Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.     

Dielectric studies of aqueous solutions of poly(L-glutamic acid)

The dielectric features of poly(L -glutamic acid) are studied by the Fourier synthesized pseudorandom noise method in a time domain combined with a four-electrode cell. Polymer concentration dependence, the effect of the solvent viscosity, salt effects, and pH dependence are studied concomitantly with measurements of CD. A helix-to-coil transition occurs near pH 5.6 for a salt-free solution; at higher pH values, the polymer has an ionized random-coil conformation, and at lower pH, it has a deionized α-helical conformation. When it is in the ionized random-coil conformation, with the usual features of an electrolytic polymer, the solution shows a relaxation spectrum with a large dielectric increment at low frequencies. In the deionized α-helical state, no distinct relaxation curves are obtained, which does not deny the existence of a permanent peptide dipole. The pH dependence of the dielectric increment does not mainly correspond to the conformational change from helix to coil, but rather corresponds to the change of chain expansion on account of a charge–charge interaction under low ionic strength, which is conceived of by a viscosity measurement.     

Helical formation of a 13-residue C-peptide analogue of ribonuclease A in sodium dodecyl sulfate solution

The conformation of a 13-residue C-peptide analogue of ribonuclease A——in surfactant solutions was studied by CD. The CD spectrum of the peptide in excess NaDodSO₄ solution was typical for a helical conformation; the spectrum appeared to be virtually independent of pH (2.5–6) and temperature (3–25°C). Analysis of the CD data indicated a helicity of about 65–70% with no α-sheet and β-turn; this corresponded to 8 or 9 residues in the helical form or slightly more than two turns of α-helix. This compares with an average of about one turn of α-helix for the C-peptide analogue in water at pH 4.7 and 7°C. The conformation of the peptide in cationic surfactant, dodecyl ammonium chloride, and nonionic surfactant, dodecyl heptaoxyethylene ether, solution resembled that in water. We concluded that the C-peptide analogue can develop a maximum helicity close to the corresponding segment in ribonuclease A in hydrophobic environment provided by the clustering of NaDodSO₄ molecules to the cationic side groups of the peptide, except that the end effects may destabilize two or three residues each at both ends of the helix. Thus, in the interior of a protein molecule this hydrophobic effect may overshadow the charged-group effect than can be explained by the helix dipole model for the helical segments on the exterior of the protein molecule.     

Mechanical properties of poly(glutamic acid) in aqueous solution

The interaction between ionizable carboxyl groups and the conformation of poly-(glutamic acid) (PGA) in aqueous solution were investigated by the mechanical method. The dynamic rigidity of the PGA solution has a maximum value at the pH corresponding to about 50% neutralization point. This may be due to establishing of a maximum attractive force by proton/charge fluctuation between ionizable carboxyl groups at that pH. The dynamic viscosity has a sharp change in the region of pH 5.5–6.5. It is suggested that this behavior is due to the helix–coil transition.     

Solution conformation of an immunogenic peptide from HRV2: comparison with the conformation found in a complex with a Fab fragment of an anti-HRV2 neutralizing antibody

The conformation of a [15]-peptide (H-VKAETRLNPDLQPTE-NH₂) from VP2 of rhinovirus HRV2 complexed with a Fab fragment was previously shown by X-ray crystallographic studies to be similar to the one found in the corresponding region of HRV1A. Antibodies raised against this peptide bind to and neutralize HRV2. In order to identify structural features preserved in solution that may explain the ability of this short peptide to mimic the structure of the protein surface, the peptide has been studied by NMR in aqueous solution as well as under denaturing conditions. The peptide is shown to be a random coil in solution. However, the sequence forming a 3₁₀ helix in the complex is biased into a helical conformation according to NOE intensity data as well as from urea and pH titrations. This sequence adopts the same conformation in an unrelated protein. NOE data suggest that a β-turn found in the complex may be sampled in solution. Also, Glu4, interacting with Arg6 in the crystal, has a reduced pK_a value in solution. It is concluded that the local structure present in the random coil state of VP2(156–170) contains enough information to direct the production of antibodies that bind to and neutralize HRV2. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Amino-terminal extension analogs of methionine-enkephalin

Five NH₂-terminal extension analogs of methionine-enkephalin have been synthesized by solid phase methodology and their morphinomimetic activity determined in the guinea pig ileum-myenteric plexus preparation. Relative to methionine-enkephalin which corresponds to β-LPH-(61–65) and arbitrarily assigned a potency value of 100, β-LPH-(60–65), β-LPH-(59–65), β-LPH-(58–65), β-LPH-(57–65) and β-LPH-(56–65) have potencies of 90, 7, 4.4, 0.3 and 0.11 respectively.     

Conformation and aggregation of melittin: effect of pH and concentration of sodium dodecyl sulfate

The conformation of melittin, a surface-active polypeptide, in solution was studied by CD spectra between 190 and 240 nm. The molecule was essentially unordered (possibly with a trace of helix) in water without salt at neutral pH. Upon deprotonation of four of the six cationic groups at pH 12 the polypeptide became partially helical (about 35%). The addition of NaDodSO₄ to an aqueous melittin solution first caused the solution to become turbid but it became clear again in excess surfactant solution. The conformational changes depended on the molar NaDodSO₄/melittin ratio, R. With R from 2.34 to 23.4, the melittin solution was turbid and the polypeptide conformation was probably a mixture of α-helix and β-sheets. This was supported by the ir spectrum of the turbid solution, which indicated the presence of both conformations. With R = 46.8 or 468 (1 or 10 mM NaDodSO₄) the polypeptide conformation was characteristic of an α-helix, about 70–80% of the molecule, regardless of whether the surfactant was above or below its critical micelle concentration. This compared well with the x-ray results of 92% helix in crystals. The lower helicity of melittin in NaDodSO₄ solution might be attributed to the end effects that destabilize the first and last turn of an helix at its N- and C-terminus, respectively.     

Cell-free translation of human pheochromocytoma messenger RNA yields protein(s) containing methionine-enkephalin

Cell-free translation of messenger RNA extracted from a human pheochromocytoma yields protein(s) of apparent molecular weight >70,000 daltons which contain the pentapeptide methionine-enkephalin. It is estimated that 0.8–1.0% of the total pheochromocytoma mRNA codes for the methionine-enkephalin-containing protein, based on percent incorporation of [³⁵S]methionine into methionine-enkephalin during cell-free translation. These results demonstrate that human pheochromocytoma mRNA contains the message for a high-molecular weight methionine-enkephalin-containing protein or proteins, presumably the methionine-enkephalin precursor molecule(s).     

Investigation of the pH‐dependence of dye‐doped protein–protein interactions

Proteins can dramatically change their conformation under environmental conditions such as temperature and pH. In this context, Glycoprotein's conformational determination is challenging. This is due to the variety of domains which contain rich chemical characters existing within this complex. Here we demonstrate a new, straightforward and efficient technique that uses the pH‐dependent properties of dyes‐doped Pig Gastric Mucin (PGM) for predicting and controlling protein–protein interaction and conformation. We utilize the PGM as natural host matrix which is capable of dynamically changing its conformational shape and adsorbing hydrophobic and hydrophilic dyes under different pH conditions and investigate and control the fluorescent properties of these composites in solution. It is shown at various pH conditions, a large variety of light emission from these complexes such as red, green and white is obtained. This phenomenon is explained by pH‐dependent protein folding and protein–protein interactions that induce different emission spectra which are mediated and controlled by means of dye–dye interactions and surrounding environment. This process is used to form the technologically challenging white light‐emitting liquid or solid coating for LED devices.     

Mesoscopic structure of pectin in solution

Mesoscopic structure of pectin with different molecular characteristics was investigated by means of small angle X‐ray scattering (SAXS), electrokinetic measurements and data modelling. The influence of a broad range of pH (2–7) on chain conformation in the dilute and semi‐diluted regime was investigated. Scattering data and concomitant analysis revealed two length scales at all environmental conditions studied. pH showed greater influence at acidic values (pH 2.0) enhancing the globular component of the structure due to association of galacturonic acid residues. Double logarithmic scattering intensity plots revealed fractal dimensions of 1.9 ± 0.2 in the low‐q regime and 1.5 ± 0.2 in the high q‐region, irrespectively of the specific environment. Increase in branching of RG‐I regions of the polysaccharide chains enhanced the compact conformation irrespectively of the pH or concentration. The present work shows that radical changes in pectin conformation can be induced only under strongly acidic conditions a finding that has important consequences in tailoring the technological performance of these biopolymers.     

Assignment of the 270 MHz nuclear magnetic resonance spectrum of somatostatin in water

The proton nuclear magnetic resonance spectrum of the 14 residue peptide hormone somatostatin in D₂O at 270 MHz has been assigned by comparing the spectra of synthetic analogs with those of the native peptide. Extensive difference double resonance studies of all somatostatins and pH titrations confirmed all assignments. ³J_NHCH values and conventional NMR hydrogen bonding studies confirm the existence of preferred secondary conformations but not with a predominant conformation possessing a β-turn in either of the sequences 7–8–9–10 or 8–9–10–11. More extensive data treatment is needed before the actual conformation(s) of somatostatin is elucidated, but several NMR criteria for conformations are proposed.     

Crystal structure of methionine-enkephalin

The crystal structure of methionine-enkephalin has been determined by X-ray crystallography. There are two independent pentapeptides in the asymmetric unit and both display extended backbone conformations with their side chains arranged alternately below and above the backbone plane. The two molecules form a hydrogen-bonded head-to-tail dimer similar in conformation to one dimeric pair of leucine-enkephalin molecules in a previously reported crystal structure.     

Hydrodynamical properties of recombinant spider silk proteins: Effects of pH,salts and shear,and implications for the spinning process

We have investigated the effect of pH, salts and shear on the hydrodynamical diameter of recombinant major ampullate (MA) rMaSpI silk proteins in solution as a function of time using ¹H solution NMR spectroscopy. The results indicate that the silk proteins in solution are composed of two diffusing populations, a high proportion of “native” solubilized proteins and a small amount of high molecular weight oligomers. Similar results are observed with the MA gland content. Salts help maintaining the proteins in a compact form in solution over time and inhibit aggregation, the absence of salts triggering protein assembly leading to a gel state. Moreover, the aggregation kinetics of rMaSpI at low salt concentration accelerates as the pH is close to the isoelectric point of the proteins, suggesting that the pH decrease tends to slow down aggregation. The data also support the strong impact of shear on the spinning process and suggest that the assembly is driven by a nucleation conformational conversion mechanism. Thus, the adjustment of the physicochemical conditions in the ampulla seems to promote a stable, long term storage. In addition, the optimization of protein conformation as well as their unfolding and aggregation propensity in the duct leads to a specifically organized structure. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 582–593, 2013.     

Influence of methanol on the rotational diffusion of poly(L-lysine) in the helix–coil transition

¹³C spin-lattice relaxation times of poly(L -lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the ¹³C^α and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix–coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the C^α-H^α vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ? 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.     

A Raman spectroscopic study of hen egg yolk phosvitin: structures in solution and in the solid state

Laser Raman spectroscopy has been employed to study the structure of the hen egg yolk protein phosvitin in H2O and D2O solutions at neutral and acidic pH (pD) and in the solid state. The Raman data indicate an unusual conformation for phosvitin in neutral aqueous solution, which is deficient in both alpha-helix and conventional beta-sheet conformations. This unusual pH 7 structure is, however, largely converted to a beta-sheet conformation in strongly acidic media (pH less than 2). beta-Sheet is also the predominant secondary structure for phosvitin in the solid state, obtained by lyophilization of the protein from aqueous solution at neutral pH. The imidazolium rings of histidyl residues remain significantly protonated near neutrality, which suggests substantial elevation of the pK for imidazolium ring ionizations of phosvitin in aqueous solution. This may result from extensive ion-pair interactions involving positively charged histidines and negatively charged phosphoserines, which are prevalent in the phosvitin sequence. The present results suggest that antiparallel beta-sheets may not be the secondary structure most characteristic of native phosvitin (physiological pH), even though beta-sheet is the predominant conformation for phosvitin in acidic solutions (pH 1.5) and in the lyophilized solid. Phosvitin appears to be the first protein for which the major component to the Raman amide I band is centered near 1685 cm-1, which is 10-40 cm-1 higher than proteins heretofore examined in aqueous solution by Raman spectroscopy.     

Non-Watson-Crick structures in oligodeoxynucleotides: self-association of d(TpCpGpA) stabilized at acidic pH

The 1H NMR spectrum of the tetradeoxynucleotide d(TpCpGpA) was examined as a function of temperature, pH, and concentration. At pH 7 and above the solution conformation for this oligodeoxynucleotide appears to be a mixture of random coil and Watson-Crick duplex. At 25 degrees C, a pH titration of d(TpCpGpA) shows that distinct conformational changes occur as the pH is lowered below 7.0. These conformational changes are reversible upon readjusting the pH to neutrality, indicating the presence of a pH-dependent set of conformational equilibria. At 25 degrees C, the various conformational states in the mixture are in rapid exchange on the NMR time scale. Examination of the titration curve shows the presence of distinct conformational states at pH greater than 7, and between pH 4 and pH 5. At pH less than 4, a third conformational state is present. When the pH titration is repeated at 5 degrees C, the conformational equilibria are in slow exchange on the NMR time scale; distinct signals from each conformational state are observable. The stable conformational state present between pH 4 and pH 5 represents an ordered conformation of d(TpCpGpA) which dissociates to a less ordered structure upon raising the temperature. This ordered conformation does not result from an intramolecular rearrangement, as is shown by by spectra obtained by varying oligodeoxynucleotide concentration at constant pH. The ordered conformation differs from the Watson-Crick helix, as is shown from nuclear Overhauser enhancement experiments, as well as chemical shift data. An ordered conformation for d(TpCpGpA) was previously reported [Reid, D. G., Salisbury, S. A., Brown, T., & Williams, D. H. (1985) Biochemistry 24, 4325-4332].(ABSTRACT TRUNCATED AT 250 WORDS)     

pH Variation of midpoint potential for three photosynthetic bacterial cytochromes c′. A link between physical and functional properties

Midpoint redox potential (E_M) versus pH curves are reported over the pH range 5 to 10 for the cytochromes c′ from three species of purple photosynthetic bacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris and Chromatium vinosum. In each case, theoretical curves are fitted to the data and pK values for the reduced (pH 5–5.5) and oxidized (pH 8–8.5) forms of the protein are found to influence the midpoint redox potentials. The oxidized form pK values in each case are found to correlate with previously determined pK values for variation in physical and/or spectroscopic properties. This correlation of functional and physical observables is discussed in terms of a possible mechanism of control of midpoint redox potential through heme iron-ligand bonding as moderated by the protein conformation in response to solution conditions. The reduced form pK values are discussed in terms of a mechanism which would alter the polarity of the heme environment, thereby influencing redox potentials.     

Paramagnetic relaxation enhancement‐assisted structural characterization of a partially disordered conformation of ubiquitin

Nuclear magnetic resonance (NMR) is a powerful tool to study three‐dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N₁ and N₂ and plus another folded conformation, I, in which some amide group signals of residues 33–41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well‐converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure‐induced loss of backbone amide group signals at residues 28, 33, 36, and 39–41 like the wild‐type, with a similar but smaller effect on CαH and CβH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N₂ conformation, although the amide groups at residues 33–41 have more heterogeneous conformations and more contact with water, which differ from the N₁ and N₂ states. The PRE‐assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.     

The kinetics of the alkaline dissociation of myosin.

The rates of two processes in alkaline (pH 10.5–11.5) myosin solutions at 0 °C have been investigated: production of ionized tyrosine residues and production of light subunits. The progressive absorbance change is shown to result from a first-order irrevocable exposure to solvent and subsequent ionization of 40% of the tyrosine residues. Extrapolation to zero time gives the spectrophotometric ionization curve for native myosin; the pK of the abnormal tyrosines exceeds 12. Similarly, extrapolation to infinite time gives the curve for denatured myosin; the pK of the normal tyrosines (and of all tyrosines after denaturation) is 11.0–11.6. From the pH dependence of the rate, it is found that activation requires ionization of six residues and that their pK is much greater than 11.3. The rate of production of subunits was determined by fractionating the reaction mixture and determining the weight of light subunits produced. The process is also first order. Within experimental error, the rate constants for these two processes are equal. We conclude that they have the same rate-determining step. The data are consistent with either of two simple possible mechanisms. These are a rapid conformation change, followed by rate-determining subunit dissociation, followed by a rapid, irrevocable conformation change; or, a rapid conformation change, followed by a rate-determining, irrevocable conformation change, followed by rapid subunit dissociation.     

Ligand Binding Turns Moth Pheromone-binding Protein into a pH Sensor: EFFECT ON THE ANTHERAEA POLYPHEMUS PBP1 CONFORMATION*

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the “open” (bound) conformation at high pH but switches to the “closed” (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of ∼1:1. Mutation of His⁷⁰ and His⁹⁵ to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.