Peptide models for inherited neurodegenerative disorders: conformation and aggregation properties of long polyglutamine peptides with and without interruptions.

Several neurodegenerative diseases are caused by expansion of polyglutamine repeats in the affected proteins. In spino-cerebellar ataxia type 1 (SCA1), histidine interruptions have been reported to mitigate the pathological effects of long glutamine stretches. To understand this phenomenon, we investigated the conformational preferences of peptides containing both the uninterrupted polyglutamine stretches and those with histidine interruption(s) as seen in SCA1 normals. Our study suggests that substitution of histidines by glutamines induces a conformational change which results in decreased solubility and increased aggregation. Our findings also suggest that all the polyglutamine peptides with and without interruption(s) adopt a beta-structure and not random coil.     

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.     

Conformational change of poly(L-lysine) induced by lipid vesicles of dilauroylphosphatidic acid

The effect of negatively charged dilauroylphosphatidic acid (DLPA) vesicles on the conformation of poly(L-lysine) was investigated by circular dichroism measurements. DLPA vesicles induced a conformational change of poly(L-lysine) from the random coil to beta-structure in 5 mM Tes, pH 7.0. The fraction of induced beta-structure (F beta) was determined via a procedure of curve fitting of the observed spectra to the reference spectra. F beta increased linearly with the molar ratio, r, of DLPA to lysine residues up to r congruent to 0.7, and reached a saturation value of 1 at r greater than 1. Within the range 0.7 less than or equal to r less than or equal to 1, precipitation occurred. The effect of dilution of the negative charge on vesicle membranes was examined by mixing DLPA with dilauroylphosphatidylcholine (DLPC). Although the beta-structure of poly(L-lysine) was also induced by mixed vesicles, the saturation value of F beta decreased with decreasing DLPA content in mixed vesicles. The variation in saturation value of F beta with the composition of mixed vesicles was interpreted in terms of the change in average distance between DLPA head groups in mixed vesicles.     

Secondary structural changes of non-reduced and reduced ribonuclease A in solutions of urea, guanidine hydrochloride and sodium dodecyl sulfate

The secondary structures of ribonuclease A (RNAase A) before and after reduction of the disulfide bridges and blockage of the thiol groups with iodoacetamide were examined in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate (SDS). The relative proportions of alpha-helix, beta-structure, and disordered structure were estimated by the curve-fitting method of circular dichroism (Chen, Y.H., Yang, J.T. and Chau, K.H. (1974) Biochemistry 13, 3350-3359). The native RNAase A, with the disulfide bridges intact, contained 19% helix and 38% beta-structure. Reduction of its disulfide bridges led to a decrease in the proportion of these structures to 9% for the alpha-helix and 17% for the beta-structure. The non-reduced RNAase A resisted unfolding in low concentrations of urea and guanidine hydrochloride. The beta-structure which remained after reduction appeared to be stable even in solutions of 6 M guanidine and 9 M urea. A considerable amount of the beta-structure in both the non-reduced and the reduced RNAase A remained unaffected by high concentrations of SDS.     

Occurrence of D-histidine residues in antimicrobial poly(arginyl-histidine), conferring resistance to enzymatic hydrolysis

The antimicrobial peptide poly(arginyl-histidine) is secreted by the ergot fungus Verticillium kibiense. We previously showed that poly(arginyl-histidine) from the fungus inhibits the growth of certain microorganisms more effectively than that chemically synthesized from the L-form of arginine and histidine, implying some substantial differences between the fungal and synthetic peptides. To elucidate what causes such differences, we here investigated the structural features of the fungal peptides. The acid hydrolysates of the fungal peptide contained d-histidine. When synthetic poly(L-arginyl-D-histidine) mimicking the fungal peptide was added to the culture of Salmonella typhimurium together with poly(L-arginyl-L-histidine), poly(L-arginyl-D-histidine) was not easily degraded during the incubation compared with poly(L-arginyl-L-histidine). We concluded that the d-form of histidine residues in the fungal peptide prolongs the life of the peptide leading to the enhancement of antimicrobial activity.     

Changes in the secondary structure of apolipoprotein B-100 after Cu2+-catalysed oxidation of human low-density lipoproteins monitored by Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectra have been obtained of human low-density lipoproteins (LDL) in H2O and 2H2O buffers. The absorption bands are assigned to vibrations of the lipid and apolipoprotein B-100 components. The analysis of second-derivative spectra allowed an assignment of individual protein bands to alpha-helical, random, coil or beta-structure and beta-turn conformations. Changes in the FTIR spectra after Cu2+-catalysed oxidation of the LDL particles indicate that the structure of apolipoprotein B-100 becomes less ordered, with some alterations of alpha-helical and beta-turn conformation. The main beta-structure absorption at 1620 cm-1 is unaffected by oxidation. Taking into account the resistance to oxidation and the slow H-2H exchange it is suggested that the beta-structure is hidden from external factors whereas other structures are mostly present on the surface of the LDL particle. Oxidation affects mainly the surface region of apolipoprotein B-100 and leads to a structural rearrangement which consequently changes the receptor specificity of the LDL.     

Thermodynamics of the coil <==> beta-sheet transition in a membrane environment

Biologically important peptides such as the Alzheimer peptide Abeta(1-40) display a reversible random coil <==>beta-structure transition at anionic membrane surfaces. In contrast to the well-studied random coil left arrow over right arrow alpha-helix transition of amphipathic peptides, there is a dearth on information on the thermodynamic and kinetic parameters of the random coil left arrow over right arrow beta-structure transition. Here, we present a new method to quantitatively analyze the thermodynamic parameters of the membrane-induced beta-structure formation. We have used the model peptide (KIGAKI)(3) and eight analogues in which two adjacent amino acids were substituted by their d-enantiomers. The positions of the d,d pairs were shifted systematically along the three identical segments of the peptide chain. The beta-structure content of the peptides was measured in solution and when bound to anionic lipid membranes with circular dichroism spectroscopy. The thermodynamic binding parameters were determined with isothermal titration calorimetry and the binding isotherms were analysed by combining a surface partition equilibrium with the Gouy-Chapman theory. The thermodynamic parameters were found to be linearly correlated with the extent of beta-structure formation. beta-Structure formation at the membrane surface is characterized by an enthalpy change of DeltaH(beta)=-0.23 kcal/mol per residue, an entropy change of DeltaS(beta)=-0.24 cal/mol K residue and a free energy change of DeltaG(beta)=-0.15 kcal/mol residue. An increase in temperature induces an unfolding of beta-structure. The residual free energy of membrane-induced beta-structure formation is close to that of membrane-induced alpha-helix formation.     

Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.     

Secondary structure of charge isomers of myelin basic protein before and after phosphorylation

Human myelin basic protein (MBP) was fractionated into several of its charge isomers (components). Of these, the secondary structures of four isomers before and after phosphorylation have been studied by circular dichroism (CD). None of the four showed any alpha-helical structure. All of the components showed varying amounts of beta-structure, random structure, and turns. Component 1 (C-1), the most cationic of the components, showed 13%; component 2 (C-2) had 19%; C-3, 17%; and C-4, 24% of beta-structure. Each of the four components was phosphorylated with protein kinase C, from human brain. The extent of phosphorylation varied considerably from 2.8 +/- 0.6 mol of PO4/mol of protein in C-1 to 5.2 +/- 0.8 mol of PO4/mol of protein in C-4. The effect of phosphorylation on the secondary structure was to induce beta-structure in all the components. The largest change in beta-structure was in C-1 and the least in C-4. The surprising result is that although the components were phosphorylated to different extents, the amount of beta-structure in all four components increased to a final proportion of 35-40%. Treatment of phosphorylated C-1 with acid phosphatase removed 50% of the total radioactivity. Although the remainder represented approximately 1 mol of PO4/mol of protein, the proportion of beta-structure was unaltered. We concluded that a single phosphorylation site identified as residues 5-13 represented a critical size for stabilization of beta-structure of MBP in solution and that phosphorylation at the other sites had little influence on secondary structure.     

Oligomerization and structural changes of the pore-forming Pseudomonas aeruginosa cytotoxin.

Pseudomonas aeruginosa produces a pathogenic factor, the 29-kDa pore-forming protein cytotoxin. Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on intermolecular disulfide bridges. SDS induced tetramer, hexamer and mainly pentamers that were resistant to reducing conditions, indicating an additional oligomerization mechanism. Functional oligomerization after incubation with different membranes resulted in an oligomer of approximately 145 kDa that was identified as the pentamer by comparison with the SDS-induced oligomers. Covalent modification with diethylpyrocarbonate showed that histidine residues are indispensable for functional pentamerization. Pentamer formation was not influenced by the lipid composition of the liposomes tested, indicating that rising membrane fluidity did not increase oligomerization. The secondary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% beta-sheet, 20% beta-turn, 10% alpha-helix and 20% remaining structure. Contact with detergent micellae or liposomes induced a reorganization of beta-structure associations, as observed by attenuated total reflection-Fourier transform infrared spectroscopy. Electron microscopy and principle component analysis of the cytotoxin monomer demonstrated a tapered molecule of 11 nm in length and a maximum width of 3.5 nm. These results classify the cytotoxin as a pore-forming toxin, rich in antiparallel beta-structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureus alpha-toxin.     

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.     

Crystal structures of [Met5] and [(4-bromo)Phe4,Met5]enkephalins: formation of a dimeric antiparallel beta-structure

The crystal structure of [(4-bromo)Phe4,Met5]enkephalin (Tyr-Gly-Gly-(4-bromo)-Phe-Met) shows two independent molecular conformations. The molecules are arranged in parallel in a head-to-tail fashion and form an antiparallel beta-sheet structure involving intermolecular hydrogen bonds. This dimeric beta-structure is also observed in the [Met5]enkephalin crystal, in spite of their different crystal packing environments, which shows the energetic stability of this molecular conformation. The three-dimensional similarity between the dimeric beta-structure and the beta-turn form is discussed in the relation to the opioid delta and mu receptors.     

Intramolecular alpha-helix-beta-structure-random coil transition in polypeptides. I. Equilibrium case

The present paper is devoted to the study of the conformational transition of polypeptides which are capable of forming alpha-helix, beta-structure and random coil conformations with the finite homogeneous chain model. The experimental results on the surfactant-induced conformational change of poly(L-lysine) can be well described by the present model assuming cooperative binding of the surfactant ions to the polypeptide side groups.     

Low-ultraviolet circular dichroism spectroscopy of sequential peptides 1-63, 64-95, 96-128, and 129-168 derived from myelin basic protein of rabbit

Four sequential peptides (sequences 1-63, 64-95, 96-128, and 129-168) derived from rabbit myelin basic protein by thrombic cleavage were examined by low-ultraviolet circular dichroism spectroscopy in 0.5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH approximately 7.2) containing 0-92% trifluoroethanol (TFE). In the absence of the alcohol, all of the peptides contained a significant amount (17-29%) of beta-structure. In the presence of relatively low concentrations (up to 30%) of TFE, all of the peptides except 96-128 adopted considerable alpha-helix (16-33%). This involved a transition from the beta-structure in peptide 1-63 and transitions from the nonordered structure in peptides 1-63, 64-95, and 129-168. Furthermore, additional alpha-helix formed in peptide 1-63 between 30% and 92% TFE at the expense of nonordered structure, whereas the alpha-helix formation above 50% TFE in peptide 129-168 resulted largely from a beta-structure----alpha-helix transition. With the exception of the 129-168 peptide, approximately 65-100% of the maximum level of beta-structure persisted throughout the entire range of TFE concentration. In the case of peptide 129-168, however, most of the beta-structure was converted to alpha-helix and nonordered structure at 75% TFE. While the present results support our previous assignments of beta-structure- and alpha-helix-forming regions to specific amino acid sequences of the basic protein, they also demonstrate that the beta-structure----alpha-helix transitions evidenced at various concentrations of TFE were influenced to a considerable degree by the length of the peptide, presumably due to the presence or absence of interactions between noncontiguous portions of the myelin basic protein polypeptide chain.     

Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules

A (poly)histidine tag was fused to either the N- or the C-terminus of L-lactate dehydrogenase (LDH) of Bacillus stearothermophilus to facilitate purification and immobilization of these enzymes. The C-terminally tagged enzyme displayed lower activity compared both to the wild-type and to the N-terminally tagged variant. The reason for this loss of activity was investigated by affinity chromatography of the enzymes on a 5'-AMP-Sepharose resin and by size-exclusion chromatography. The C-terminally tagged enzyme could be separated into an inactive, unbound fraction and an active, bound fraction. Further differences between the C-terminally tagged enzyme and the N-terminally tagged and wild-type LDH were observed on size-exclusion chromatography of the three enzymes. These data suggest that the introduction of a "his-tag" at the C-terminus may induce misfolding of the LDH and serve as a warning that the introduction of a (poly)histidine tag can produce unforseen changes in a protein.     

Methylated poly(L-lysine): conformational effects and interactions with polynucleotides

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of epsilon-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally-induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO4)2. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from beta, to 50% alpha, to random coil at the maximum methylation. Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)n.(dAdT)n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)n.(dC)n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative psi-spectra are induced, which, in the case of (dGdC)n.(dGdC)n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration. It is suggested that the biological effects of methylation proteins may be evoke by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

The histidine kinase CpHK2 has impact on spore germination, oxidative stress and fungicide resistance, and virulence of the ergot fungus Claviceps purpurea

Histidine kinases are important mediators for adaptation of bacteria and plants to environmental signals. Genome analyses of filamentous fungi have revealed the presence of a high number of potential hybrid histidine kinase (HK)-encoding genes; the role of most of these potential sensors is so far unclear, though some members of the class III histidine kinases were shown to be involved in osmostress responses. Here we present a functional analysis of cphk2 , a histidine kinase-encoding gene in the biotrophic grass pathogen Claviceps purpurea . The putative product of cphk2 (CpHK2) was shown to group within family X of fungal HKs and it had high homology to the oxidative stress sensors SpMAK2/3 of Schizosaccharomyces pombe . Analysis of a cphk2 deletion mutant indicated that this histidine kinase is involved in spore germination, sensitivity to oxidative stress and fungicide resistance. In addition, virulence of the D cphk2 mutant on rye was significantly reduced compared with the wild-type strain, even if the conidial titre was adjusted to the lower germination rate. This is the first report of a role for a class X histidine kinase in a filamentous fungus.     

Low-ultraviolet circular dichroism spectroscopy of oligopeptides 1-95 and 96-168 derived from myelin basic protein of rabbit

Myelin basic protein (MBP) is a major protein constituent of the myelin sheath of the central nervous system, where it is believed to have functional alpha-helical segments. One element of the function of the protein might be "conformational adaptability" of specific regions of its amino acid sequence, since the purified protein appears to be largely devoid of ordered structure. To pursue this question, low-ultraviolet circular dichroism (CD) spectroscopy was conducted on the sequential thrombic peptides 1-95 and 96-168 of the protein in the presence of 0-92% trifluoroethanol (TFE), a solvent known to promote stable secondary structures in polypeptides. The series of CD spectra of the oligopeptides were subjected to a computerized best-fit analysis of four peptide conformations, the alpha-helix, beta-structure, beta-turn, and nonordered form. Agreement between experimental and best-fit composite spectra was achieved when standard CD curves of peptide conformations were derived from known theoretical spectra and experimental spectra of polypeptides. In dilute buffer alone, oligopeptides 1-95 and 96-168 evidence no alpha-helix but significant beta-structure (18% and 23%, respectively), as well as a predominant, extended nonordered conformation. However, the two parts of the protein differed in conformational adaptability. From 0% to 30% TFE, 96-168 exhibited concomitant transitions to 10% helix and 32% beta-structure from the nonordered form. In contrast, in 10-30% TFE, 1-95 underwent a transition to approximately 21% helix with partial loss of beta-structure as well as nonordered form; higher concentrations of TFE (40-75%) promoted additional transitions to both helix and beta-structure (totaling 33% and 25%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)