Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.     

Metal selectivity of clusters in rabbit liver metallothionein.

In mammalian metallothioneins the metals are organized in two adamantane-type clusters with three and four metal ions which are tetrahedrally coordinated by thiolate ligands. The metal selectivity of the metal-thiolate clusters in rabbit liver metallothionein has been studied by offering two ions, i.e. Co(II)/Cd(II), Zn(II)/Cd(II) or Co(II)/Zn(II), to the metal-free protein. The heterogeneous metal complexes thus formed were characterized by electronic absorption, magnetic circular dichroism. 113Cd-NMR and EPR spectroscopy. In the case of Co/Cd-metallothionein, homometallic cluster occupation occurs, with the Cd(II) ions bound exclusively to the four-metal cluster. In contrast, heterometallic clusters were formed for both Zn/Cd- and Co/Zn-metallothionein. Based on evidence from corresponding inorganic structures of adamantane metal-thiolate cages, it is suggested that the major factor governing the cluster type is the protein structure perturbation due to the cluster volume variations. Thus, while metal thiolate affinities are important in the folding process, size-match selectivity is the dominant factor in the metal-loaded protein.     

Spectroscopic characterization of native and Co(II)-substituted zucchini mavicyanin

Mavicyanin from zucchini peelings has been characterized by electronic absorption, circular dichroism (CD), magnetic circular dichroism (MCD), resonance Raman (RR), and electron paramagnetic resonance (EPR) spectra. The electronic absorption, CD, MCD, and EPR spectra are appreciably similar to those of stellacyanin from lacquer, in which the tetrahedral Cu center has a donor set composed of four amino acid residues [2 histidine (His), cysteine (Cys), and glutamine (Gln)]. Under neutral conditions, mavicyanin and stellacyanin show intense blue bands at 599 and 604?nm, respectively. However, the RR spectrum of mavicyanin between 300 and 450?cm^–1, which is believed to originate from the predominant Cu–S stretching vibration, is remarkably different from that of stellacyanin. This might be due to a slight distortion of the tetrahedral Cu(II) center toward tetragonal geometry in mavicyanin. Moreover, the d–d transition bands of Co(II)-substituted mavicyanin are slightly blue-shifted compared with those of Co(II)-substituted stellacyanin. This finding also suggests a difference in distortion between these tetrahedral Co(II) centers in spite of the same donor sets.     

Mechanism of formation of a productive molten globule form of barstar

Structural analysis of the initial steps in protein folding is difficult because of the swiftness with which these steps occur. Hence, the link between initial polypeptide chain collapse and formation of secondary and other specific structures remains poorly understood. Here, an equilibrium model has been developed for characterizing the initial steps of folding of the small protein barstar, which lead to the formation of a productive molten globule in the folding pathway. In this model, the high-pH-unfolded form (D form) of barstar, which is shown to be as unstructured as the urea-denatured form, is transformed progressively into a molten globule B form by incremental addition of the salt Na(2)SO(4) at pH 12. At very low concentrations of Na(2)SO(4), the D form collapses into a pre-molten globule (P) form, whose volume exceeds that of the native (N) state by only 20%, and which lacks any specific structure as determined by far- and near-UV circular dichroism. At higher concentrations of Na(2)SO(4), the P form transforms into the molten globule (B) form in a highly noncooperative transition populated by an ensemble of at least two intermediates. The B form is a dry molten globule in which water is excluded from the core, and in which secondary structure develops to 65% and tertiary contacts develop to 40%, relative to that of the native protein. Kinetic refolding experiments carried out at pH 7 and at high Na(2)SO(4) concentrations, in which the rate of folding of the D form to the N state is compared to that of the B form to the N state, indicate conclusively that the B form is a productive intermediate that forms on the direct pathway of folding from the D form to the N state.     

Folding kinetics and structure of OEP16

The chloroplast outer membrane contains different, specialized pores that are involved in highly specific traffic processes from the cytosol into the chloroplast and vice versa. One representative member of these channels is the outer envelope protein 16 (OEP16), a cation-selective high conductance channel with high selectivity for amino acids. Here we study the mechanism and kinetics of the folding of this membrane protein by fluorescence and circular dichroism spectroscopy, using deletion mutants of the two single tryptophanes Trp-77-->Phe-77 and Trp-100-->Phe-100. In addition, the wild-type spectra were deconvoluted, depicting the individual contributions from each of the two tryptophan residues. The results show that both tryptophan residues are located in a completely different environment. The Trp-77 is deeply buried in the hydrophobic part of the protein, whereas the Trp-100 is partially solvent exposed. These results were further confirmed by studies of fluorescence quenching with I(-). The kinetics of the protein folding are studied by stopped flow fluorescence and circular dichroism measurements. The folding process depends highly on the detergent concentration and can be divided into an ultrafast phase (k > 1000 s(-1)), a fast phase (200-800 s(-1)), and a slow phase (25-70 s(-1)). The slow phase is absent in the W100F mutant. Secondary structure analysis and comparision with closely related proteins led to a new model of the structure of OEP16, suggesting that the protein is, in contrast to most other outer membrane proteins studied so far, purely alpha-helical, consisting of four transmembrane helices. Trp-77 is located in helix II, whereas the Trp-100 is located in the loop between helices II and III, close to the interface to helix III. We suggest that the first, very fast process corresponds to the formation of the helices, whereas the insertion of the helices into the detergent micelle and the correct folding of the II-III loop may be the later, rate-limiting steps of the folding process.     

The kinetics of bovine growth hormone folding are consistent with a framework model

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.     

Reassessment of the electronic circular dichroism criteria for random coil conformations of poly(L-lysine) and the implications for protein folding and denaturation studies

The circular dichroism (CD) spectra of poly(L-lysine) in water and ethanediol/water (2:1) solutions in the temperature range -110 to 85 degrees C are presented. The results combined with vibrational CD data are interpreted in terms of a two-state conformational equilibrium with a left-handed trans polyproline II conformation being preferred at low temperatures. The relevance of these studies to the CD criteria for random-coil conformations, the study of helix-coil transitions and protein/peptide folding is pointed out.     

Spectroscopic rationalization of the separation abilities of decaproline chiral selector in dichloromethane-isopropanol solvent mixture

A chiral column, with decaproline as the chiral selector, has broad chiral selectivity. To understand the separation mechanism of this chiral column, multiple spectroscopic techniques, including optical rotation, electronic circular dichroism, infrared absorption and vibrational circular dichroism, have been used here to study the conformation of the decaproline oligomer in isopropanol(IPA)/dichloromethane(DCM) mixtures. These studies indicate that decaproline oligomer adopts polyproline II conformation in IPA/DCM solvent system (0% IPA approximately 100% IPA). Hydrogen bonding interactions between C=O groups of decaproline and IPA molecules increase as the content of IPA in the solvent mixture increases up to 60% and become less significant from then onwards. These spectroscopic observations are found to have a good correlation with the enantiomeric separation of racemic 2,2,2-trifluoro-1-[10-(2,2,2-trifluoro-1-hydroxy-ethyl-anthracen-9-yl]-ethanol by the decaproline column.     

The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: electronic and vibrational circular dichroism studies

The interactions were studied of DNA with the nonhistone chromatin protein HMGB1 and histone H1 in the presence of manganese(II) ions at different protein to DNA and manganese to DNA phosphate ratios by using absorption and optical activity spectroscopy in the electronic [ultraviolet (UV) and electronic circular dichroism ECD)] and vibrational [infrared (IR) and vibrational circular dichroism (VCD)] regions. In the presence of Mn2+, the protein-DNA interactions differ from those without the ions and cause prominent DNA compaction and formation of large intermolecular complexes. At the same time, the presence of HMGB1 and H1 also changed the mode of interaction of Mn2+ with DNA, which now takes place mostly in the major groove of DNA involving N7(G), whereas interactions between Mn2+ and DNA phosphate groups are weakened by histone molecules. Considerable interactions were also detected of Mn2+ ions with aspartic and glutamic amino acid residues of the proteins.     

Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies

Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.     

Ligation of the diiron site of the hydroxylase component of methane monooxygenase. An electron nuclear double resonance study.

Electron nuclear double resonance (ENDOR) spectroscopy is used to probe the coordination of the mixed valence (Fe(II).Fe(III)) diiron cluster of the methane monooxygenase hydroxylase component (MMOH-) isolated from Methylosinus trichosporium OB3b. ENDOR resonances are observed along the principal axis directions g1 = 1.94 and g3 = 1.76 from at least nine different protons and two different nitrogens. The nitrogens are strongly coupled and appear to be directly coordinated to the cluster irons. The ratio of their superhyperfine coupling constants is roughly 4:7, which equals the ratio of the spin expectation values of the Fe(II) and Fe(III) in the ground state and suggests that at least one nitrogen is coordinated to each iron of the mixed valence cluster. Moreover, the superhyperfine and quadrupole coupling constants assigned to the Fe(III) site (AN = 13.6 MHz, PN = 0.7 MHz) are comparable with those observed for semimethemerythrin sulfide (AN = 12.1 MHz, PN = 0.7 MHz), for which the nitrogen ligands are histidines. At least three of the coupled protons exchange slowly when MMOH- is incubated in D2O, and 2H ENDOR resonances are subsequently observed. These observations are also consistent with histidine ligation of the iron cluster. On addition of the inhibitor dimethyl sulfoxide (Me2SO) to MMOH- the EPR spectrum sharpens and shifts dramatically. Only one set of 14N ENDOR resonances is observed with frequencies equal to those assigned to the Fe(III)-histidine resonances of uncomplexed MMOH- suggesting that the nitrogen coordination to the Fe(II) site is altered or possibly lost in the presence of Me2SO. 2H ENDOR resonances are observed in the presence of d6-Me2SO indicating that the inhibitor Me2SO binds near or possibly to the diiron cluster. In contrast, no 2H ENDOR resonances are observed from d4-methanol upon addition to MMOH-. Thus, the changes observed in the EPR spectrum of MMOH- upon addition of methanol may result from binding to a site away from the diiron cluster or from bulk solvent effects on the protein structure.     

Folding and membrane insertion of the pore-forming peptide gramicidin occur as a concerted process

Many antibiotic peptides function by binding and inserting into membranes. Understanding this process provides an insight into the fundamentals of both membrane protein folding and antibiotic peptide function. For the first time, in this work, flow-aligned linear dichroism (LD) is used to study the folding of the antibiotic peptide gramicidin. LD provides insight into the combined processes of peptide folding and insertion and has the advantage over other similar techniques of being insensitive to off-membrane aggregation events. By combining LD data with conventional measurements of protein fluorescence and circular dichroism, the mechanism of gramicidin insertion is elucidated. The mechanism consists of five separately assignable steps that include formation of a water-insoluble gramicidin aggregate, dissociation from the aggregate, partitioning of peptide to the membrane surface, oligomerisation on the surface and concerted insertion and folding of the peptide to the double-helical form of gramicidin. Measurement of the rates of each step shows that although changes in the fluorescence signal cease 10 s after the initiation of the process, the insertion of the peptide into the membrane is actually not complete for a further 60 min. This last membrane insertion phase is only apparent by measurement of LD and circular dichroism signal changes. In summary, this study demonstrates the importance of multi-technique approaches, including LD, in studies of membrane protein folding.     

Kinetic study on the successive four-step reduction of Cyt c3

A detailed kinetic study on the successive four-step reduction of cyt c3, which has four heme units in a single protein, III4 leads to III3II leads to III2II2 leads to III II3 leads to II4, was carried out by stopped-flow electronic spectroscopy (SF-UV) and stopped-flow circular dichroism spectroscopy (SF-CD). Based on the absorbance change vs. time and the ellipticity change vs. time at the characteristic CD, together with the electronic absorption of the enzyme, rate constants for the successive four electron transfer steps, k1-k4, were successfully estimated by computer simulation. The rate constants of the four steps (k1 = 19.8 s-1, k2 = 11.9 s-1, k3 = 8.9 s-1, and k4 = 1.6 s-1; 8.0 10(-4) M Na2S2O4) are quite different from the statistical values (4: 3: 2: 1), thus excluding the possibility of random reduction of hemes of equal reactivities. Instead, each heme has its own reactivity, probably dependent on its local environment. The value of k3 is somewhat higher than the statistical value, indicating the existence of an autoacceleration effect, although small. This autoacceleration is most probably due to a unique heme-heme and/or heme-environment interaction since unusual CD and electronic absorptions were observed at 350-400 nm at about the time corresponding.     

Global changes of chlorophyll excitonic interactions in photosystem II during thermal denaturation

Photosystem II (PSII) is a multisubunit chlorophyll-binding enzyme that absorbs light to catalyze water oxidation and plastoquinone reduction. Chlorophyll excitonic interaction changes in PSII were studied by absorption and circular dichroism spectra from 25 degrees C to 80 degrees C, and protein subunit denaturation was monitored by differential scanning calorimetry. A four-stage process of chlorophyll excitonic interaction change was observed being correlated with the denaturation of protein subunits.     

Spectroscopic studies on Neurospora copper metallothionein

The spectral properties of Neurospora copper metallothionein were investigated and compared with those of the Cu(I)-2-mercaptoethanesulfonic acid complex. In both cases, the absorption spectra are rather similar, showing a characteristic shoulder at approximately 250 nm. However, marked differences were observed in their emissive properties. Thus, only metallothionein emits detectable luminescence in solution, but both the copper protein and the Cu(I) complex are luminescent at 77 K. The circular dichroism spectrum of Neurospora copper metallothionein shows several Cotton extrema attributable to asymmetry in metal coordination. The influence of HgCl2 and p-(chloromercuri)benzoate on the spectral properties of metallothionein was also investigated. The two mercurials exerted a pronounced effect on the electronic absorption, chiroptical, and emissive properties of the protein. Spectroscopic titrations followed by gel filtration experiments indicate that two mercurials can be bound per metallothionein molecule without loss of copper. This binding is responsible for the disappearance of the emissive properties of metallothionein and for the distinct changes in its electronic absorption and circular dichroism spectra. From these data, it is suggested that the Cu(I) ions are coordinated to the cysteinyl residues in the form of a single metal cluster.     

The plant metallothionein 2 from Cicer arietinum forms a single metal-thiolate cluster

The plant metallothionein 2 from Cicer arietinum (chickpea; cicMT2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by a linker region 41 amino acids in length. This metallothionein thus differs significantly from the well-studied vertebrate forms. A synthetic gene encoding cicMT2 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. For the first time, an in-depth spectroscopic characterization of cicMT2 in the presence of divalent metal ions is performed showing a binding capacity for five Zn(II), Cd(II), or Co(II) ions and the typical features of metal-thiolate clusters. Based on proteolytic digestion experiments, the cluster arrangement formed by the divalent metal ions and the cysteine thiolate groups connects the amino-terminal with the carboxy-terminal cysteine-rich region. The cluster formation process, put into effect with the addition of the fourth metal ion to the apo protein, was investigated using the characteristic shift of absorption bands observed in the UV/Vis spectra upon titration with Co(II). The pH-dependent Zn(II)- and Cd(II)-thiolate cluster stability is one of the highest observed for plant MTs so far, but lower than that usually found in vertebrate metallothioneins. The dependence of the pH stability on the ionic strength of the solution is more pronounced for the Cd(II)- than for the Zn(II)-form of the protein.     

Spectroscopic properties of the cobalt(II)-substituted alpha-fragment of rabbit liver metallothionein

The C-terminal segment of rabbit liver metallothionein 1 (alpha-fragment) containing four paramagnetic Co(II) ions was obtained by stoichiometric replacement of the originally bound diamagnetic Cd(II) ions. The latter form was prepared by limited proteolysis with subtilisin as described previously [Winge, D. R., & Miklossy, K. A. (1982) J. Biol. Chem. 257, 3471-3476]. Electronic absorption, magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) measurements were employed to monitor the stepwise incorporation of Co(II) ions into the metal-free fragment. Absorption and MCD spectra of the apofragment containing the first 3 Co(II) equiv show the typical features of tetrahedral tetrathiolate Co(II) coordination. However, in the d-d region only small changes in the visible and no apparent change in the near-infrared region are discernible when the fourth Co(II) is bound. This unusual spectral behavior was not seen in Co(II) substitution of native metallothionein [Vasák, M., & K?gi, J. H. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6709-6713] and may indicate a different cluster geometry. In the charge-transfer region, the binding of all 4 Co(II) equiv is accompanied by characteristic increments of the thiolate S----Co(II) bands. As in the formation of Co(II)7-metallothionein, the development of the charge-transfer and EPR spectral properties upon binding of the first 2 Co(II) equiv to the apofragment is indicative of isolated, noninteracting tetrahedral tetrathiolate Co(II) complexes. The binding of the additional Co(II) ion is accompanied by a red shift in the charge-transfer region and by the dramatic loss of paramagnetism in the EPR spectra, both diagnostic of the formation of metal-thiolate cluster structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal-thiolate clusters in neuronal growth inhibitory factor (GIF)

Human neuronal growth inhibitory factor (GIF) is a metallothionein-like protein specific to the central nervous system, which has been linked to Alzheimer's disease. In this article a short overview of the biological and structural properties of native Cu4,Zn3-GIF are described. Moreover, metal-thiolate clusters formed in the synthetic beta-domain (residues 1-32) and the alpha-domain (residues 32-68) both with native CuI and ZnII, and as a spectroscopic probe also with Cd(II) are discussed. The cluster formation was followed by electronic absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and 113Cd NMR spectroscopy and, in the special case of Cu(I) complexes, by luminescence spectroscopy at 77 K. These structural features are compared with those of recombinant Zn7- and 113Cd7-GIF. The structural studies suggest the existence of distinct MeII4S11 and MeII3S9 clusters located in the mutually interacting alpha- and beta-domains, respectively, of Cd7-GIF. In addition, evidence for a highly dynamic and flexible structure of this protein is presented.