Circular dichroism and conformation of human alpha1-antitrypsin.

The solution conformation of alpha 1-antitrypsin from human blood plasma was studied by the circular dichroism (CD) probe. The CD spectra revealed in this glycoprotein approximately 16-20% of alpha-helix, the rest of the main polypeptide chain possessing the pleated sheet (beta) and the aperiodic structures. The conformation was stable between pH 4.7 and 8.8. Reversible change in conformation was observed at pH 10.3, and more dratic denaturation occurred at pH 11.6. The environment of the side chain chromophores was strongly affected by acid at pH 2.5, whereas the main chain conformation was changed slightly. A drastic change in the CD spectra, indicating denaturation, was observed in 3.5 M guanidine hydrochloride. Sodium dodecyl sulfate was effective in disorganizing the tertiary structure and in enhancing the helix content. The phenylalanine band fine structure was observed in the native protein and also after denaturation with acid, guanidine hydrochloride and sodium dodecyl sulfate.     

Circular dichroism and conformational properties of modeccin,a toxic protein

According to its circular dichroism (CD) spectrum, modeccin, a toxic lectin from the roots of the South African plantModecca digitata, is structurally similar to the ricins and abrins. In nearly neutral and weakly alkaline solutions (pH 7.6–9.0) the CD spectra of modeccin displayed a positive CD band at 190–195 nm and a negative band at 210–220 nm, indicating the presence of some α-helix and β-sheet structures. In the near-ultraviolet zone, we observed positive CD bands at 232 and 245 nm and weak negative bands at 285 and 293 nm. In more strongly alkaline solutions of pH 9.5–10.2 the CD bands in the farultraviolet zone were not affected, but the CD band at 232 nm diminished and the CD band at 245 nm was enhanced. These transitions were reversible. At pH 11.2–11.5 the CD band at 232 nm disappeared completely, and the CD bands in the far-ultraviolet diminished. The CD bands at 285 and 293 nm were affected very little by the alkali, and these bands were assigned to buried tryptophan side chains. Sodium dodecyl sulfate and 2,2,2-trifluoroethanol disorganized the tertiary structure of modeccin and reconstructed the secondary structure into a new form with a higher helix content than in the native protein.     

Circular dichroism and the conformational properties of soybean beta-amylase

The conformational properties of soybean β-amylase were investigated by the circular dichroism probe and measurement of enzyme activity. The enzyme exhibited a positive circular dichroism band at 192 nm, a negative band at 222 nm, and a shoulder near 210 nm. Analysis of the spectrum in the far ultraviolet zone indicated the presence of approximately 30% of α helix and 5–10% of β-pleated sheet, the rest of the polypeptide main chain possessing aperiodic structure. In the near ultraviolet reagion, the enzyme protein showed at least six positive peaks at 259, 265, 273, 281, 292, and 297 nm. The positive bands at 292 and 297 nm remained unaltered on acetylation of the enzyme by N-acetylimidazole and were assigned to tryptophanyl chromophores. These bands were affected in intensity in the presence of maltose or cycloheptaamylose, which indicates that some tryptophan residues are situated at the binding sites. The native conformation of soybean β-amylase was found to be sensitive to pH variation (below pH 5 and above pH 10), sodium dodecyl sulfate, guanidine hydrochloride, and heating to 50–55 °C. Complete disorganization of the secondary structure was attained by 6 m guanidine hydrochloride. Sodium dodecyl sulfate was effective in disturbing the tertiary structure of the enzyme but did not affect significantly the secondary structure. Enzymatic inactivation was paralleled by the decrease of circular dichroism bands in the near ultraviolet region as produced by the denaturants. It is concluded that the uniquely folded structure of the enzyme contains some less rigid domains and a rigid core stabilized by hydrophobic interactions, electrostatic interactions, and hydrogen bonds.     

Tyrosine sulfation in precursors of collagen V

Radioactive labeling of p-collagens V, collagens V, and, to a small extent, of procollagen V occurred when [35S]sulfate was incubated with tendons or primary tendon cell cultures, or blood vessels and crops of 17- to 19-day-old chick embryos, or with lung slices from neonatal rats. Most or all of this label is in the form of 1 or more sulfated tyrosine residues/chain of p alpha 1(V), alpha 1(V), p alpha 1'(V), alpha 1'(V), p alpha 2(V), and alpha 2(V), and it remains attached through purification by dialysis, ammonium sulfate precipitation, CsCl-GdnCl2 equilibrium buoyant density and velocity sedimentations, ion-exchange chromatography, and sodium dodecyl sulfate gel electrophoresis. Radioactive tyrosine sulfate was identified in alkaline hydrolysates of these collagen V chains, after labeling the tissues with either [35S]sulfate or [3H]tyrosine, by electrophoretic and chromatographic comigration with a tyrosine sulfate standard. Tunicamycin A1, which inhibits the attachment of N-linked complex carbohydrate, did not interfere with the sulfation process. The tyrosine sulfate is located in a noncollagenous domain, which is probably adjacent to the amino end of the collagen helix, and is retained throughout the physiological proteolytic processing of procollagens V. After digestion with Staphylococcus aureus V8 protease, 35S-labeled p alpha 1(V) and alpha 1(V) chains gave the same map of labeled peptides, and this differed from the map given by p alpha 1'(V) and alpha 1'(V) chains. Little sulfation of p alpha 2(V) and alpha 2(V) chains occurs. The implications of these observations for the structure and properties of procollagens V and their derivatives are considered.     

Conformational transitions of non-helical proteins effected by dodecyl sulfate. Circular dichroism of alpha1-acid glycoprotein, Bence Jones protein, carbonic anhydrase B, deoxyribonuclease, pepsinogen, and plasminogen.

Circular dichroism (CD) of serum alpha1-acid glycoprotein, urinary Bence Jones protein, human carbonic anhydrase B, deoxyribonuclease from bovine pancreas, porcine pepsinogen, and plasminogen from human serum was tested in the absence and presence of 0.005-0.05 M sodium dodecyl sulfate. It was found that in all cases the CD spectra of these proteins were modified by the dodecyl sulfate into spectra indicating the presence of a moderate content of alpha-helix. The transitions were enhanced by addition of acid (pH 2.1-4.4) in all cases tested. Comparison of the various proteins with respect to the amount of reconstruction of the main chain conformation showed that the amount of helix formed depended on the amino acid composition of the protein. Rigidity due to cross-linking by disulfide bridges is the strongest deterrant to the conformational change of the main chain. The CD bands of the native proteins in the 250-350 nm spectral zone were extinguished by sodium dodecyl sulfate, and new weak bands were observed the positions of which corresponded approximately to those of the native proteins. In all cases, except the carbonic anhydrase B, the bands of thus denatured proteins were negative.     

The Full-Length Mu-Opioid Receptor: A Conformational Study by Circular Dichroism in Trifluoroethanol and Membrane-Mimetic Environments

The secondary structure content of the recombinant human mu-opioid receptor (HuMOR) solubilized in trifluoroethanol (TFE) and in detergent micelles was investigated by circular dichroism. In both conditions, this G protein-coupled receptor adopts a characteristic alpha-helical structure, with minima at 208 and 222 nm as observed in the circular dichroism spectra. After deconvolution of spectra, the alpha-helix contents were estimated to be in the range of 50% in TFE and in sodium dodecyl sulfate at pH 6. These values are in accordance with the predicted secondary structure content determined for the mu-opioid receptor. A pH-dependent effect was observed on the secondary structure of the receptor solubilized in detergents, which demonstrates the essential role of ionic and hydrophobic interactions on the secondary structure. Circular dichroism spectra of EGFP-HuMOR, a fusion protein between the enhanced green fluorescent protein (EGFP) and the mu-opioid receptor, and EGFP solubilized in TFE were also analyzed as part of this study.     

Effect of temperature and pH on the β–helix transition of poly(L-lysine) in sodium dodecyl sulfate solution

The conformational phase diagram of poly(L -lysine) (4.6 × 10^?4 M, residue) in sodium dodecyl sulfate (1.6 × 10^?2 M) solution was constructed from circular dichroism results at various temperatures and pH's. Poly(L -lysine)–sodium dodecyl sulfate complexes undergo a β–helix transition upon raising the pH of the solution. The transition pH tends to shift downward at elevated temperatures. No helix–β transition can be detected for poly(L -lysine) in sodium dodecyl sulfate solution (pH > 11) even after 1-hr heating at 70°C. This is in marked contrast with uncharged poly(L -lysine) solution without sodium dodecyl sulfate, which is converted into the β-form upon mild heating of the solution above 50°C.     

Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum

The structure of phospholamban, a 30-kDa oligomeric protein integral to cardiac sarcoplasmic reticulum, was probed using ultraviolet absorbance and circular dichroism spectroscopy. Purified phospholamban was examined in three detergents: octyl glucoside, n-dodecyloctaethylene glycol monoether (C12E8) and sodium dodecyl sulfate (SDS). Ultraviolet absorption spectra of phospholamban reflected its aromatic amino acid content: absorption peaks at 275-277 nm and 253, 259, 265 and 268 nm were attributed to phospholamban's one tyrosine and two phenylalanines, respectively. Phospholamban phosphorylated at serine 16 by the catalytic subunit of cAMP-dependent protein kinase exhibited no absorbance changes when examined in C12E8 or SDS. Circular dichroism spectroscopy at 250-190 nm demonstrated that phospholamban possesses a very high content of alpha-helix in all three detergents and is unusually resistant to denaturation. Dissociation of phospholamban subunits by boiling in SDS increased the helical content, suggesting that the highly ordered structure is not dependent upon oligomeric interactions. The purified COOH-terminal tryptic fragment of phospholamban, containing residues 26-52 and comprising the hydrophobic, putative membrane-spanning domain, also exhibited a circular dichroism spectrum characteristic of alpha-helix. Circular dichroism spectra of phosphorylated and dephosphorylated phospholamban were very similar, indicating that phosphorylation does not alter phospholamban secondary structure significantly. The results are consistent with a two-domain model of phospholamban in which each domain contains a helix and phosphorylation may act to rotate one domain relative to the other.     

Conformational changes induced in ionized poly(L-arginine) and poly(L-histidine) by sodium dodecyl sulfate

Circular dichroism spectra have been obtained for cationic poly(L -arginine) and poly(L -histidine) in aqueous solutions containing varying amounts of sodium dodecyl sulfate. The detergent induces a disorder-order transition in both polypeptides. In each case the transition is cooperative and occurs when the ratio of detergent to amino acid residue is near unity. The ordered structure formed by poly(L -arginine) is readily identifiable as an α helix. Poly(L -histidine) appears to form a β structure in which the 211-nm electronic absorption band of the imidazole group exhibits significant rotatory strength.     

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

Circular dichroism spectra have been obtained for tri(L -lysine), tetra(L -lysine), and penta(L -lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high-molecular-weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10^?4 M the penta(L -lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L -lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L -lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L -lysine) by sodium dodecyl sulfate.     

Solution conformations of the pituitary opioid peptide dynorphin-(1–13)

Circular dichroism spectra have been measured for dynorphin-(1–13) in water and in solutions of sodium dodecyl sulfate and L-α-lysophosphatidylcholine (palmitoyl). Spectra in water have the features expected for a peptide containing little, if any, order. Small changes are brought about by L-α-lysophosphatidylcholine (palmitoyl), but the resulting spectrum retains the characteristics expected for a random coil. In contrast, sodium dodecyl sulfate produces significant changes which are those expected for induction of α helical content. Quantitative analysis of the circular dichroism spectra suggests the conformation changes from about 5% helix in water to 17% helix in sodium dodecyl sulfate. These results from experiment are in excellent agreement with those obtained from our formulation of the configuration partition function. This formulation predicts a change in helical content from 1% to 19%. The ordering influence is felt most strongly by those residues immediately following the enkephalin sequence.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Conformational transitions of monellin, an intensely sweet protein.

Conformational transitions of monellin, an intensely sweet protein from the berries of Dioscoreophyllum cumminsii, were studied by the circular dichroism (CD) probe. According to the CD spectra, monellin has a low content of the helical structure and a significant amount of the pleated sheet (beta) conformation. The native conformation was found to be sensitive to alkali, sodium dodecyl sulfate, and guanidine-HC1, but it was stable in acid (e.g. pH 2.4) as shown by CD and persistence or the disappearance of sweet taste. The main chain conformation of the alkali-denatured monellin (pH 10.9) was restored upon acidification (pH 3.3) of the alkaline solutions. The tertiary structure, however, was not completely restroed, as indicated by CD in the 230-300 nm spectral zone, although the sweet taste reappeared. If the pH of a neutral solution was raised to 9.6, the CD in the near ultraviolet was significantly altered, though the sweet taste persisted. This indicates that a slight conformational change did not interfere with the effects on the taste buds. While sodium dodecyl sulfate readily disorganized the tertiary structure, the main chain was reconstructed by this reagent into a new form of higher helix content than in the native macromolecule. Reconstruction into a modified conformation of higher helix content was achieved also with 50% ethanol. The main chain conformation was not affected by 25% ethanol which produced slight changes in the CD at 230-260 nm zone and did not abolish the sweet taste.     

The effects of the side chains of hydrophobic aliphatic amino acid residues in an amphipathic polypeptide on the formation of alpha helix and its association

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.     

Presence of an amphipathic helical segment and its relationship to biological potency of calcitonin analogs

The conformational properties of a number of calcitonin analogs were studied by circular dichroism. The ability of dimyristoylphosphatidylglycerol, lysophosphatidylcholine or sodium dodecyl sulfate to induce the formation of more highly ordered structures in these peptides was also assessed by circular dichroism. In all cases sodium dodecyl sulfate induced the largest change in the circular dichroism spectra of the peptides. Salmon calcitonin and its analogs were slightly more helical in the presence of the anionic phospholipid than in the presence of the zwitterionic detergent lysophosphatidylcholine while the reverse is true for human calcitonin and its analogs. Some of the calcitonin analogs convert turbid suspensions of phosphatidylglycerol to a clear solution from which the phospholipid is no longer readily sedimentable by centrifugation. Several of the physical properties of these peptides could be correlated with their biological activity. Generally peptides which showed no hypocalcemic activity had the least negative mean residue ellipticities at 222 nm. Only biologically active analogs were able quantitatively to solubilize dimyristoyl-phosphatidylglycerol and in this solubilized form the peptides have a higher helical content. More active derivatives exhibit larger increases in helix content in the presence of this phospholipid. Inactive analogs had the least negative mean residue ellipticities at 222 nm in the presence of lysophosphatidylcholine or sodium dodecyl sulfate. Thus, the ability of a calcitonin analog to form structures of higher helical content in the presence of amphiphiles is a requirement for the analog to exhibit high potency in assays of biological activity.     

Sodium dodecyl sulfate-bovine plasma albumin complex. Structural transition from native to relatively compact globule forms with mobile side chains in acidic region--N-F transition

The acid-induced isomerization (the N-F transition) and expansion of sodium dodecyl sulfate-bovine plasma albumin complex (ADm; m, molar ratio of added sodium dodecyl sulfate to bovine plasma albumin; O less than or equal to m less than or equal to 12) were studied by measuring CD-resolved secondary structure, fluorescence polarization and life-time of tryptophyl fluorophors, acid-titration with the electrostatic correction for the surface potential, 1H-n.m.r. spectra and cross relaxation time between irradiated and observed protons. The immobilization of tryptophyl fluorophors observed in the F-form of AD0 was suppressed in the F-form of AD10. The acidtitration analysis of AD12 showed non salt-bonding between carboxylate groups and cationic side chains in the F-form, as in the case of AD0, indicating charged side chains being presumably mobile. 1H-n.m.r. spectra and cross relaxation times between irradiated and observed protons in the F-form of AD10 indicated the increase in the local motion. On the other hand, AD10 and AD12 did not show any significant change in the CD-resolved secondary structure in the N-F transition region. The F-form of AD10 or AD12 may therefore be the moltenglobule state which has secondary structure similar to the N-form of the complexes with fluctuating tertiary structure (side chains).     

Secondary structure of substance P bound to liposomes in organic solvents and in solution from Raman and CD spectroscopy.

The membrane lipid phase may be an important mediator of the peptide-receptor interaction. In order to understand the mechanism of this interaction, it is important to know the peptide structure, not only in the hydrophobic lipid bilayer environment, but also at the bilayer surface and in solution. To investigate this problem we have measured the secondary structure of the 11-residue neuropeptide substance P (SP) and its fragments in aqueous solutions, in membrane mimetic solvents, and associated with lipid bilayers using Raman and CD spectroscopy. Raman and CD spectra of SP bound to liposomes indicate a less than 20% helix content. We interpret these results to indicate that SP contains virtually no helix when bound to negatively charged liposomes. These spectra are similar to spectra of peptides in type I and III beta-turns. SP forms between 10 and 30% (1-3 residues) helical structure in sodium dodecyl sulfate micelles and less than 10% helix in methanol and trifluoroethanol. The binding of SP to negatively charged liposomes significantly changes the structure of the lipid acyl chains, decreasing order in some cases and increasing it in others. Raman spectra of SP in water indicates that SP near 30 mM forms an ensemble of structures in water that is distinct from completely unfolded peptide and from the aggregated beta-sheet form observed in saline solutions. We conclude from our CD results that methods used to quantitate secondary structure from CD spectra of short peptides cannot be used to distinguish between very short helical segments and beta-turns.     

Circular dichroism studies on the alpha-D-galactopyranosyl binding lectin isolated from the seeds of Bandeiraea simplicifolia.

The conformation of the alpha-D-galactopyranosyl binding lectin isolated from Bandeiraea simplicifolia seeds has been investigated over a broad range of pH in the presence of various solvents by circular dichroism (CD) spectroscopy in the region 200-300 nm. Analyses of the spectra obtained on the native protein show the lectin to contain a considerable proportion of beta structure (30-40%). The native conformation was found to be largely insensitive to changes in pH, but was influenced by sodium dodecyl sulfate or trifluoroethanol. Alterations in conformation in the presence of these agents were reflected in the CD spectra and show the presence of alpha helix under these conditions. These changes in conformation are accompanied by a loss in polysaccharide-precipitating activity. The protein is irreversibly denatured in 8 M urea. Neither removal of the intrinsic calcium ions from the protein nor addition of methyl alpha-D-galactopyranoside induces any appreciable change in the CD spectra of the protein although the former treatment abolishes the polysaccharide-precipitating capacity of the lectin. The conformational data obtained in the present study are compared with data available from conformational studies of other lectins and leads to the hypothesis that most lectins probably contain beta structure as the predominant conformational feature.     

A circular dichroism study of the structure of Apis mellifera melittin

The secondary structure of the polypeptide melittin has been examined employing circular dichroism. In water or dilute buffer at pH 7, this protein is approximately 25–30% helical. Helicity is increased to near 50% by increasing the pH to 9 or above. High concentrations of phosphate buffer dramatically increase mean residue ellipticity at 222 nm to a value near the maximum expected for a totally helical molecule. Dilute solutions of sodium dodecyl sulfate have the same effect. In contrast, the secondary structure of melittin is little affected by bromide ion, calcium ions, or dihexanoyl-phosphatidylcholine.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.