The Intramolecular Impact to the Sequence Specificity of B→A Transition: Low Energy Conformational Variations in AA/TT and GG/CC Steps

Abstract It is well known, that local B→A transformation in DNA is involved in several biological processes. In vitro B?A transition is sequence-specific. The physical basis of this specificity is not known yet. Here we analyze the effect of intramolecular interactions on the structural behavior of the GG/CC and AA/TT steps. These steps exemplify sequence specific bias to the B- or A-form structure. Optimization of potential energy of the molecular systems composed of an octanucle-otide, neutralized by Na(+) and solvated with TIP3P water molecules in rectangular box with periodic boundary conditions gives the statistically representative sets of low energy structures for GG/CC and AA/TT steps in the middle of the diverse flanking sequences. Permissible 3D variations of GG/CC and AA/TT, and correlation of the relative motion of base pairs in these steps were analyzed. AA/TT step permits high variability for low energy conformers in the B-form DNA and small variability for low energy conformers in the A-form DNA. In contrast GG/CC step permits high variability for low energy conformers in the A-form DNA and small variability for low energy conformers in the B-form DNA. The relative motion of base pairs in GG/CC step is high correlated, while in AA/TT step this correlation is notably less. Atom-atom interactions inside-the-step always favors the B-form and their component - stacking interactions (atomatom interactions between nucleic bases) is crucial for the duplex stabilization. Formation of the A-form for both steps is a result of interactions with the flanking sequences and water-cation environment in the box. The average energy difference between conformations presenting B-form and A-form for the GG/CC step is high, while for the AA/TT step it is rather low. Thus, intramolecular interactions in GG/CC and AA/TT steps affect the possible conformational diversity ("conformational entropy") of the A- and B- type structures of DNA step. This determines the known bias of the A-form DNA depending on the enrichment of sequences with GG/CC. If structural tuning during the process of protein-DNA complex formation lead to the local B→A transformation of DNA, it is largely directed by high conformational diversity of GG/CC step in the A-form. In such a case the presence in the target site of both kinds of examined steps ensures the reversible character of ligand binding.     

The transitions between left- and right-handed forms of poly(dG-dC).

The circular dichroism study of water/trifluoroethanol (TFE) solutions of poly(dG-dC) has revealed the following: The polynucleotide is present as a B form up to a TFE content of 60% (v/v) or less. Then, a cooperative transition into a left-handed Z form occurs. Within the region of 66-78% TFE, a continuous non-cooperative change is going on which can be attributed to an intrafamily transition within the family of Z forms. At last, in the interval of 80-84% TFE, a second cooperative transition, probably, Z - A is realized. Both transitions, Z - A and Z - B, show slow kinetics (10-60 min) while the direct transitions from the A to B form taking less than 10 sec. The length of cooperativity for the B - Z transition, Vo = 25 base pairs was estimated using spermine molecules. Spermine was found to induce the B to Z transition in the (dG-dC) sequences even in the absence of TFE which might be biologically interesting.     

Differences in the effects of TFE on the folding pathways of human stefins A and B.

Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses

Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1-73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1-73) using NMR methods and describe the 2.1-A x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15-93). These results identify conserved dsRNA binding surfaces on both NS1A-(1-73) and NS1B-(15-93) that are very different from those indicated in earlier "working models" of the complex between dsRNA and NS1A-(1-73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an approximately 45 degrees angle relative to the axes of helices alpha2/alpha2'. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.     

Peculiarities of the B to A transition of the lambda phage regulatory site OR3 and of its fragment

Conformations of the synthetic deoxyoligonucleotide 17 base pairs long, which is an OR3 operator of lambda phage, and of its 9-b.p. fragment were studied by the circular dichroism method (CD). The regions of stability of the double-stranded state were determined for these duplexes. A comparison of the CD spectra for these oligonucleotides with the CD for a lengthy DNA showed the conformation of these short DNA pieces to belong to the B-family. A cooperative change in the CD spectra is observed in trifluoroethanol (TFE) solutions at a TFE concentration specific for each oligonucleotide, which is supposed to stem from a B to A transition. The length of the fragment was found to affect the ability for the B-A transition. The transition into the A form is hindered by 13% TFE for the short 9-nucleotide in comparison with the 17-nucleotide. We suggest that this is due to the B form stabilization by terminal base pairs (B-phility of the ends).     

B-form and A-form DNA in an electric field

The structures of B-form and A-form DNA are studied in 0–70 and 70–80% ethanol solutions, respectively, in an electric field, using linear dichroism. The limiting reduced linear dichroism data of B-form DNA are chain length dependent in 0% ethanol solution. However, there is no such chain length dependence of the limiting reduced linear dichroism of the A-form. Our reslts also suggest that (1) the transition moments at 260 nm lie within the plaxe of the DNA bases, (2) the two allomorphs (A and B forms) of the long chain DNA in solution in the electric field are like the respective classica forms.     

Peculiarities of the B to A Transition of the λ Phage Regulatory Site OR3 and of Its Fragment

Abstract

Conformations of the synthetic deoxyoligonucleotide 17 base pairs long, which is an OR3 operator of λ phage, and of its 9-b.p. fragment were studied by the circular dichroism method (CD). The regions of stability of the double-stranded state were determined for these duplexes. A comparison of the CD spectra for these oligonucleotides with the CD for a lengthy DNA showed the conformation of these short DNA pieces to belong to the B-family.

A cooperative change in the CD spectra is observed in trifluoroethanol (TFE) solutions at a TFE concentration specific for each oligonucleotide, which is supposed to stem from a B to A transition. The length of the fragment was found to affect the ability for the B-A transition. The transition into the A form is hindered by 13% TFE for the short 9-nucleotide in comparison with the 17-nucleotide. We suggest that this is due to the B form stabilization by terminal base pairs (B-phility of the ends).     

Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.     

Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit

The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.     

Does trifluoroethanol affect folding pathways and can it be used as a probe of structure in transition states?

Nonaqueous co-solvents, particularly 2,2,2-trifluoroethanol (TFE), have been used as tools to study protein folding. By analyzing FKBP12, an alpha/beta-protein that folds with two-state kinetics, we have been able to address three key questions concerning the use of TFE. First, does TFE perturb the folding pathway? Second, can the observed changes in the rate of folding and unfolding in TFE be attributed to a change in free energy of a single state? Finally, can TFE be used to infer information on secondary structure formation in the transition state? Protein engineering experiments on FKBP12, coupled with folding and unfolding experiments in 0% and 9.6% TFE, conclusively show that TFE does not perturb the folding pathway of this protein. Our results also suggest that the changes in folding and unfolding rates observed in 9.6% TFE are due to a global effect of TFE on the protein, rather than the stabilization of any elements of secondary structure in the transition state. Thus, studies with TFE and other co-solvents can be accurately interpreted only when combined with other techniques.     

In vitro study of stability and amyloid-fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-alpha-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.     

Photochemical probing of the B--a conformational transition in a linearized pUC19 DNA and its polylinker region

We induced the B-to-A conformational transition by ethanol in a linearized pUC19 DNA. A primer extension method was used in combination with UV light irradiation to follow the transition, based on pausing of DNA synthesis due to the presence of damaged bases in the template. Primer extension data highly correlated with the results of another method monitoring the B-A transition, i.e. inhibition of restriction endonuclease cleavage of UV light-irradiated DNA. Primer extension enabled us to locate damaged nucleotides within the region of interest. Most damaged nucleotides were located in B-form trimers, exclusively containing both pyrimidine bases (TTC, TCT, CTC, and CTT), and in a cytosine tetramer. The amount of damaged bases decreased in the course of B-A transition. Some of the damage even disappeared in the A-form, which mainly concerns the C(4) and C(3) blocks. The cleavage was nearly restored in the A-form within this region (Eco88I). On the contrary the decrease of damage was less significant with thymine dimers, only dropping to 50-60% of the B-form level. Consequently, the cleavage with EcoRI and HindIII remained mostly as before the transition (75% and 60% of uncleaved DNA preserved). We found significant differences in the B- and A-form pattern of UV light-damaged bases within the same region (polylinker) of DNA embedded within long (plasmid) or short (127 bp fragment) DNA molecules. The B-A transition of the fragment was found less cooperative than with linearized plasmid, which was confirmed by both CD spectroscopy and restriction cleavage inhibition.     

Mechanochemical study of NaDNA and NaDNA-netropsin fibers in ethanol-water and trifluoroethanol-water solutions.

Highly oriented calf-thymus NaDNA fibers, prepared by a wet-spinning method, were complexed with netropsin in ethanol-water and trifluoroethanol (TFE)-water solutions. The relative fiber length, L/L0, was measured at room temperature as a function of ethanol or TFE concentration to obtain information on the B-A conformational transition. The B-A transition point and transition cooperativity of the fibers were calculated. The binding of netropsin to NaDNA fibers was found to stabilize B form and to displace the B-A transition to higher ethanol concentration, as indicated by its elongational effect on the fiber bundles. An increased salt concentration was found to reduce netropsin binding. In netropsin-free ethanol solution, the dissociation of bound netropsin from the DNA fibers was observable. Pure B-NaDNA fibers were found to be more stable in TFE solution than in ethanol solution. This was interpreted as being due to a different steric factor and a larger polarity of TFE compared with ethanol, resulting in its smaller capacity to reduce the water activity and dielectric constant of the medium in the immediate vicinity of DNA fibers. Therefore, the effect of netropsin binding on the B-A transition of NaDNA fibers became less obvious in TFE solution. In another series of experiments, L/L0 was measured as a function of temperature to obtain information on the helix-coil transition, or melting, as well as the B-A transition of NaDNA and NaDNA-netropsin fibers. The melting temperature and helix-coil transition width were calculated from the melting curves. A phenomenological approach was used to describe the melting behavior of the fibers in and around the B-A transition region. The effect of netropsin on the melting of DNA fibers was attributed mainly to the stabilization of B-DNA and to a higher melting cooperativity in the B-DNA region.     

Effect of trifluoroethanol on the conformational stability of a hyperthermophilic esterase: a CD study

The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of alpha-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v(-1)), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 degrees C for native AFEST, but becomes 85 degrees C at 1.4 M TFE (10% v v(-1)), and 56 degrees C at 2.8 M TFE (20% v v(-1)). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.     

Comparative docking of dual conformations in human fatty acid synthase thioesterase domain reveals potential binding cavity for virtual screening of ligands

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4’ phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (?.164 kcal/mol) and B (?.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: ?5.872 kcal/mol and B form: ?5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.     

Destabilisation of native tertiary structural interactions is linked to helix-induction by 2,2,2-trifluoroethanol in proteins

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all β-sheet protein, cardiotoxin analogue 111 (CTX III) from the Taiwan cobra (Naja naja atra) is studied. It is found that high concentrations ( > 80% v/v) of TFE induced a β-sheet to -helix structural transition. It is found that in denatured and reduced CTX III (rCTX III) helical conformation is induced even upon addition of low concentrations ( > 10% v/v) of TFE. Using three other proteins, namely, ribonuclease A (RNase A), lysozyme and -lactalbumin, it is been observed that helix-induction by TFE is intricately linked to drastic destabilization of native tertiary structural interactions in the proteins.     

Structure analysis of a fusogenic peptide sequence from the sea urchin fertilization protein bindin

The structure of "B18", an 18-residue fusogenic peptide from the sea urchin fertilization protein bindin, was investigated in several membrane-mimicking environments with circular dichroism and nuclear magnetic resonance spectroscopy. The fully conserved peptide sequence represents the minimal functional part of the 24 kDa protein, which can bind to membranes and induce fusion of lipid vesicles. The B18 peptide undergoes a coil-helix transition in the presence of TFE, showing a transient tendency to self-associate. Its NMR structure in 30% TFE exhibits two helical regions at either side, connected by a flexible loop. In DPC and SDS detergent micelles, this loop becomes distinctly bent, presumably due to the high degree of curvature of the micelles. The loop contains a histidine-rich motif for binding zinc, which is required for the fusogenic function of the peptide. Therefore, we monitored the structural response of B18 and of recombinant bindin toward this ion. Like TFE, and in a mutually cooperative manner, zinc induces a partially helical structure in both the peptide and the protein. Complex formation via the histidine residues rigidifies the flexible loop and is accompanied by self-association of the molecules. The data suggest that the zinc-bound functional state is a continuous amphipathic alpha-helix, bearing some resemblance to a leucine zipper. Two hydrophobic patches on one face could favorably penetrate into a membrane, while two arginines on the other face could interact with lipid phosphate groups. The three-dimensional model of the B18 sequence thus contributes to a better understanding of peptide-induced vesicle fusion in general, and of the lipid-protein interactions of sperm bindin in particular.     

Trifluoroethanol-induced beta --> alpha transition in beta-lactoglobulin: hydration and cosolvent binding studied by 2H, 17O, and 19F magnetic relaxation dispersion

Alcohols, such as 2,2,2-trifluoroethanol (TFE), have been shown to induce a cooperative transition to an open helical structure in many proteins, but the underlying molecular mechanism has not been identified. Here, we employ the technique of magnetic relaxation dispersion (MRD) to study the TFE-induced beta --> alpha transition of beta-lactoglobulin at pH 2.4. Unlike traditional techniques that focus on protein secondary structure, the MRD method directly monitors the solvent, providing quantitative information about preferential solvation and solvent penetration and about the overall size and structural integrity of the protein. In this multinuclear MRD study, we use the (2)H and (17)O resonances to examine hydration and the (19)F resonance to study TFE. The transformation from the native to the helical state via an intermediate state at 300 K is found to be accompanied by a progressive expansion of the protein and loss of specific long-lived hydration sites. The observation of (17)O and (19)F dispersions from the helical state shows that water and TFE penetrate the protein. The MRD data indicate a strong accumulation of TFE at the surface as well as in the interior of the protein. At 277 K, BLG is much less affected by TFE, remaining in the native state at 16% TFE, but adopting a nonnative structure at 30% TFE. This nonnative structure is not penetrated by long-lived water molecules. The implications of these findings for the mechanism of TFE-induced structural transformations are discussed.