The effect of digging on development of adult characters in blowflies Calliphora erythrocephala

Digging delays expansion after emergence of adult Calliphora. If flies are kept digging for 12 to 14 hr they lose the capacity to expand and, if they are then reared for 10 days, do not develop the normal adult corpus allatum, ovaries, and cuticle. In particular, ultrastructural examination of the resilin tendon of the pleurotergal muscle shows that development is arrested at a stage similar to that in the late pharate adult but the resilin is not cross-linked. It is suggested that bursicon release is irreversibly inhibited in flies that fail to expand normally.     

Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme.

Chemical shifts of resonances of specific protons in the 1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded "molten globule" state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.     

β‐amyloid model core peptides: Effects of hydrophobes and disulfides

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of β‐amyloid (Aβ21–30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aβ, and variants of these either cyclized or with an N‐terminal Cys. While Aβ21–30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aβ16–34 led to formation of typical amyloid fibrils. NMR showed no long‐range nuclear overhauser effect (nOes) in Aβ21–30, Aβ16–34, or their variants, however. Serial ¹H‐¹⁵N‐heteronuclear single quantum coherence spectroscopy, ¹H‐¹H nuclear overhauser effect spectroscopy, and ¹H‐¹H total correlational spectroscopy spectra were used to follow aggregation of Aβ16–34 and Cys‐Aβ16–34 at a site‐specific level. The addition of an N‐terminal Cys residue (in Cys‐Aβ16–34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aβ16–34 and Cys‐Aβ16–34, according to which Cys‐Aβ16–34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.     

Structural Lability of Barley Stripe Mosaic Virus Virions

Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.     

Constraining ribosomal RNA conformational space

Despite the potential for many possible secondary-structure conformations, the native sequence of ribosomal RNA (rRNA) is able to find the correct and universally conserved core fold. This study reports a computational analysis investigating two mechanisms that appear to constrain rRNA secondary-structure conformational space: ribosomal proteins and rRNA sequence composition. The analysis was carried out by using rRNA–ribosomal protein interaction data for the Escherichia coli 16S rRNA and free energy minimization software for secondary-structure prediction. The results indicate that selection pressures on rRNA sequence composition and ribosomal protein–rRNA interaction play a key role in constraining the rRNA secondary structure to a single stable form.     

Hormonal control of resilin deposition in the blowfly, Calliphora erythrocephala

The deposition of the resilin tendon in the blowfly Calliphora erythrocephala was investigated in normal and in various experimental conditions. The results showed that the weight of the protein resilin that is deposited is controlled by diet as well as by the hormone secreted by the medial neurosecretory cells.Endocrinologically abnormal Calliphora adults deposited a tendon with normal ultrastructure but showed signs of premature ageing while Calliphora fed on a sugar diet deposited a tendon with abnormal ultrastructure.     

Acceleration of the folding of acylphosphatase by stabilization of local secondary structure

The addition of trifluoroethanol or hexafluoroisopropanol converts the apparent two-state folding of acylphosphatase, a small alpha/beta protein, into a multistate mechanism where secondary structure accumulates significantly in the denatured state before folding to the native state. This results in a marked acceleration of folding as revealed by following the intrinsic fluorescence and circular dichroism changes upon folding. The folding rate is at a maximum when the secondary-structure content of the denatured state corresponds to that of the native state, while further stabilization of secondary structure decreases the folding rate. These findings indicate that stabilization of intermediate structure can either enhance or retard folding depending on its nature and content of native-like interactions.     

Raman scattering of chymotrypsinogen A, ribonuclease, and ovalbumin in the aqueous solution and solid state

The Raman spectra of three globular proteins, beef pancreas chymotrypsinogen A, beef pancreas ribonuclease, and hen egg white ovalbumin have been obtained in the solid state and aqueous solution. X-ray diffraction and circular dichroism evidence have indicated that these proteins have a low α-helical content and a large fraction of the residues in the unordered and β-sheet conformation. The frequencies and intensities of the amide I and amide III lines are consistent with assignments based on the Raman spectra of polypeptides. The intense amide III lines observed in all the spectra would be expected for proteins with a low fraction of the residues in the α-helical conformation. Several spectra changes occur upon dissolution of the proteins in water and may be associated with further hydration of the proteins. The spectrum of thermally denatured chymotrypsinogen is presented. A 3 cm^–1 decrease in the frequency of the amide I line of the protein dissolved in D₂O upon heating was observed. This observation is consistent with a denaturation mechanism allowing only slight changes in the secondary structure but an increase in solvent penetration upon going from the native to the reversibly denatured state.     

1H-NMR relaxation studies of native and thermally denatured lysozyme solutions: an isotope dilution experiment

1H-NMR relaxation times are reported for native and thermally denatured lysozyme aqueous solutions measured as the function of the proton mole fraction in the sample. A two-exponential character of proton longitudinal relaxation function was observed for native lysozyme solutions: the fast component was attributed to the non-exchangeable protein protons, the slow one to water protons. Purely exponential decay of longitudinal magnetization was observed for the thermally denatured samples. This has been explained in terms of a fast spin exchange model. The contributions of the protein protons to the water proton relaxation rate in native and thermally denatured samples were determined, too.     

Circular dichroism of cysteine proteinases from papaya latex. Evidence of differences in the folding of their polypeptide chains

Two forms of proteinase Ω were isolated from a commercial preparation of chymopapain (EC 3.4.22.6) by means of cation-exchange liquid chromatography. Their circular dichroism (CD) spectra in the 182–320 nm region indicated that the two forms possess closely related structures. For comparison, we also recorded the CD spectra of chromatographically purified samples of papain (EC 3.4.22.2) and the most abundant form of chymopapain. According to the qualitative criteria proposed by Manavalan and Johnson ((1983) Nature 305, 831–832), the spectral characteristics of papain correctly indicate that this protein belongs to the α + β class. Proteinase Ω is also placed in the α + β category, while chymopapain seems to be an α/β protein. Qualitative estimation of secondary structures yielded contents of helices and parallel ß-sheet that were higher in the case of chymopapain. Thus, the results of this work suggest that there are some differences in the folding pattern of chymopapain with respect to the other two proteinases. This proposal seems unexpected when the high amino acid sequence identity among these enzymes is considered.     

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

Circular dichroism and the secondary structure of the ROF2 protein from Arabidopsis thaliana

The protein ROF2 from the plant Arabidopsis thaliana acts as a heat stress modulator, being involved in the long-term acquired thermotolerance of the plant. Here we investigate the relationship between the biological function and the structure of ROF2, inferred by circular dichroism (CD) spectroscopy. The far-UV CD spectra, analyzed with the CDPro and DICHROWEB program packages, yield the percentages of α-helices, β-sheets, unordered regions, turns and poly(Pro)II-helices in the secondary structure of ROF2. According to the analysis, the percentages of the structural elements of ROF2 are about 40% for β-sheets, 30% for unordered regions, 17% for turns, 10% for poly(Pro)II-helices and 3% for α-helices. The near-UV CD spectra suggest that ROF2 proteins can associate, forming super-secondary structures. Our CD experiments performed at temperatures between 5 °C and 97 °C indicate that the thermal denaturation of ROF2 caused by a raise in temperature up to 55 °C is followed by a thermal refolding of the protein as the temperature is raised further. The new secondary structure, acquired around 65 °C, remains stable up to 97 °C. The structural stability of ROF2 at high temperatures might play an important role in the experimentally observed thermotolerance of Arabidopsis thaliana.     

Partial analysis of the bands seen in circular dichroism spectra of green and blue-green algal cells and thylakoids

A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.     

Cooperative Unfolding of Residual Structure in Heat Denatured Proteins by Urea and Guanidinium Chloride

The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, α-chymotrypsinogen A and α-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (ΔG ⁰ _X→D) associated with the process heat denatured (X) state ↔ GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ΔG ⁰ _N→X (native (N) state ↔ X state), ΔG ⁰ _X→D and ΔG ⁰ _N→D (N state ↔ D state), and (c) there is not a good enthalpy difference between X and D states, which is the reason for the absence of endothermic peak in DSC scan for the transition, X state ↔ D state.     

Flow-induced structural transition in the beta-switch region of glycoprotein Ib

The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this article, we present a molecular-dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue β-switch region of platelet glycoprotein Ibα, for which crystal structures of its N-terminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a β-hairpin. Interaction between glycoprotein Ibα and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to β-hairpin transition in glycoprotein Ibα is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop→β-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side-group packing step.     

Conformational study of calf brain tubulin

The conformation of calf brain tubulin has been monitored by circular dichroism, optical rotatory dispersion, and spectrophotometric titration as a function of pH, temperature, ligand concentrations, and denaturants. At pH 7, calf brain tubulin maintains its structural integrity between 5 and 37 °C as determined by circular dichroism. Furthermore, the presence of MgCl ₂ up to 1.6 × 10^?2m does not induce any observable changes in the circular dichroism spectra, nor does 10^?4m CaCl₂. With increasing pH, the spectral data can best be described as a gradual loosening of the secondary structure between pH 7 and 9. Both spectral and titrimetric data suggest a major unfolding of tubulin between pH 9 and 10. The apparent pK of tyrosine shifts from 10.85 to 9.98 upon transferring from buffer to 6 m guanidine hydrochloride, indicating that at least 14 of the 15 tyrosine groups are not fully accessible to protons in the native protein. The single disulfide bridge in calf brain tubulin helps to maintain a domain which is highly resistant to unfolding by denaturants.     

Backbone resonance assignments of a promiscuous aminoglycoside antibiotic resistance enzyme; the aminoglycoside phosphotransferase(3′)-IIIa

The aminoglycoside phosphotransferase(3′)-IIIa (APH) is a promiscuous enzyme and renders a large number of structurally diverse aminoglycoside antibiotics useless against infectious bacteria. A remarkable property of this ~31 kDa enzyme is in its unusual dynamic behavior in solution; the apo-form of the enzyme exchanges all of its backbone amide protons within 15 h of exposure to D ₂ O while aminoglycoside-bound forms retain ~40% of the amide protons even after >90 h of exposure. Moreover, the number of observable peaks and their dispersion in HSQC spectra varies with each aminoglycoside, rendering the resonance assignments very challenging. Therefore, the binary APH–tobramycin complex, which shows the largest number of well-resolved peaks, was used for the backbone resonance assignments (Cα, C, N, H, and some Cβ) of this protein (BMRB-16337).     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations

Two different, theoretical studies of intramolecular proton-proton distances in polypeptide chains are described. Firstly, the distances between amide, C^α and C^β protons of neighbouring residues in the amino acid sequence, which correspond to the sterically allowed values for the dihedral angles φ_i, ψ_i and χ_i¹, were computed. Secondly, the frequency with which short distances occur between amide, C^α and C^β protons of neighbouring and distant residues in the amino acid sequence were statistically evaluated in a representative sample of globular protein crystal structures. Both approaches imply that semi-quantitative measurements of short, non-bonding proton-proton distances, e.g. by nuclear Overhauser experiments, should present a reliable and generally applicable method for sequential, individual resonance assignments in protein ¹H nuclear magnetic resonance spectra. Similar calculations imply that corresponding distance measurements can be used for resonance assignments in the side-chains of the aromatic amino acid residues, asparagine and glutamine, where the complete spin systems cannot usually be identified from through-bond spin-spin coupling connectivities.     

Improved sequence-based prediction of protein secondary structures by combining vacuum-ultraviolet circular dichroism spectroscopy with neural network

Synchrotron-radiation vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy can significantly improve the predictive accuracy of the contents and segment numbers of protein secondary structures by extending the short-wavelength limit of the spectra. In the present study, we combined VUVCD spectra down to 160 nm with neural-network (NN) method to improve the sequence-based prediction of protein secondary structures. The secondary structures of 30 target proteins (test set) were assigned into alpha-helices, beta-strands, and others by the DSSP program based on their X-ray crystal structures. Combining the alpha-helix and beta-strand contents estimated from the VUVCD spectra of the target proteins improved the overall sequence-based predictive accuracy Q(3) for three secondary-structure components from 59.5 to 60.7%. Incorporating the position-specific scoring matrix in the NN method improved the predictive accuracy from 70.9 to 72.1% when combining the secondary-structure contents, to 72.5% when combining the numbers of segments, and finally to 74.9% when filtering the VUVCD data. Improvement in the sequence-based prediction of secondary structures was also apparent in two other indices of the overall performance: the correlation coefficient (C) and the segment overlap value (SOV). These results suggest that VUVCD data could enhance the predictive accuracy to over 80% when combined with the currently best sequence-prediction algorithms, greatly expanding the applicability of VUVCD spectroscopy to protein structural biology.