\text{mole}$. A comparison with the kinetic parameters obtained for A · U oligomer helices shows a specific influence of the C₆ loop due to the stacking tendency of the cytosine residues. This intluence is preferentially reflected in the relatively low value of the rate constant of helix formation.     

Removal of the 5‐nitro‐2‐pyridine‐sulfenyl protecting group from selenocysteine and cysteine by ascorbolysis

We previously reported on a method for the facile removal of 4‐methoxybenzyl and acetamidomethyl protecting groups from cysteine (Cys) and selenocysteine (Sec) using 2,2′‐dithiobis‐5‐nitropyridine dissolved in trifluoroacetic acid, with or without thioanisole. The use of this reaction mixture removes the protecting group and replaces it with a 2‐thio(5‐nitropyridyl) (5‐Npys) group. This results in either a mixed selenosulfide bond or disulfide bond (depending on the use of Sec or Cys), which can subsequently be reduced by thiolysis. A major disadvantage of thiolysis is that excess thiol must be used to drive the reaction to completion and then removed before using the Cys‐containing or Sec‐containing peptide in further applications. Here, we report a further advancement of this method as we have found that ascorbate at pH 4.5 and 25 °C will reduce the selenosulfide to the selenol. Ascorbolysis of the mixed disulfide between Cys and 5‐Npys is much less efficient but can be accomplished at higher concentrations of ascorbate at pH 7 and 37 °C with extended reaction times. We envision that our improved method will allow for in situ reactions with alkylating agents and electrophiles without the need for further purification, as well as a number of other applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.