Phosphorylation loops in synthetic peptides of the human neurofilament protein middle-sized subunit

Peptides containing 13 and 39 amino acid residues and serine-side-chain-phosphorylated (P) analogues thereof, corresponding to human neurofilament protein middle-sized subunit (NF-M), have been synthesized in order to localize the phosphorylation site of this protein. The secondary structure of the nonphosphorylated peptides, determined by circular dichroism (CD) measurements, predicted secondary structural calculations and energy conformational calculations, was suggested to be a series of alternating type I (III) -turns and 3₁₀ or -helices. By contrast, the phosphorylated peptides exhibit a unique conformation, probably due to salt bridges between the phosphoserine and the lysine residues. This has provided the first clear evidence that phosphorylation induces conformational changes among these synthetic peptides and presumably, in NF proteins as well. These phosphorylation loops might be the major recognition sites of the neurofilament protein-directed kinases.     

Biophysical characterization of longer forms of amyloid beta peptides: possible contribution to flocculent plaque formation

Alzheimer's disease is characterized by amyloid deposits in the parenchyma and vasculature of the brain. The plaques are mainly composed of amyloid beta (Abeta) peptides ending in residues 40 and 42. Novel longer Abeta peptides were found in brain homogenates of mouse models of Alzheimer's disease and human brain tissue of patients carrying the familial amyloid precursor protein V717F mutation. The biophysical characteristics of these longer Abeta peptides and their role in plaque formation are not understood. We chose to focus our studies on Abeta peptides ending in residues Ile45, Val46 and Ile47 as these peptides were identified in human brain tissue. A combination of circular dichroism and electron microscopy was used to characterize the secondary and tertiary structures of these peptides. All three longer Abeta peptides consisted mainly of a beta-sheet secondary structure. Electron microscopy demonstrated that these beta-structured peptides formed predominantly amorphous aggregates, which convert to amyloid fibres over extended time periods. As these longer peptides may act as seeds for the nucleation of fibrils composed predominantly of shorter amyloid peptides, these interactions were studied. All peptides accelerated the random to beta-structural transitions and fibril formation of Abeta40 and 42.     

Electronic and vibrational optical activity of several peptides related to neurohypophyseal hormones: disulfide group conformation

Electronic and vibrational optical activity of the set of neurohypophyseal hormones and their analogs was investigated to clarify the S-S bond solution conformation. The selected compounds include oxytocin (I), lysine vasopressin (II), arginine vasopressin (III), and their analogs (IV-IX), differing widely in their pharmacological properties. We have extended the already known electronic circular dichroism data by new information provided by vibrational circular dichroism (VCD) and Raman optical activity (ROA). The use of VCD brought additional details on three-dimensional structure of the chain reversal in the ring moiety and on its left handedness. Furthermore, Raman scattering and ROA allowed us to deduce the sense of the disulfide bond torsion.     

Synthesis and CD studies of an 88-residue peptide containing the main receptor binding site of HTLV-I SU-glycoprotein

Essential HTLV-I biological functions, like host-cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg¹⁴⁷-Leu²³⁴] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid-phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non-random conformation of at least 70–80% of the synthetic peptide. Investigation of the 3D-structure of the synthetic peptide will provide useful information for future vaccine and drug-design strategies. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci.3: 347–353 No. of Figures: 5. No. of Tables: 0. No. of References: 23     

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation.

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.     

310-Helices,Helix Screw Sense and Screw Sense Reversal in the Dehydro-peptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe

The pentapeptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe, containing two dehydro-phenylalanine (ΔPhe) residues, has been synthesized and its structure investigated. In the crystalline state, the molecule adopts a right-handed 3₁₀-helical conformation stabilized by two intramolecular hydrogen bonds between CO of Val¹ and NH of ΔPhe⁴, and between CO of ΔPhe² and NH of Val⁵, respectively. NMR measurements are consistent with the presence of 3₁₀-helical structures also in acetonitrile and dimethylsulphoxide solution: the distances between backbone protons estimated from NOE connectivities are in overall agreement with those observed in the solid state; the chemical shifts of the amide protons show the smaller temperature coefficients for the NHs that in solid state are involved in intramolecular hydrogen bonds. The CD spectra in acetonitrile, chloroform, methanol and dimethylsulphoxide display exciton couplets of bands corresponding to the ΔPhe electronic transition at 280nm; the sign of the bands is consistent with the presence of helical structures having a prevalent left-handed screw sense. Addition of 1,1,1,3,3,3-hexafluoro- propan-2-ol gives rise to the gradual appearance of a couplet of opposite sign, suggesting the helix reversal from left-handed sense to right-handed sense. The conformational behaviour is discussed on the basis of the specific sequence of the peptide.     

Binding of rationally designed non-natural peptides to the human leukocyte antigen HLA-B*2705

High-affinity ligands of non-peptidic nature, binding to the class I major histocompatibility complex protein HLA B*2705 whose expression is strongly linked to the pathogenesis of the autoimmune disease ankylosing spondylitis, should give way to a selective immunotherapy by blocking or antagonising the interaction with autoreactive T cell clones. Here we present experimental data on the binding of modified peptides, designed to optimally bind to HLA-B*2705 by filling a hydrophobic binding pocket (pocket D) with nonencoded aromatic amino acids. Three peptides with altered side chains (alpha-naphthylalanine, beta-naphthylalanine and homophenylalanine) in position 3 were synthesised. The thermal denaturation profiles of the HLA protein in complex with the modified peptides, monitored by circular dichroism spectroscopy, showed a significant shift towards higher melting temperatures with respect to the parent T cell epitope. The proposed binding mode of the nonnatural peptides was checked by site-directed mutagenesis of the pocket D, hypothesised to accommodate the large hydrophobic side chains. Reducing the size and depth of the pocket by mutating Leu l56 into Trp only affects the binding of the non-natural ligands, thus providing experimental evidence that the nonnatural peptide amino acids bind as predicted to the host MHC protein. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein.

Two equilibrium intermediates have previously been observed in the urea denaturation of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded beta/alpha barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth beta-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of betaalphabeta supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four betaalpha units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in alphaTS requires the participation of hydrophobic residues in multiple beta-strands that span the entire sequence.     

Synthesis and characterization of cyclic peptides that are β‐helical in trifluoroethanol

We show that three designed cyclic d ,l ‐peptides are β‐helical in TFE—a solvent in which the archetypal β‐helical peptide, gA, is unstructured. This result represents an advance in the field of β‐helical peptide foldamers and a step toward achieving β‐helical structure under a broad range of solvent conditions. We synthesized two of the three peptides examined using an improved variant of our original CBC strategy. Here, we began with a commercially available PEG–PS composite resin prefunctionalized with the alkanesulfonamide ‘SCL’ linker and preloaded with glycine. Our new conditions avoided C‐terminal epimerization during the CBC step and simplified purification. In addition, we present results to define the scope and limitations of our CBC strategy. These methods and observations will prove useful in designing additional cyclic β‐helical peptides for applications ranging from transmembrane ion channels to ligands for macromolecular targets. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Trichotoxin A-40, a new membrane-exciting peptide. Part A. Isolation,characterization and conformation

The new polypeptide antibiotic trichotoxin A-40 is isolated by chloroform/methanol extraction from the dry mycelium of Trichoderma viride NRRL 5242. The lipophilic peptide is purified by chromatography on Kieselgel H-60 and reverse-phase chromatography on Lichrosorb RP-8. The new antibiotic differs in amino acid composition and various chemical and physicochemical properties from similar peptides such as trichotoxin A, the suzukacillins or alamethicins. The amino acid composition is (Pro)₁ (Gly)₁ (Ala)₂ (Leu)₂ (Aib)₁₀ (Glx)₂. (Aib, α-aminoisobutyric acid.) The antibiotic has a carboxyl group which can be esterified by diazomethane, which results in slightly enhanced membrane-modifying activities.The peptide exhibits a right-handed α-helical conformation increasing about two-fold from aqueous to lipophilic media as shown by solvent-dependent circular dichroism measurements. Most of the ¹³C-NMR resonances can be assigned unequivocally and amino acids situated in the α-helical part show characteristic shift differences from those in the non-helical regions. No β-phenylalaninol residue could be identified by ¹³C-NMR and ultraviolet spectroscopy, as can be for alamethicins and suzukacillins. A pronounced hemolytic action is found on human erythrocytes, which develops at micromolar concentrations. Trichotoxin A-40 induces a voltage-dependent ionic conductance in bilayer lipid membranes and it can serve as a new pore-forming model system for structure/activity studies in membrane excitation by peptides.     

Delineation via site-directed mutagenesis of the carboxyl-terminal region of human choriogonadotropin beta required for subunit assembly and biological activity.

The choriogonadotropin beta subunit is unique in the human glycoprotein hormone family in containing a carboxyl-terminal extension, with four sites of O-glycosylation, that is not present in the other beta subunits. We have used site-directed mutagenesis to define boundaries on the extent to which truncations can be made at the COOH terminus without abolishing subunit assembly and biological activity. Two COOH-terminal deletion mutant chains of human choriogonadotropin beta, des(93-145) and des(101-145), were prepared and expressed in Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. The heterologous gonadotropin, bovine alpha-human choriogonadotropin des(101-145) beta, formed a heterodimer and, when assayed with transformed murine Leydig cells in vitro, competed with the binding of standard human choriogonadotropin and stimulated both cAMP and progesterone production, albeit with a reduced potency relative to bovine alpha-human choriogonadotropin beta wild type. In contrast, human choriogonadotropin des(93-145) beta, expressed under identical conditions in the presence of bovine alpha, failed to form heterodimer and thus exhibited no competitive binding and was without effect on cAMP and progesterone levels. Consequently, removal of the putative determinant loop region of the beta subunit (residues 93-100), which is believed to be important in determining receptor specificity, abolishes association with alpha. Hence, in addition to its possible role as a receptor determinant, this region of the molecule appears to be critical for proper folding or subunit interaction. The truncated form of human choriogonadotropin beta lacking residues 101-145 is the shortest form of the subunit yet described that retains biological activity. Moreover, these results demonstrate that the proposed disulfide between Cys-26 and Cys-110 is not required for subunit assembly or for receptor binding and subsequent intracellular signaling.     

Synthesis of new modified truncated peptides and inhibition of glycogen phosphorylase

The first solution state structural analysis (NMR) of the C‐terminal sequence of human G_L that binds to glycogen phosphorylase a (GPa), PEWPSYLGYEKLGPYY‐NH₂ ( 1 ), showed it to be in a random coil conformation. This was supported by molecular dynamics simulation (modelled in solution) using NAMD 2.6. The conformational ambiguity of the peptide makes the structural arrangement of the peptide (and internal residues) strongly dependent on the environment. Thirteen tetra‐peptide fragments of the C‐terminal sequence, YEKLG‐NH₂, and the corresponding tri‐ and di‐peptide sequences were used in a fragment screen against GPa. Compound 2 (H‐GPYY‐NH₂) did not give an IC₅₀ value, whereas PEWPSYLGYEKLGPYY‐NH₂ ( 1 ) displayed an IC₅₀ of 34 µM against GPa. Truncated peptides derived from 1 , (EKL‐NH₂, EKLG‐NH₂, and AcEKNH₂) inhibited GPa (21%, 32%, 63%, respectively at 22 mM ). These studies suggest key residues within the peptide chain have additional molecular interactions with GPa. The interaction of intra‐sequence residues in combination with the terminal residues of PEWPSYLGYEKLGPYY with GPa may form the basis for the design of new inhibitors of GPa. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

FtsZ polymer-bundling by the Escherichia coli ZapA orthologue, YgfE, involves a conformational change in bound GTP

Cell division is a fundamental process for both eukaryotic and prokaryotic cells. In bacteria, cell division is driven by a dynamic, ring-shaped, cytoskeletal element (the Z-ring) made up of polymers of the tubulin-like protein FtsZ. It is thought that lateral associations between FtsZ polymers are important for function of the Z-ring in vivo, and that these interactions are regulated by accessory cell division proteins such as ZipA, EzrA and ZapA. We demonstrate that the putative Escherichia coli ZapA orthologue, YgfE, exists in a dimer/tetramer equilibrium in solution, binds to FtsZ polymers, strongly promotes FtsZ polymer bundling and is a potent inhibitor of the FtsZ GTPase activity. We use linear dichroism, a technique that allows structure analysis of molecules within linear polymers, to reveal a specific conformational change in GTP bound to FtsZ polymers, upon bundling by YgfE. We show that the consequences of FtsZ polymer bundling by YgfE and divalent cations are very similar in terms of GTPase activity, bundle morphology and GTP orientation and therefore propose that this conformational change in bound GTP reveals a general mechanism of FtsZ bundling.     

Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli.

The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex.     

同步辐射圆二色谱及其在糖生物学及生物分子中的应用

同步辐射圆二色谱与普通圆二色谱相比,特点在于向真空紫外波段（〈200nm）拓展,以及同步辐射所提供的高强度紫外和真空紫外光源。糖的圆二色谱结构主要在200nm以下。蛋白质和核酸在200nm以下的真空紫外范围,也具有丰富的光谱结构。因此向真空紫外拓展,伴随新的电子跃迁,对应新的光谱结构,包含更丰富的结构信息,确定的结构种类就越多和越精确。同步辐射高强度的真空紫外光源,是获得高质量真空紫外圆二色谱数据的保证,为糖及糖蛋白、蛋白质和核酸研究提供了溶液中结构探测新的实验方法。综述同步辐射圆二色谱特点及其在结构生物学中的应用,以及新发展的蛋白质圆二色谱数据库（PCDDB）。介绍已对外开放的北京同步辐射实验室同步辐射圆二色谱探测,及其在蛋白质、糖和核酸研究中的应用,以及基于微流控混合芯片的亚毫秒动态探测发展。     

Studies of human transcortin at different pHs: circular dichroism, polymerisation and binding affinity.

The transcortin we have used in this work is extremely pure. This was shown by the polymerisation observed at pH 4. This polymerisation is never observed with an impure form of transcortin [4]. Moreover, since it is known that the presence of cortisol in the binding site is an essential condition to the activity of purified transcortin [5], it appears that a correlation between the secondary structure and the biological activity of the transcortin exists. The results we have obtained are summarized below: (1) The inhibition of the transcortin binding capacity essentially takes place between pH 5 and 4. (2) A reorganisation of the structure of the protein moiety is observed between pH 6.5 and 5.9. (3) A decrease of the helicity ratio is observed between pH 5 and 4. It appears therefore that, in the limits of experimental accuracy of CD measurements to determine the amount of beta-structure, no appreciable change of binding activity is taking place after the appearance of a large percentage of beta-structure between pH 6.5 and 6. On the other hand, the sudden decrease of protein activity at low pH is likely to be correlated with the disappearance of a well-defined helical region. Other biochemical and physical experiments would be of course necessary, in order to precise this first observation of a structure-function relationship in transcortin.     

Chiral resolution of alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene, a novel antiplatelet compound.

alpha,alpha'-Bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, a novel antiplatelet agent, was resolved into three isomers A, B, and C, on a chiral alpha 1-acid glycoprotein analytical column using a mobile phase of 0.025 M phosphate buffer containing 0.025 M tetrabutylammonium hydrogen sulfate, at a pH of 6.5. The effect of molarity, temperature, pH, flow rate, and organic modifiers on the enantioselectivity was examined. Based on circular dichroic spectra at 220 nm, A and C appear to be the (-)- and (+)-enantiomers, respectively, and B the meso diastereomer. Attempts at resolution using Pirkle type columns gave unsatisfactory results. It appears that both hydrophobic and polar interactions between the compound and the stationary phase are important determinants of resolution.     

Yeast-derived recombinant human insulin-like growth factor I: Production, purification, and structural characterization

Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted fromSaccharomyces cerevisiae using a yeast -factor leader to direct secretion. However, approximately 10–20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr²⁹ was the site of glycosylation. Site-directed mutagenesis was used to convert Thr²⁹ to Asn²⁹. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in -turns.     

Tightly bound 2-azido-adenine nucleotides at catalytic and noncatalytic sites of the rat liver F1 ATPase label adjacent tryptic peptides of the beta subunit

UV irradiation of rat liver F1 ATPase, previously exposed to Mg2+ and [beta, gamma-32P]-2-azido-ATP and separated from medium nucleotides, covalently modifies two tyrosine residues in adjacent tryptic peptides of the beta subunit. This results from the occupancy by 2-azido-ATP or 2-azido-ADP of two distinct types of nucleotide binding sites, the catalytic and noncatalytic sites. The two modified peptides are identical to the ones modified by 2-azido-adenine nucleotides in the beef heart F1 ATPase. Both catalytic and noncatalytic sites are labeled when the ATPase is exposed to [beta-32P]-2-azido-ADP in the presence or the absence of 5'-adenylyimidodiphosphate (AMP-PNP), showing that two distinct types of ADP binding sites are present on the liver enzyme. Similar incorporation of 2-azido-adenine nucleotides is obtained when membrane-bound rat liver F1 ATPase is incubated with Mg2+ and [beta, gamma-32P]-2-azido-ATP.