pH-Induced Molten Globule State of <Emphasis Type="Italic">Rhizopus niveus</Emphasis> Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R _h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.     

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.     

Interaction of amphipathic polypeptides with phospholipids: characterization of conformations and the CD of oriented beta-sheets

The effects of interaction with phospholipids on the conformation of the alternating copolymer, poly(Leu-Lys), and the random copolymer poly(Leu⁵⁰, Lys⁵⁰) have been investigated by CD and ir spectroscopy. Poly(Leu-Lys) undergoes a partial unordered → β-sheet transition in solution in the presence of lysolecithin. On addition of lysolecithin plus cholate, an unordered → α -helix transition is observed. In films deposited from these solutions, poly(Leu-Lys) adopts the anti-parallel β-sheet conformation, as in aqueous solutions at moderate ionic strength. Polarized ir spectra showed that the plane of the β-sheet in such films deviates from the plane of the film by no more than 14°. The random copolymer, poly(Leu⁵⁰, Lys⁵⁰), is α-helical in the presence of lysolecithin and lysolecithin plus cholate, regardless of whether the sample is a solution or a film. CD measurements on the poly(Leu-Lys) films provide information about the component of the CD tensor for light propagating normal to the plane of the β-sheet. These measurements show (1) a negative n → π* CD band (214 nm maximum) with higher intensity than the average CD for isotropic solution; and (2) a positive band in the π → π* region (195 nm maximum), which is weaker than that in the isotropic spectrum.     

The random coil → β transition of copolymers of L-lysine and L-isoleucine: Potentiometric titration and circular dichroism studies

Copolymers of L -lysine and L -isoleucine [poly(L -Lys^f,L -Val^{1 ? f})] containing 4–15% isoleucine were investigated using potentiometric titration and circular dichroism (CD) spectroscopy. With increasing isoleucine content, β-sheet formation is favored over α-helix formation at high pH and room temperature. The fraction of β-sheet present, as a function of pH, calculated from titrations of poly(L -Lys^85.2,L -Ile^14.8), agreed well with data obtained from CD studies for the same copolymer. Thermodynamic parameters were determined from titrations using the method of Zimm and Rice; the partial free energy (ΔG°_{C → β}) at 25° for the coil-to-β-sheet transition for isoleucine was estimated to be ?515 cal/mol; from the temperature dependence of free energy, the partial entropy (ΔS°_cβ), and the partial free enthalpy (ΔH°_{c → β}) of the coil → β transition for isoleucine is estimated to be 2.6 e.u. and 260 cal/mol, respectively. The partial thermodynamic parameters obtained for lysine are in good agreement with literature values. It is concluded from these studies that isoleucine has a very high potential for a β-sheet formation.     

Resolution of the effects induced by W?→?F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.     

Poly(L–lysine)–DNA interactions in NaCl solutions: B → C and B → ψ Transitions

Thermal denaturation and circular dichroism (CD) properties of poly(L -lysine)–DNA complexes vary greatly when these complexes are prepared differently, that is, whether by NaCl-gradient dialysis starting from 2.0 M NaCl or by direct mixing at low salt. These differing properties were investigated in more detail by examining complexes, made by direct mixing in the presence of various concentrations of NaCl, both before and after the NaCl was dialyzed out of the complex solution. The precipitation curves of DNA due to polylysine binding indicate that such binding is noncooperative at zero salt; from 0.1 up to 1.0 M NaCl they exhibit varying degrees of cooperatively. Starting from zero salt, as the NaCl concentration used for complex formation is increased, both the CD and the melting properties of the complexes are shifted from those of directly mixed at zero salt to those of reconstitution: in the CD spectra there is a gradual shift from a B → C transition to a B → ψ transition; thermal denaturation results show a gradual increase in the melting temperatures of both free DNA (t_m) and polylysine-bound DNA (t′_m). The progressive shift from B → C to B → ψ suggests a close relationship between these two transitions. Large aggregates of the complexes do not warrant the appearance of ψ-type CD spectra: ψ-spectra have been obtained in the supernatants of polylysine–DNA complexes made and measured at 1.0 M NaCl while slightly perturbed CD spectra in B → C transition have been observed in turbid solutions of fully covered complexes made at very low salt. If the complexes are made at intermediate salts and dialyzed to a very low salt, although up to 60% of the DNA is still bound by polylysine, the CD spectra of the complexes are shifted back to the B-type CD characteristic of pure DNA.     

Dynamics and dimension of an amyloidogenic disordered state of human β2-microglobulin

Human β₂-microglobulin (β₂m) aggregation is implicated in dialysis-related amyloidosis. Previously, it has been shown that β₂m adopts an ensemble of partially unfolded states at low pH. Here we provide detailed structural and dynamical insights into the acid unfolded and yet compact state of β₂m at pH 2.5 using a host of fluorescence spectroscopic tools. These tools allowed us to investigate protein conformational dynamics at low micromolar protein concentrations in an amyloid-forming condition. Our equilibrium fluorescence data in combination with circular dichroism data provide support in favor of progressive structural dissolution of β₂m with lowering pH. The acid unfolded intermediate at pH 2.5 has high 8-anilinonaphthalene, 1-sulfonic acid (ANS)-binding affinity and is devoid of significant secondary structural elements. Using fluorescence lifetime measurements, we have been able to monitor the conformational transition during the pH transition from the native to the compact disordered state. Additionally, using time-resolved fluorescence anisotropy measurements, we have been able to distinguish this compact disordered state from the canonical denatured state of the protein by identifying unique dynamic signatures pertaining to the segmental chain mobility. Taken together, our results demonstrate that β₂m at pH 2.5 adopts a compact noncanonical unfolded state resembling a collapsed premolten globule state. Additionally, our stopped-flow fluorescence kinetics results provide mechanistic insights into the formation of a compact disordered state from the native form.     

pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

Acid unfolding pathway of conalbumin (CA), a monomeric glycoprotein from hen egg white, has been investigated using far- and near-UV CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe 1-anilino-8-napthalene sulfonate (ANS) and dynamic light scattering (DLS). We observe pH-dependent changes in secondary and tertiary structure of CA. It has native-like α-helical secondary structure at pH 4.0 but loss structure at pH 3.0. The CA existed exclusively as a pre-molten globule state and molten globule state in solution at pH 4.0 and pH 3.0, respectively. The effect of pH on the conformation and thermostability of CA points toward its heat resistance at neutral pH. DLS results show that MG state existed as compact form in aqueous solutions with hydrodynamic radii of 4.7 nm. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states.     

Urea induced unfolding of F isomer of human serum albumin: a case study using multiple probes

The human serum albumin is known to undergo N <==> F (neutral to fast moving) isomerization between pH 7 and 3.5. The N < ==> F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher KSV of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).     

Alteration of lacto-series glycolipid glycosyltransferase activities in human colonic adenocarcinoma DLD-1 cells after culture in N,N-dimethylformamide-containing medium

Human colonic adenocarcinoma DLD-1 cells were grown under conditions which induce characteristics of differentiated cells using medium containing 0.8% N,N-dimethylformamide in order to study alterations in glycosphingolipid glycosyltransferase activities during this process. Analysis of biosynthetic reactions involved in lacto-series antigen synthesis revealed no changes in the specific activities of either β1→4galactosyltransferase or α1→3/4fucosyltransferase with N,N-dimethylformamide treatment. However, a dramatic decrease of from 14- to 20-fold in the β1→3N-acetylglucosaminyltransferase activity was observed in the treated cells. This enzyme catalyzes the rate-limiting step in lacto-series core chain synthesis. This is consistent with the pattern of regulation of lacto-series antigen expression found to occur during oncogenesis in human colonic mucosa (Holmes EH, Hakomori S, Ostrander GK: J Biol Chem 262:15649, 1987). Total glycolipids from untreated and N,N-dimethylformamide-treated cells were isolated and subjected to TLC immunostain analysis and solid phase radioimmunoassay with a series of monoclonal antibodies specific for lacto-series-based carbohydrate antigens. A decrease of about 2-fold or less in the quantity of lacto-series antigens was observed as a consequence of N,N-dimethylformamide treatment in both neutral glycolipid and ganglioside fractions. The results suggest that only very low levels of β1→3N-acetylglucosaminyltransferase activity are required for the steady state expression of significant levels of lacto-series based glycolipids and that modulation of its activity levels by N,N-dimethylformamide treatment in DLD-1 cells represents a convenient in vitro system for studying aspects of regulation of lacto-series antigen expression.     

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies

The sequential polypeptides (L -Arg-X-Gly)_n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine-rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene-4-sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L -Arg-X-Gly)_n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β-structure. Characteristics of β-turn are excluded from the poly(L -Arg-L -Leu-Gly), which assumed the most pronounced helical conformation. The poly(L -Arg-L -Val-Gly) exerts a significant preference to the β-turn structure compared to that of poly(L -Arg-L -Ala-Gly). Thus the probability for helical, β-structure or β-turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X-residue side chain (β-branching). We concluded that the prepared sequential arginine-containing polypeptides are plausible models for histone fractions, f₃ and f₂α₁.     

Conformational transitions of calmodulin as studied by vacuum-uv CD

CD measurements were made for calmodulin and its calcium (Ca²⁺) complexes at different ionic strengths and Ca²⁺ concentrations. Calmodulin at an ionic strength of 0.00M and in the absence of Ca²⁺ exists as an α-helical protein with a negligible amount of β-sheet. An increase in ionic strength, whether or not Ca²⁺ is present, increases α-helix at the expense of “other” (coil) structure. The changes in β-sheet and β-turns are insignificant. Binding of Ca²⁺ at low ionic strength occurs in stages with at least one folding intermediate before attaining the final stable state. Binding of Ca²⁺ at an ionic strength of 0.165M causes only a slight increase in α-helix, so that the secondary structure of the protein depends on ionic strength and is insensitive to the nature of the cation (i.e., Ca²⁺). Thus, the activation of calmodulin by Ca²⁺ must be due to a structural reorientation rather than to a major secondary structural alteration. The CD estimation of secondary structure with 4 mol Ca²⁺/calmodulin (61% α-helix, 2% antiparallel β-sheet, 2% parallel β-sheet, 21% β-turns, and 14% other) is in excellent agreement with the x-ray results.     

β-Galactosidase from Lactobacillus pentosus: Purification,characterization and formation of galacto-oligosaccharides

A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U_oNPG/mg protein. The K_m, k_cat and k_cat/K_m values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 38 mM, 20 s^-1, 530 M^-1·s^-1 and 1.67 mM, 540 s^-1, 325 000 M^-1·s^-1, respectively. The temperature optimum of β-galactosidase activity was 60–65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg²⁺ ions enhanced both activity and stability significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D -Glc, β-D-Galp-(1→3)-D -Glc, β-D -Galp-(1→6)-D -Gal, β-D -Galp-(1→3)-D -Gal, and the trisaccharides β-D -Galp-(1→3)-D -Lac, β-D -Galp-(1→6)-D -Lac.     

Glycoside‐specific glycosyltransferases catalyze regio‐selective sequential glucosylations for a sesame lignan,sesaminol triglucoside

Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid‐related plant specialized metabolites. (+)‐Sesamin and (+)‐sesamolin are major hydrophobic lignans, whereas (+)‐sesaminol primarily accumulates as a water‐soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6‐O‐glucosidic linkages [i.e. (+)‐sesaminol 2‐O‐β‐d ‐glucosyl‐(1→2)‐O‐β‐d ‐glucoside‐(1→6)‐O‐β‐d ‐glucoside]. We previously reported that the 2‐O‐glucosylation of (+)‐sesaminol aglycon and β1→6‐O‐glucosylation of (+)‐sesaminol 2‐O‐β‐d ‐glucoside (SMG) are mediated by UDP‐sugar‐dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2‐O‐glucosylation of SMG and (+)‐sesaminol 2‐O‐β‐d ‐glucosyl‐(1→6)‐O‐β‐d ‐glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside‐specific glycosyltransferases (GGTs) and co‐ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG‐deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar–sugar β1→6‐O‐glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two‐hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane‐associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane‐bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β‐O‐glucosylation sequence of STG biosynthesis is β1→2‐O‐glucosylation of SMG by UGT94AG1 followed by UGT94AA2‐mediated β1→6‐O‐glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio‐selectivity of sequential glucosylations catalyzed by GGTs.     

Random-phase calculation of the low-energy circular dichroism in glucans

The CD of the long-wavelength electronic transition in the α-(1 → 4)-linked glucan dimer (maltose) and polymer (amylose), and in the β-(1 → 6)-linked dimer (gentiobiose) and polymer (pustulan), are calculated in the random-phase approximation using time-dependent Hartree theory. This long-wavelength transition (180–190 nm) is assumed to be localized on the linkage oxygen atom and to be of a σ*/3 s ← n character. The zerothorder σ*/3s ← n magnetic-dipole transition moment is coupled to the CC, CO, and CH bond density of states via a polarizability approximation. We assume this coupling is dominated energetically by the electric-dipole, electric-dipole interaction terms in the context of Schellman's μ_e-μ_m coupling mechanism of rotatory power. The CD is calculated as a function of rotation about the two single bonds of the linkage oxygen atom and also as a function of rotation about the C(5)-C(6) bond. For maltose, four rotational isomers are considered, resulting from combinations of the gg and gt C(6)H₂OH group rotational isomers. The calculated CD values were then Boltzmann-averaged over an empirical potential, and the resulting CD was found to compare satisfactorily with experiment. In the case of the polymers, only structures having periodicity (helicity) were examined.     

Cross-Reactive Polysaccharides from Trypanosoma cruzi and Fungi (Especially Dactylium dendroides)1

ABSTRACT. Cross-reactivity between fungal and Trypanosoma cruzi polysaccharides, owing to common residues of β-D-galactofuranose, β-D-galactopyranose, and α-D-mannopyranose, was demonstrated by using a) rabbit immune sera against T. cruzi epimastigotes and b) sera from patients with Chagas’disease. Several chagasic (Ch) sera precipitated partly purified galactomannans from Aspergillus fumigatus and from T. cruzi epimastigotes and also the galactoglucomannan from Dactylium dendroides. Reaction of one Ch serum with T. cruzi galactomannan (GM) was completely inhibited by synthetic β-D-Galf-(1 → 3)-Me α-D-Manp, and that of another Ch serum with a purified D. dendroides galactoglucomannan (GGM) was partly inhibited by (1 → 6)-linked (81%) or by (1 - 3)-linked (33%) β-D-Galf-Me α-D-Manp. The β-D-Galf-(1 → 3)-α-D-Manp epitope was present in both T. cruzi and D. dendroides polysaccharides. Rabbit anti-T. cruzi antisera precipitated A. fumigatus GM, T. cruzi antigenic extracts containing the lipopeptidophosphoglycan (LPPG), T. cruzi alkali-extracted GM, a synthetic GM, and D. dendroides GGM. Weak reactivities were obtained for a Torulopsis lactis-condensi GM containing β-D-Galp terminal residues and for baker's yeast mannan with α-D-Manp-(1 - 3)-α-D-Manp-(1- → 2)-α-D-Manp-(1 → 2) side chains. An anti-LPPG rabbit serum precipitated D. dendroides GGM—a reaction inhibited (82%) by β-D-Galf-(1 → 3)-Me α-D-Manp and, less efficiently, by a (1 → 5)-linked β-D-Galf-tetrasaccharide. Sera from mice immunized with D. dendroides whole cells reacted with CL-strain trypomastigotes as shown a) by indirect immunofluorescence, b) by a Staphylococcus adherence test, but were not lytic. Mice immunized with D. dendroides were not protected against a challenge with virulent T. cruzi trypomastigotes.     

Structure of Succinoglucan: Fragmentation by Partial Acid Hydrolysis

Succinoglucan, a succinylated polysaccharide produced by Alcaligenes faecalis var. myxogenes 10C3, was partially hydrolyzed with acid. Fractionation of the neutral oligosaccharides gave cellobiose, gentiobiose, laminaribiose, laminaritriose, 6-O-β-laminaribiosylglucose, 6-O-β-laminaritriosylglucose, and 3-O-β-cellobiosylgalactose, confirming the previous results that the polysaccharide consists of β-(l→3)-linked, (1→4)-linked and (1 →6)-linked d-glucose residues, and β-(1→3)-linked d-galactose residues.

Possible structural features of succinoglucan were discussed on the basis of the above and previous results obtained by Smith degradation.     

Vacuum ultraviolet circular dichroism spectrum of beta-turn in solution.

The vacuum ultraviolet circular dichroism spectrum of an isolated 4 → 1 hydrogen bonded β-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at ? 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the β-turn conformation. This spectrum, particularly the presence of a strong negative band around 180 nm and a large ratio $\text{[θ]}{}_{201}\text{[θ]}{}_{225}$, can be taken as a characteristic feature of the isolated β-turn conformation. These CD spectral features can thus be used to distinguish the β-turn conformation from the β-structure in solution.     

Circular dichroism of type 13 β-turn in linear tripeptides containing L-proline and D-alanine

The linear tripeptides tBoc-L-Prolyl-D-alanyl-L-leucine and tBoc-L-prolyl-D-alanyl-L-valine have been shown, from circular dichroism (CD) and infrared spectral data, to take up the 4 → 1 hydrogen bonded β-turn conformation in organic solvents. The CD spectra of these tripeptides in trifluoroethanol exhibit a positive n → π^★ band around 220 nm contrary to the usual negative band observed for the type II β-turn. The observed CD spectra of the tripeptides provide the first examples of those predicted theoretically by Woody for peptides containing L,D sequences and adopting the Venkatachalam type 13 β-turn. This conformation is seen to revert to the type II β-turn when the N-terminal protecting group is acetyl or when the C-terminal residue is glycine. These data are shown to have a direct bearing on the interpretation of the CD spectra of globular proteins.     

Dynamic fluorescence in copper proteins Selected examples

Summary The fluorescence properties of three copper proteins, namely human superoxide dismutase,Pseudomonas aeruginosa azurin andThiobacillus versutus amicyanin have been studied. All these proteins show a non-exponential decay of fluorescence, though the tryptophanyl residues responsible for the emission are very differently located in the three proteins. All the three decays can be fitted by at least two lifetimes or better with one or two lorentzian-shaped, continuous distributions of lifetime. In each case the removal of copper affects the quantum yield of fluorescence without affecting the shape of the emission.