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1.
The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase
Howes BD Feis A Raimondi L Indiani C Smulevich G 《The Journal of biological chemistry》2001,276(44):40704-40711
The extent to which the structural Ca(2+) ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca(2+) ion. The Fe(III) heme states are modified upon Ca(2+) depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca(2+)-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca(2+) depletion are easily reversed. Contrary to the native protein, the Ca(2+)-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the nu(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca(2+) depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca(2+)-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca(2+) depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca(2+) ion as that which is lost upon Ca(2+) depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties. 相似文献
2.
The endogenous calcium ions of horseradish peroxidase C are required to maintain the functional nonplanarity of the heme
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Horseradish peroxidase C (HRPC) binds 2 mol calcium per mol of enzyme with binding sites located distal and proximal to the heme group. The effect of calcium depletion on the conformation of the heme was investigated by combining polarized resonance Raman dispersion spectroscopy with normal coordinate structural decomposition analysis of the hemes extracted from models of Ca(2+)-bound and Ca(2+)-depleted HRPC generated and equilibrated using molecular dynamics simulations. Results show that calcium removal causes reorientation of heme pocket residues. We propose that these rearrangements significantly affect both the in-plane and out-of-plane deformations of the heme. Analysis of the experimental depolarization ratios are clearly consistent with increased B(1g)- and B(2g)-type distortions in the Ca(2+)-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca(2+)-depleted HRPC and of significant changes in the relative contributions of three of the six lowest frequency deformations. Most noteworthy is the decrease of the strong saddling deformation that is typical of all peroxidases, and an increase in ruffling. Our results confirm previous work proposing that calcium is required to maintain the structural integrity of the heme in that we show that the preferred geometry for catalysis is lost upon calcium depletion. 相似文献
3.
Resonance Raman spectroscopy is used to probe the effect of calcium depletion on the heme group of horseradish peroxidase C at pH 8. Polarized Raman spectra are recorded with an argon ion laser at eight different wavelengths to provide a sound database for a reliable spectral decomposition. Upon calcium depletion, the spectrum is indicative of a predominantly pentacoordinated high spin state of the heme iron coexisting with small fractions of hexacoordinated high and low spin states. The dominant quantum mixed spin state of native ferric horseradish peroxidase, which is characteristic for class III peroxidases, is not detectable in the spectrum of the enzyme with partial distal Ca(2+) depletion. The quenching of the quantum mixed spin state and the predominance of the pentacoordinated high spin state are likely to arise from distortions induced by distal calcium depletion, which translates into a weaker Fe-N(epsilon)(His) bond and a more tilted imidazole. A correlation is proposed between the lower enzyme activity and the elimination of the pentacoordinated quantum mixed state upon Ca(2+) depletion. 相似文献
4.
Verdín J Pogni R Baeza A Baratto MC Basosi R Vázquez-Duhalt R 《Biophysical chemistry》2006,121(3):163-170
Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted. 相似文献
5.
Horseradish peroxidase C is a class III peroxidase whose structure is stabilized by the presence of two endogenous calcium atoms. Calcium removal has been shown to decrease the enzymatic activity of the enzyme and significantly affect the spectroscopically detectable properties of the heme, such as the spin state of the iron, heme normal modes, and distortions from planarity. In this work, we report on normal mode analysis (NMA) performed on models subjected to 2 ns of molecular dynamics simulations to describe the effect of calcium removal on protein collective motions and to investigate the correlation between active site (heme) and protein matrix fluctuations. We show that in the native peroxidase model, heme fluctuations are correlated to matrix fluctuations while they are not in the calcium-depleted model. 相似文献
6.
Chen Y Naik SG Krzystek J Shin S Nelson WH Xue S Yang JJ Davidson VL Liu A 《Biochemistry》2012,51(8):1586-1597
MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and M?ssbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and M?ssbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity. 相似文献
7.
The endogenous calcium ion (Ca2+) in horseradish peroxidase (HRP) was removed to cause substantial changes in the proton NMR spectra of the enzyme in various oxidation/spin states. The spectral changes were interpreted as arising from the substantial alterations in the heme environments, most likely the heme proximal and distal sides. The comparative kinetic and redox studies revealed that these conformational changes affect the reduction process of compound II, resulting in the decrease of the enzymatic activity of HRP. It is also revealed from the ESR spectrum and the temperature dependences of the NMR and optical absorption spectra of the Ca2+-free enzyme that the heme iron atom of the Ca2+-free enzyme is in a thermal spin mixing between ferric high and low spin states, in contrast to that of the native enzyme. These results show that Ca2+ functions in maintaining the protein structure in the heme environments as well as the spin state of the heme iron, in favor of the enzymatic activity of HRP. 相似文献
8.
Medda R Padiglia A Longu S Bellelli A Arcovito A Cavallo S Pedersen JZ Floris G 《Biochemistry》2003,42(29):8909-8918
A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca(2+) was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca(2+). Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca(2+) ions, except for a significant perturbation of the Fe(3+) inner sphere geometry, as detected by EPR measurements. We propose that Ca(2+) binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca(2+) level. 相似文献
9.
The tightly bound calcium of MauG is required for tryptophan tryptophylquinone cofactor biosynthesis
Shin S Feng M Chen Y Jensen LM Tachikawa H Wilmot CM Liu A Davidson VL 《Biochemistry》2011,50(1):144-150
The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. The crystal structure of the MauG-preMADH complex revealed the presence of a Ca(2+) in proximity to the two hemes [Jensen, L. M. R., Sanishvili, R., Davidson, V. L., and Wilmot, C. M. (2010) Science 327, 1392-1394]. This Ca(2+) did not readily dissociate; however, after extensive treatment with EGTA or EDTA MauG was no longer able to catalyze TTQ biosynthesis and exhibited altered absorption and resonance Raman spectra. The changes in spectral features are consistent with Ca(2+)-dependent changes in heme spin state and conformation. Addition of H(2)O(2) to the Ca(2+)-depleted MauG did not yield spectral changes characteristic of formation of the bis-Fe(IV) state which is stabilized in native MauG. After addition of Ca(2+) to the Ca(2+)-depleted MauG, full TTQ biosynthesis activity and reactivity toward H(2)O(2) were restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca(2+) binding to Ca(2+)-depleted MauG indicated a two-step mechanism. Ca(2+) initially reversibly binds to Ca(2+)-depleted MauG (K(d) = 22.4 μM) and is followed by a relatively slow (k = 1.4 × 10(-3) s(-1)) but highly favorable (K(eq) = 4.2) conformational change, yielding an equilibrium dissociation constant K(d,eq) value of 5.3 μM. The circular dichroism spectra of native and Ca(2+)-depleted MauG were essentially the same, consistent with Ca(2+)-induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure. These results are discussed in the context of the structures of MauG and heme-containing peroxidases. 相似文献
10.
Carvalho AS Neves-Petersen MT Petersen SB Aires-Barros MR Pinho e Melo E 《Biochimica et biophysica acta》2005,1747(1):99-107
Horseradish peroxidase A1 can refold to a native-like structure without binding calcium, originating a Ca2+-depleted native state as previously demonstrated. Thermal unfolding studies of horseradish peroxidase anionic 1 (HRPA1) have shown that calcium ions present during refolding lead to the appearance of a misfolded conformational state, which cannot incorporate the heme group. This calcium-induced conformational state, ICa2+, is less stable than the native state and has distinct secondary and tertiary structures as probed by far-UV and visible circular dichroism and tryptophan fluorescence. The fraction of ICa2+ increases exponentially with increasing calcium concentration. The ICa2+ state is formed during refolding after calcium binding to the unfolded state, as reconstitution of HRPA1 from its apoprotein reveals that the affinity of the apoprotein to protoporphyrin IX is higher in the presence of calcium. If calcium is added after refolding only, the majority of HRPA1 molecules retain their native conformation, thus confirming the binding of calcium to the unfolded state. 相似文献
11.
Lohmiller T Cox N Su JH Messinger J Lubitz W 《The Journal of biological chemistry》2012,287(29):24721-24733
Ca(2+) is an integral component of the Mn(4)O(5)Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S(2)' state of the Ca(2+)-depleted cluster from spinach is examined by X- and Q-band EPR and (55)Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca(2+) removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S(2)' state in spinach PS II is very similar to the native S(2) state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca(2+) a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y(Z) and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel (55)Mn(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca(2+)-depleted PS II. Mn(2+) titration, monitored by (55)Mn ENDOR, revealed a specific Mn(2+) binding site with a submicromolar K(D). Ca(2+) titration of Mn(2+)-loaded, Ca(2+)-depleted PS II demonstrated that the site is reversibly made accessible to Mn(2+) by Ca(2+) depletion and reconstitution. Mn(2+) is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn(4)O(5)Ca cluster during photoassembly and/or D1 repair. 相似文献
12.
The quantum mixed-spin heme state of barley peroxidase: A paradigm for class III peroxidases.
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B D Howes C B Schiodt K G Welinder M P Marzocchi J G Ma J Zhang J A Shelnutt G Smulevich 《Biophysical journal》1999,77(1):478-492
Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state. 相似文献
13.
The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca(2+) ions. Earlier studies suggested a critical role of one of the bound Ca(2+) ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca(2+)-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca(2+)-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca(2+), perturbs a H-bonding network connecting this Ca(2+) to the heme crevice that involves the distal water. The measured effects of Ca(2)(+) depletion can be interpreted as supporting a structural role for the distal Ca(2+) and for its enhanced significance in finetuning the protein structure to optimize enzyme activity. 相似文献
14.
Indiani C Feis A Howes BD Marzocchi MP Smulevich G 《Journal of inorganic biochemistry》2000,79(1-4):269-274
A spectroscopic study of soybean peroxidase (SBP) has been carried out using electronic absorption, resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopy in order to determine the effects of temperature on the heme spin state. Upon lowering the temperature a transition from high spin to low spin is induced in SBP resulting from conformational changes in the heme cavity, including a contraction of the heme core, the reorientation of the vinyl group in position 2 of the porphyrin macrocycle, and the binding of the distal His to the Fe atom. Moreover, the combined analysis of the data derived from the different techniques at both room and low temperatures demonstrates that at low temperature the quantum-mechanically admixed spin state (QS) of SBP has RR frequencies different from those observed for the QS species at room temperature. 相似文献
15.
Pintus F Mura A Bellelli A Arcovito A Spanò D Pintus A Floris G Medda R 《The FEBS journal》2008,275(6):1201-1212
A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron-protoporphyrin IX pentacoordinated with a histidine 'proximal' ligand as heme prosthetic group. In addition, the purified peroxidase contained 1 mole of endogenous Ca(2+) per mole of enzyme, and in the presence of excess Ca(2+), the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni(2+) causes reversible inhibition, whereas, in the presence of excess Ca(2+), Ni(2+) leads to an increase of the catalytic activity of Euphorbia peroxidase. UV/visible absorption spectra show that the heme iron remains in a quantum mechanically mixed-spin state as in the native enzyme after addition of Ni(2+), and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni(2+). In the presence of H(2)O(2) and in the absence of a reducing agent, Ni(2+) decreases the catalase-like activity of Euphorbia peroxidase and accelerates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni(2+) affects the H(2)O(2)-binding site and inhibits the formation of compound I. In the presence of excess Ca(2+), Ni(2+) accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni(2+)-binding sites with opposite functional effects. 相似文献
16.
Mura A Longu S Padiglia A Rinaldi AC Floris G Medda R 《International journal of biological macromolecules》2005,37(4):205-211
The changes in the heme environment and overall structure occurring during reversible thermal inactivation and in denaturant guanidinium of Euphorbia characias latex peroxidase (ELP) were investigated in the presence and absence of calcium ions. Native active enzyme had an absorption spectrum typical of a quantum-mixed spin ferric heme protein. After 40 min at 60 degrees C ELP was fully inactivated showing the spectroscopic behavior of a pure hexacoordinate low-spin protein. The addition of Ca2+ to the thermally inactivated enzyme restored its native activity and its spectroscopic features, but did not increase the stability of the protein in guanidinium. It is concluded that, in Euphorbia peroxidase, Ca2+ ion play a key role in conferring structural stability to the heme environment and in retaining active site geometry. 相似文献
17.
Echalier A Brittain T Wright J Boycheva S Mortuza GB Fülöp V Watmough NJ 《Biochemistry》2008,47(7):1947-1956
A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity. This material was used to carry out the first integrated biochemical, spectroscopic and structural investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca2+ ion (K = 3 x 10(10) M-1) found at the domain interface of both the fully oxidized and mixed-valence forms of the enzyme is absolutely required for catalytic activity. Reduction of the electron-transferring (high-potential) heme in the presence of Ca2+ ions triggers substantial structural rearrangements around the active-site (low-potential) heme to allow substrate binding and catalysis. The enzyme also forms a mixed-valence state in the absence of Ca2+ ions, but a combination of electronic absorption, and EPR spectroscopies suggests that under these circumstances the low potential heme remains six-coordinate, unable to bind substrate and therefore catalytically inactive. Our observations strongly suggest that the two mixed-valence forms of native PsaCCP reported previously by Foote and colleagues (Foote, N., Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J. 230, 227-237) correspond to the Ca2+-loaded and -depleted forms of the enzyme. 相似文献
18.
B D Howes H A Heering T O Roberts F Schneider-Belhadadd A T Smith G Smulevich 《Biopolymers》2001,62(5):261-267
Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state. 相似文献
19.
Carvalho AS Santos AM Neves-Petersen MT Petersen SB Aires-Barros MR e Melo EP 《Biopolymers》2004,75(2):173-186
Fluorescence, CD, and activity measurements were used to characterize the different conformational states of horseradish peroxidase A1 induced by thermal unfolding. Picosecond time-resolved fluorescence studies showed a three-exponential decay dominated by a picosecond lifetime component resulting from energy transfer from tryptophan to heme. Upon thermal unfolding a decrease in the preexponential factor of the picosecond lifetime and an increase in the quantum yield were observed approaching the characteristics observed for apoHRPA1. The fraction of heme-quenched fluorophore decreased to 0.4 after unfolding as shown by acrylamide quenching. A new unfolding pathway for HRPA1 was proposed and the effect of the low molecular weight solutes trehalose, sorbitol, and melezitose on this pathway was analyzed. Native HRPA1 unfolds with an intermediate between the native and the unfolded conformation. The unfolded conformation can refold to the native state or to a native-like conformation with no calcium ions upon cooling or can give an irreversible denatured state. The refolded conformation with no calcium ions was clearly identified in a second thermal scan in the presence of EDTA and shows secondary and tertiary structures, heme reincorporation in the cavity, and at least 59% of activity. Melezitose stabilized the refolded Ca2+-depleted protein and induced a more complex mechanism for heme disruption. The effect of sorbitol and trehalose were mainly characterized by an increase in the temperature of unfolding. 相似文献
20.
High affinity binding of Ca(2+) to alpha-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca(2+) as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca(2+). To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-A resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of alpha-helical and beta-subdomains separated by a cleft. Even though alternative cations and Ca(2+) liganding solvent molecules are absent, removal of Ca(2+) has only minor effects on the structure of the metal-binding site and a structural change was observed in the cleft on the opposite face of the molecule adjoining Tyr(103) of the helical lobe and Gln(54) of the beta-lobe. Changes include increased separation of the lobes, loss of a buried solvent molecule near the Ca(2+)-binding site, and the replacement of inter- and intra-lobe H-bonds of Tyr(103) by interactions with new immobilized water molecules. The more open cleft structure in the apo protein appears to be an effect of calcium binding transmitted via a change in orientation of helix H3 relative to the beta-lobe to the inter-lobe interface. Calcium is well known to promote the folding of LA. The results from the comparison of apo and holo structures of LA provide high resolution structural evidence that the acceleration of folding by Ca(2+) is mediated by an effect on interactions between the two subdomains. 相似文献