Hydrodynamic and spectroscopic studies of substrate binding to human recombinant deoxycytidine kinase

Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, plays a key role in the activation of therapeutic nucleoside analogues by their 5'-phosphorylation. The structure of human dCK is still not known and the current work was undertaken to determine its oligomeric and secondary structure. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 +/- 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of -11,900 +/- 300 and -12,500 +/- 300 deg x cm2 x dmol(-1), respectively, indicating the presence of approximately 40% alpha-helix and 50% beta-structure. Circular Dichroism studies in the aromatic and far-ultraviolet range and UV difference spectroscopy indicated that binding of substrates to dCK reduced its alpha-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that deoxycytidine (the phosphate acceptor) and ATP (the phosphate donor) bound to different sites on dCK and fluorescence quenching revealed bimodal binding of deoxycytidine and unimodal binding of ATP. Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates. These results are consistent with a random bi-bi kinetic mechanism of phosphorylation of dCyd with either ATP or UTP.     

Influence of secondary structure of polyglycine on some conformational properties

Values of four conformational properties, namely unperturbed dimension [r2]0, dipole moment [mu 2], mean squared optical anisotropy [gamma 2], and molar Kerr constant [mK], have been calculated for polyglycine chains allowing several combinations of the secondary structure with the aim of studying the dependence of these magnitudes on the secondary structure of the chain. Two different approaches to the secondary structure have been used. In the first, chains with all their units in a given conformation (random coil, alpha-helix or beta-sheet) are interrupted at several positions by one unit in a different conformation. In the second, chains with varying composition of two conformations alpha-helix/beta-sheet and beta-sheet/random coil were allowed and the results obtained compared with previous work for alpha-helix/random coil chains.     

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant. The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm. CD spectral analyses with three different methods (S.W. Provencher, J. Gl?ckner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y. Yang, C.-S.C. Wu, H.Z. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208-269; N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet. Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG. These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.     

Characterization of amphiphilic secondary structures in neuropeptide Y through the design, synthesis, and study of model peptides

Neuropeptide Y (NPY) has the potential to form two amphiphilic secondary structures: a polyproline II-like helix in residues 1-8, and an alpha-helix in residues 13-32. NPY dimerizes in aqueous solution and forms stable monolayers at the air-water interface, suggesting that these amphiphilic conformations are stabilized at interfaces. Furthermore, the negative molar ellipticity of monomeric NPY at 222 nm (-8500 degree cm2/dmol), suggests that hydrophobic interactions with the NH2-terminal amphiphilic structure may stabilize the alpha-helix in residues 13-32 before it binds to cell surfaces, even at physiological concentrations. In order to investigate the role of these amphiphilic structures, five NPY models with multiple substitutions in positions 13-32 have been synthesized and studied. Our data demonstrate that the surfactant properties of NPY result from its potential to form amphiphilic secondary and tertiary structures and not from specific amino acid sequences in this region. However, specific residues on the hydrophilic face of the amphiphilic alpha-helix that have been substituted in the models appear to be required to reproduce the full potency of NPY in our pharmacological assays. A possible role for the amphiphilic structures in NPY in presenting such specific determinants to cell surface receptors in the correct conformation is suggested.     

pH- and thermal-dependent conformational transition of PGAIPG, a repeated hexapeptide sequence from tropoelastin

The secondary structure of PGAIPG (Pro-Gly-Ala-IIe-Pro-Gly), a repeated hexapeptide of tropoelastin, in buffer solution of different pH was determined by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The thermal-dependent structural change of PGAIPG in aqueous solution or in solid state was also examined by thermal FTIR microspectroscopy. The conformation of PGAIPG in aqueous solution exhibited a pH-dependent structural characterization. A predominant peak at 1614 cm(-1) (aggregated beta-sheet) with a shoulder near 1560 cm(-1) (beta-sheet) appeared in pH 5.5-8.5 buffer solutions. A new broad shoulder at 1651 cm(-1) (random coil and/or alpha-helix) with 1614 cm(-1) was observed in the pH 4.5 buffer solution. However, the broad shoulder at 1651 cm(-1) was converted to a maximum peak at 1679 cm(-1) (beta-turn/antiparallel beta-sheet) when the pH shifted from 4.5 to 3.5, but the original pronounced peak at 1614 cm(-1) became a shoulder. Once the pH was lowered to 2.5, the IR spectrum of PGAIPG was dominated by major absorption at 1679 cm(-1) with a minor peak at 1552 cm(-1) (alpha-helix/random coil). The result indicates that the pH was a predominant factor to transform PGAIPG structure from aggregated beta-sheet (pH 8.5) to beta-turn/intermolecular antiparallel beta-sheet (pH 2.5). Moreover, a partial conformation of PGAIPG with minor alpha-helix/random coil structures was also explored in the lower pH buffer solution. There was no thermal-dependent structural change for solid-state PGAIPG. The thermal-induced formation of aggregated beta-sheet for PGAIPG in aqueous solution was found from 28 to 30 degrees C, however, which might be correlated with the formation of an opaque gel that turned from clear solution. The formation of aggregated beta-sheet structure for PGAIPG beyond 30 degrees C might be due to the intermolecular hydrogen bonded interaction between the hydrophobic PGAIPG fragments induced by coacervation.     

Secondary structural analysis of the Na(+),K(+)-ATPase and its Na(+) (E(1)) and K(+) (E(2)) complexes by FTIR spectroscopy

The Na(+),K(+)-ATPase is an integral membrane protein which transports sodium and potassium cations against an electrochemical gradient. The transport of Na(+) and K(+) ions is presumably connected to an oscillation of the enzyme between the two conformational states, the E(1) (Na(+)) and the E(2) (K(+)) conformations. The E(1) and E(2) states have different affinities for ligand interaction. However, the determination of the secondary structure of this enzyme in its sodium and potassium forms has been the subject of much controversy. This study was designed to provide a quantitative analysis of the secondary structure of the Na(+),K(+)-ATPase in its sodium (E(1)) and potassium (E(2)) states in both H(2)O and D(2)O solutions at physiological pH, using Fourier transform infrared (FTIR) with its self-deconvolution and second derivative resolution enhancement methods, as well as curve-fitting procedures. Spectroscopic analysis showed that the secondary structure of the sodium salt of the Na(+),K(+)-ATPase in H(2)O solution contains alpha-helix 19.8+/-1%, beta-sheet 25.6+/-1%, turn 9.1+/-1%, and beta-anti 7.5+/-1%, whereas in D(2)O solution, the enzyme shows alpha-helix 16.8+/-1%, beta-sheet 24.5+/-1.5%, turn 10.9+/-1%, beta-anti 9.8+/-1%, and random coil 38.0+/-2%. Similarly, the potassium salt in H(2)O solution contains alpha-helix 16.6+/-1%, beta-sheet 26.4+/-1.5%, turn 8.9+/-1%, and beta-anti 8.1+/-1%, while in D(2)O solution it shows alpha-helix 16.2+/-1%, beta-sheet 24.5+/-1.5%, turn 10.3+/-1%, beta-anti 9.0+/-1%, and random coil 40+/-2%. Thus the main differences for the sodium and potassium forms of the Na(+),K(+)-ATPase are alpha-helix 3.2% in H(2)O and 0.6% in D(2)O, beta-sheet (pleated and anti) 1.5% in H(2)O and random structure 2% (D(2)O), while for other minor components (turn structure), the differences are less than 1%.     

Thermal-induced changes in the secondary conformation of superoxide dismutase containing different metal ions

The thermal stability of three superoxide dismutases (SODs) with different metal ions (Mn, Cu/Zn, Fe) in the solid state was studied by a Fourier transform infrared (FT-IR) microspectroscopy combined with thermal analyzer. The IR spectra showed a maximum peak at 1652 cm(-1) for all the native SODs in the amide I band, suggesting a predominant random coil with less alpha-helix structures. By heating each sample, a shoulder at 1631 cm(-1) in the amide I band gradually appeared from 45 degrees C for Fe SOD and from 50 degrees C for Mn SOD but another shoulder at 1639 cm(-1) appeared from 50 degrees C for Cu/Zn SOD. The peak at 1631 cm(-1) is due to the intermolecular beta-sheet structure, but the peak at 1639 cm(-1) corresponds to the major intramolecular beta-sheet with less random coil structure. This reveals that in the first heating process the transformation from random coil/alpha-helix structure to beta-sheet structure initiated from around 45-50 degrees C. There was about 16-22% compositional change resulting from that transformation. However, both additional shoulders stood there and did not restore to their original spectra even with cooling to room temperature, suggesting the denaturation and irreversible properties of the solid SODs after heating. The thermal-dependent denaturation and irreversibility of Mn SOD, Cu/Zn SOD and Fe SOD were clearly evidenced by the increase in intramolecular and intermolecular beta-sheet structure.     

Pressure-induced transformation of alpha-helix to beta-sheet in the secondary structures of amyloid beta (1-40) peptide exacerbated by temperature

The effect of pressure on the conformational structure of amyloid beta (1-40) peptide (A beta(1-40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid A beta(1-40) from 1655 cm(-1) (alpha-helix) to 1647-1643 cm(-1) (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm(-1) assigned to beta-antiparallel sheet structure was also evident. Furthermore, the peak at 1540 cm(- 1) also shifted to 1527 (1529) cm(-1) in amide II band. The former was assigned to the combination of alpha-helix and random coil structures, and the latter was due to beta-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed A beta(1-40) samples were obtained from 33% to 22% for alpha-helix/random coil structures and from 47% to 57% for beta-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from alpha-helix to random coil and to beta- sheet structure. The structural transformation of the compressed A beta(1-40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of beta-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the beta-sheet structure transformed. The thermal-dependent transition temperatures of solid A beta(1-40) prepared by different pressures were near 55-60 degrees C.     

Alpha-helix to random coil transitions: determination of peptide concentration from the CD at the isodichroic point.

A method is presented for determining the concentrations of peptides and proteins having isodichroic points near 203 nm. The existence of an isodichroic point for a given substance indicates a local two-state (alpha-helix, random coil) population. The mean residue ellipticity at the isodichroic point, [theta lambda i], is, of course, independent of helix content. For a wide variety of synthetic and natural peptides, including both single helices and coiled coils, it is shown that [theta lambda i] is also essentially independent of substance and of whether the transition is induced by temperature, ionic strength, pH, chain length changes, amino acid substitution, or solvent perturbation. Averaging [theta lambda i] values culled from various laboratories gives -151 +/- 16 (SD, 7 sources) deg.cm2.mmol-1. In our laboratory, nonpolymerizable rabbit alpha-tropomyosin and two alpha-tropomyosin subsequences yield -135 +/- 10 (SD, 190 values) deg.cm2.mmol-1. Thus, given [theta lambda i] for a peptide of known concentration, it is possible to estimate the concentration of any other peptide provided that it has an isodichroic point at which the ellipticity is accurately measurable. It is then possible to calculate [theta lambda] at any other wavelength for which theta is known. It is advisable to determine [theta lambda i] for the best known peptide in one's own laboratory, since it depends on absolute instrument and cell calibrations and an absolute concentration determination.     

Conformational study of purified epoxide hydrolase from rat liver

Hepatic epoxide hydrolase (EC 3.3.2.3) was purified from phenobarbital-treated rats by ion-exchange chromatography followed by hydrophobic chromatography. The enzyme had a specific activity of 300--400 nmol min-1 mg-1 protein with benzo[a]pyrene-4,5-oxide as the substrate. Circular dichroism (CD) spectra of the purified enzyme gave two negative bands, centered at 210 nm and 222 nm, respectively. The mean residue ellipticity at 222 nm was 12,9000 deg X cm(2) X dmol(-1), which indicated the presence of about 35% alpha-helical structures. Sodium dodecyl sulfate (SDS) greatly affected the shape of the CD spectra, which were gradually shifted to the blue. This suggested a decrease in the aggregation state of the protein. Electrostatic interactions were important in the organization of the enzyme structure since the conformation was stable between pH 7.4 and pH 10. At pH-values 5.0, 6.0 and 12.0, the CD bands underwent considerable changes in both amplitude and shape. Moreover there was a good correlation between the optimal pH range of the epoxide hydrolase activity and the organization state of the protein. After membrane reconstitution with liposomes, the conformation of the enzyme was not significantly modified by the presence of dimyristoyl L-alpha-phosphatidylcholine or other phospholipids. This constancy was obtained over a wide range of molar ratios of phospholipids to protein (0--500). However, phospholipids did increase the thermal stability of the enzyme. Fluorescence measurements of diphenylhexatriene (DPH) bound to dimyristoyl L-alpha-phosphatidylcholine indicated that addition of epoxide hydrolase modified the thermal transition of the lipid phase. On the other hand, electron paramagnetic resonance (EPR) signals of the nitroxide-labelled fatty acid, 2-(14-carboxy-tetradecyl)-2-ethyl-4,4-dimethyl-3,3,-oxazolidiny-oxyl, bound to the phospholipid, indicated that the presence of the protein decreased by about 53% the correlation time of the label, suggesting that its motion had increased. In conclusion, phospholipid-epoxide hydrolase interactions enhanced the fluidity of dimyristoyl L-alpha-phosphatidylcholine liposomes without changing the secondary structure of the enzyme. Electrostatic interactions also played an important role in the conformational stability of the protein.     

Effects of cyclic GMP on the secondary structure of cyclic GMP dependent protein kinase and analysis of the enzyme's amino-terminal domain by far-ultraviolet circular dichroism

Far-UV circular dichroism spectra of bovine lung cyclic GMP dependent protein kinase (G-kinase) show that the enzyme contains alpha-helical and beta-pleated sheet elements. Binding of cyclic GMP changes the spectra in a way consistent with the induction of beta-sheet from random coil. Examination of the amino-terminal sequence of G-kinase indicates the presence of a strongly alpha-helical segment with several features in common with the leucine zipper motif. We propose that this sequence may be the important part of the dimerization domain of the enzyme. A synthetic peptide corresponding to amino acids 1-39 of G-kinase has a strongly alpha-helical CD spectrum, supporting the predicted secondary structure of this amino-terminal sequence. In contrast to the native enzyme, a structure reduced in alpha-helix was found when a constitutively active form of G-kinase, which lacks amino acids 1-77, was studied.     

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.     

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.     

Peptide-chain secondary structure of bacteriorhodopsin.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.     

Conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state 13C NMR

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local alpha-helical form is present around Gly10 whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local beta-sheet form as viewed from Gly10 and Phe22, and both beta-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from alpha-helix to beta-sheet, and from random coil to beta-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local beta-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic alpha-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel beta-sheets at pH 7.5 and a mixture of antiparallel and parallel beta-sheets at pH 3.3.     

Conformational properties of a polyglycine chain with secondary and tertiary structures of lysozyme

Unperturbed dimension mean value of r2(0), dipole moment mean value of mu2, mean squared optical anisotropy mean value of gamma 2 and molar Kerr mean value of mK constant of a polyglycine chain with the secondary and tertiary structures of lysozyme have been calculated and the results compared with polyglycine chains with the same number of repeat units but different conformations including alpha-helix, beta-sheet or random coil. Thus, the influence of secondary and tertiary structures can be investigated. The results obtained show that for mean value of r2 and mean value of gamma 2 this influence is at least of the same order of magnitude as that of the primary structure, and is much greater for mean value of mu 2 and mean value of mK.     

Secondary conformations and temperature effect on structural transformation of amyloid beta (1-28), (1-40) and (1-42) peptides

Secondary structure of three amyloid b-peptides [A beta(1-28), A beta(1-40) and A beta(1-42)] in the solid state was respectively determined by Fourier transform infrared (FT-IR) microspectroscopy. Their thermal-dependent structural transformation were also investigated by FT-IR microspectroscopy equipped with a thermal analyzer. The present result demonstrates that the solid-state A beta(1-28), A beta(1-40) and A beta(1-42) peptides showed a significant IR spectral difference in the amide I and II bands. The secondary conformation of A beta(1-28) peptide was the combination of major beta-sheet and minor alpha-helix with little random coil structures, but A beta(1-40) peptide showed the co-existence of major beta-sheet and minor random coil with little alpha-helix structures. A beta(1-42) peptide mainly consisted of the predominant b-sheet structure. Although the intact A beta(1-28), A beta(1-40) or A beta(1-42) peptide exhibits a different secondary structure, a similar beta-conformation may form after thermal treatment. A thermal-dependent transition was found for solid A beta(1-28) and A beta(1-40) peptides near 40 degrees C and 45 degrees C, respectively. There was no transition temperature for solid A beta(1-42) peptide, however, due to only a very little level of alpha-helix and random coil structure containing in the solid A beta(1-42) peptide. The thermal denaturation plays an important role in the structural transformation from alpha-helix/random coil to beta-sheet.     

Structure of glutamate decarboxylase from E. coli: spectral studies

Structure of recombinant glutamate decarboxylase (GAD alpha) was studied by optical methods and electron microscopy. The active (pH 4.6) and inert (pH 6.3) holoGAD and apoGAD were investigated. Absorption and CD spectra were recorded in the range of 190 - 500 nm. Visible spectra were resolved into the bands corresponding to individual electron transitions using lognormal curves. The structures of predominant tautomers of internal aldimines were determined as ketoenamine at pH 4.6 and enolimine at pH 6.3. CD spectra show that holoGAD and apoGAD exhibit a negative band at 204 - 245 nm and a positive band near 190 - 204 nm. The contents of the secondary structure elements were estimated on the basis of the values of the mean residue ellipticity. Evidently, the main difference between the GAD forms studied is in the content of alpha-helix and random coil. HoloGAD has 50% of alpha-helix at pH 4.6 and 67% at pH 6.3, whereas apoGAD - 17 and 27%, respectively. Thus presented data establish the essential role of pyridoxal phosphate (PLP) in the organization of the GAD secondary structure due to tightening its polypeptide chain. It seems possible, that conformational changes induced by PLP binding stabilize the protein structure and promote the assembly of subunits into macromolecule, which was confirmed by electron microscopy.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.     

Interaction of rabbit hemopexin with bilirubin

The interaction of hemopexin with bilirubin was characterized by spectrophotometric, fluorimetric and circular dichroic techniques. Hemopexin rapidly forms an equimolar complex with libirubin that has an apparent dissociation constant Kd, of 7.5.10(-7) M. The association alters the absorption band of bilirubin near 150 nm, quenches the fluorescence of tryptophan residues of hemopexin, enhances the fluorescence of bilirubin, and induces strong ellipticity extrema in bilirubin of --60 . 10(3) deg . cm2 . dmol-1 at 465 nm and +70 . 10(3) deg . cm2 . dmol-1 at 415 nm. However, the conformation-sensitive ellipticity aband at 231 nm of hemopexin is not altered. In displacement experiments using circular dichroism, heme readily replaced bound bilirubin, indicating that bilirubin and heme are bound at the same site on hemopexin. Even at molar ratios of hemopexin to albumin of 3 to 1, human serum albumin removes bilirubin from hemopexin. Hemopexin is thus unlikely to have a role in the transport of bilirubin in serum.