Folding of staphylococcal nuclease A studied by equilibrium and kinetic circular dichroism spectra

The urea-induced unfolding of staphylococcal nuclease A has been studied by circular dichroism both at equilibrium and by the kinetics of unfolding and refolding (pH 7.0 and 4.5 degrees C), as a function of Ca2+ and thymidine 3',5'-diphosphate (pdTp) concentration. The results are as follows. (1) The unfolding transition is shifted to higher concentrations of urea by Ca2+ and pdTp, and the presence of both ligands further stabilizes the protein. (2) In the first stage of kinetic refolding, the peptide ellipticity changes rapidly within the dead time of stopped-flow measurement (15 ms), indicating accumulation of a transient intermediate. This intermediate is remarkably less stable than those of other globular proteins previously studied. (3) Dependence of the folding and unfolding rate constants on urea concentration indicates that the critical activated state of folding ("transition state") has considerable structural organization. The transition state does not, however, have the capacity to bind Ca2+ and pdTp, as indicated by the effects of these ligands on the unfolding rate constant. (4) There are at least four different phases in the refolding kinetics in native conditions below 1 M urea. In the absence of pdTp, there are two phases in unfolding, while in the presence of pdTp the unfolding kinetics show a single phase. Some characteristics of the transient intermediate and of the transition state for folding are discussed.     

DNA solution conformation via infrared circular dichroism: experimental and theoretical results for B-family polymers

Infrared (vibrational) circular dichroism (VCD) has been observed for the DNA models d(CG)5, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) in the B-conformation in buffered, aqueous solution. The observed results are quantitatively interpreted in terms of the exciton model for coupled carbonyl stretching vibrational states.     

Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements

The interaction of hexamminecobalt(III), Co(NH₃)₆³⁺, with 160 and 3000–8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH₃)₆³⁺ binding up to the molar ratio [Co(NH₃)₆³⁺]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH₃)₆³⁺]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH₃)₆³⁺]/[P] = 0.3. In the case of 3000–8000 bp DNA only two processes were observed: (i) binding up to [Co(NH₃)₆³⁺]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B→Ψ transition after [Co(NH₃)₆³⁺]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH₃)₆³⁺ binding on volume and compressibility have been obtained. The binding of Co(NH₃)₆³⁺ to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH₃)₆³⁺: ΔV = 9 cm³ mol^–1 and Δκ = 33 × 10^–4 cm³ mol^–1 bar^–1. The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH₃)₆³⁺ and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.     

Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism

Kinetics of refolding and unfolding of staphylococcal nuclease and its six mutants, each carrying single or double amino acid substitutions, are studied by stopped-flow circular dichroism measurements. A transient kinetic intermediate formed within 10 ms after refolding starts possesses a substantial part of the N-domain core β-structure, whereas helices are formed at the later stages. The structure of the kinetic intermediate is less organized than the structure that is known to be formed by a nuclease 1-136 fragment. Only the refolding kinetics are affected by the mutations in all the mutants except two in which the mutations have changed the native structure. From this result and also from the locations of the mutation sites, the major N-terminal domain of the nuclease in the transition state of folding has a structure nearly identical to the native one. On the other hand, the minor C-terminal domain has previously been shown to be still disorganized in the transition state. The effects of the amino acid substitutions on the stability of the native and the transition states are in good agreement with the changes in the hydration free energy, expected for the corresponding amino acid replacements in the unfolded polypeptide. Since side chains of all the mutated residues are not accessible to solvent in the native structure, the result suggests that it is the unfolded state that is mainly affected by the mutations. © 1995 Wiley-Liss, Inc.     

The effect of manganese(II) on DNA structure: electronic and vibrational circular dichroism studies

The interaction of DNA with Mn²⁺ was studied in absorbance and optical activity in the electronic and vibrational regions. Based on the data, several stages of the interaction were identified. Con formational transition towards the C-form of DNA was observed in solution at the molar ratio Mn²⁺/DNA-phosphates between 0.1 and 1.5. The exact ratio depended on the ionic strength and increased with increasing NaCl concentration. Although manganese interacted with the phosphates and bases of DNA at higher metal concentrations, it is unlikely that direct chelation occurred. A model for the interaction between manganese ions and DNA mediated by water is suggested destabilizing the double helix and partially breaking the hydrogen bonds between the base pairs. At high Mn²⁺ concentrations DNA aggregation was observed.     

Distribution of 5-methyldeoxycytidine in products of staphylococcal nuclease digestion of nuclei and purified DNA

We have compared the distribution of 5-methyldeoxycytidine (m5dC) between staphylococcal nuclease (SN) sensitive and resistant regions of human diploid fibroblast chromatin to the corresponding distribution in purified DNA. After SN digestion of fibroblast nuclei or purified DNA, nuclease-resistant products were separated from sensitive products by perchloric acid or ethanol precipitation; the radioactively labeled nucleosides were then fractionated by high-performance liquid chromatography and quantitated. Our results indicate that m5dC is preferentially associated with SN-resistant regions of both chromatin and purified DNA. The magnitudes of these preferences in fibroblast chromatin and DNA are similar; we find that the enrichment of m5dC content in SN-resistant fractions of nuclei and DNA relative to the corresponding sensitive fractions is approximately 2-3-fold. Therefore, highly methylated regions of DNA have an intrinsic resistance to digestion by SN that is of sufficient magnitude to explain the high degree of nuclease resistance of chromatin containing highly methylated DNA.     

DNA-copper (II) complex and the DNA conformation

Spectrophotometric, sedimentation, infrared, optical rotatory dispersion (ORD), and circular dichroism (CD) methods have been used to demonstrate the structural changes in DNA induced by the interaction of copper(II) with bases and to elucidate the complex binding sites. As shown by the electrolyte-induced reversion (addition of salts) of temperature-denatured copper DNA the effectiveness of re-formation of the double-stranded structure depends on the temperature, copper(II) ion concentration, and on the base composition of the DNA. Exposure of heat-denatured copper DNA to higher temperatures decreases the reversion effect on addition of electrolyte. The results indicate that a greater fraction with a cooperative transition appears on heating DNA to 80 or 100°C at a Cu²⁺/DNA-P ratio of 2 : 1 than at a Cu²⁺/DNA-P ratio of 1 : 1. With AT-rich copper DNA, reversion to the native DNA structure was not observed. Selective methylation of guanine residues in DNA also affects the electrolyte-induced reversion, indicating the importance of GC pairs for copper(II) binding and the reversion to the native structure. Temperature-denatured copper DNA shows an increased sedimentation coefficient Which decreases again after electrolyte-induced reversion. This change in s is reduced by selective methylation of DNA. Complex formation between copper(II) and the bases is accompanied by a conformational change of the DNA double-helical structure as demonstrated by ORD and CD experiments. The ORD profile of GC-rich DNA is much more affected by copper(II) than that of AT-rich ones. Even at very low copper(II) concentrations, e.g., at 0.02 and 0.2 Cu²⁺/DNA-P, the ORD and CD measurements exhibit conformational changes of the DNA secondary structure at room temperature. By comparing the infrared spectra of deoxynucleosides with that of DNA of different GC content it has been shown that both guanine and cytosine are involved in the formation of the complex of copper(II) with DNA. N-7 and O at C-6 in guanine and N-3 as well as O of C-2 in cytosine are discussed as the most probable binding sites in DNA. A binding model for the coordination of the copper(II) ion between guanine and cytosine of the opposite strands is suggested. The results are in good agreement with the assumptions and predictions made by Eichhorn and Clark about the complexing of copper(II) with DNA. The recent proposal made by Schreiber and Daune about an interaction of the type guanine–Cu²⁺–guanine cannot be excluded as an additional kind of coordination of copper(II) in DNA.     

Protein conformation of potato (Solanum tuberosum) lectin determined by circular dichroism.

The structure of potato (Solanum tuberosum) lectin, which is a hydroxyproline-rich glycoprotein, has been investigated by circular dichroism. The spectra of the native lectin, and of the oxidized, reduced and carboxymethylated and deglycosylated derivatives were examined, as was a hydroxyproline-rich glycopeptide and its deglycosylated derivative. It is concluded that the lectin contains about 35% polyproline II conformation, 34% type II beta-turn and 31% irregular conformation. No indications were found for the presence of alpha-helix or beta-sheet conformations. The polyproline II conformation is heat-stable, but is markedly destabilized by deglycosylation. The type II beta-turn is destabilized by cleavage of disulphide bonds.     

Interactions of Hg(II) ions with DNA as revealed by CD measurements.

The circular dichroism spectra of Hg(II) complexes with native calf thymus DNA, chemically methylated Streptomyces chrysomallus DNA and with Ag(I)-DNA complexes were measured in the region of 220 - 340 nm. As a main result a conversion of the conservative CD spectrum of DNA to a distinct nonconservative type of CD spectrum for the complexes occurs with increasing Hg(II) concentration. The CD spectra of the Hg(II) complexes as well as some additional arguments strongly support the idea, that DNA in the complex undergoes a structural transition to a more condensed state with 4 -like character.     

Changes in poly[d(T-G).d(C-A)] chirality due to Hg(II)-binding: circular dichroism (CD) studies

The conformation of poly[d(T-G).d(C-A)] in aqueous solution (0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 6.9) was studied by circular dichroism (CD) spectroscopy in the ultraviolet. The conformation of the polynucleotide, as reflected by its chiroptical signature, changes in a highly cooperative fashion in the presence of Hg(ClO4)2. The CD changes signal transitions first from the B to a modified B-state (B*), or to a non-B structure termed X, and finally to a form that is presumably Z. The alterations are totally reversible subsequent to the removal of mercury with the help of a suitable complexing agent such as sodium cyanide, indicating that mercuration does not disrupt Watson-Crick hydrogen bonding to any extent.     

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.     

Fe(III) and Cu(II) conalbumin visible circular dichroism spectra.

The single polypeptide chain of conalbumin strongly binds two Fe(III) or two Cu(II) ions to yield intense absorption in the visible region similar to that shown by the related protein transferrin. Comparison of the metal-ion-binding sites in the two proteins is made by exploiting the sensitivity to ligand geometry of circular dichroism (CD). For the Fe(III) proteins strong similarities of the CD spectra outweigh marginal differences. For Cu(II) conalbumin an additional negative extremum near 506 nm appears between two positive ones at 634 and 410 nm suggesting greater subtraction of oppositely signed CD components leading to lesser magnitudes for the two positive peaks than are found in Cu(II)-transferrin. The two Fe(III)-binding sites within conalbumin are compared by noting the strong similarities of the CD and MCD of proteins with Fe(III) in one site and Ga(III) in the other site, and vice versa, with the protein containing Fe(III) in both sites. Due to features of the amino acid sequences of the single protein chains, the four strong metal ion binding sites in conalbumin and transferrin cannot be identical in all particulars, yet CD spectra of their metal ion complexes are closely similar. From a study of model phenolate complexes and the wavelength maxima of visible absorption in the Fe(III), Cu(II), and Co(III) proteins near 465, 440, and 405 nm, respectively, these strong absorption bands are identified as ligand to metal ion electron-transfer transitions. It is suggested that tyrosyl residues are the donors in the electron transfer transitions and that they lock in the metal ions after being keyed into position by binding of bicarbonate or other anions.     

Copper(II)-DNA denaturation. II. The model of DNA denaturation

In a continuing study of the denaturation of DNA as brought abought about by Cu(II) ions, results are presented for the dependence of T_m and τ (the terminal relaxation time) on ionic strength, pH, reactant concentrations, and temperature. Maximum stability of the double helix, as reflected by the longest relaxation times and highest T_m values, was observed between pH 5.3 and 6.2. Outside this range both T_m and τ decreased sharply. A second, faster relaxation time was deduced from the kinetic cureves. The apparent activation energies of the rapid and slow (“terminal”) relaxations were found to be 12 and 55 kcal/mole, respectively. Several lines of evidence led to the conclusions that (1) the rate-determining step in DNA denaturation, when occurring in the transition region, is determined by chemical events and (2) the interactions which are disrupted kinetically in the rate-determining step are those which account for the major portion of the thermal (T_m) stability of helical DNA.     

Ca(2+)-induced changes of surfactin conformation: a FTIR and circular dichroism study

Previous NMR studies on surfactin proposed two gamma or beta-turn-containing conformers while recent CD studies described beta-sheets and alpha-helices in surfactin. Since these data were not obtained in the same conditions, the conformation of surfactin was reinvestigated by FTIR spectroscopy, a diagnostic method for beta-sheets. In trifluoroethanol, the FTIR spectra of surfactin and its diester are compatible with gamma and/or beta-turn(s) and the differences in their CD spectra show the importance of the Glu(1) and Asp(5) COOH groups in stabilizing the lipopeptide conformation. The calcium-induced spectral changes of both lipopeptides suggest a first binding of the divalent ions to the surfactin COOH groups (until calcium-lipopeptide mole ratio reached 1) followed by bulk conformational changes (at higher mole ratios). In Tris buffer at pH 8.5, the FTIR amide I band shape, without the typical 1610-1628 and 1675-1695 cm(-1) bands, ascertains the absence of beta-sheets.