Lactoperoxidase folding and catalysis relies on the stabilization of the alpha-helix rich core domain: a thermal unfolding study

Lactoperoxidase (LPO) belongs to the mammalian peroxidase family and catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates at the expense of hydrogen peroxide. Despite the complex physiological role of LPO and its potential involvement in carcinogenic mechanisms, cystic fibrosis and inflammatory processes, little is known on the folding and structural stability of this protein. We have undertaken an investigation of the conformational dynamics and catalytic properties of LPO during thermal unfolding, using complementary biophysical techniques (differential scanning calorimetry, electron spin resonance, optical absorption, fluorescence and circular dichroism spectroscopies) together with biological activity assays. LPO is a particularly stable protein, capable of maintaining catalysis and structural integrity up to a high temperature, undergoing irreversible unfolding at 70 degrees C. We have observed that the first stages of the thermal denaturation involve a minor conformational change occurring at 40 degrees C, possibly at the level of the protein beta-sheets, which nevertheless does not result in an unfolding transition. Only at higher temperature, the protein hydrophobic core, which is rich in alpha-helices, unfolds with concomitant disruption of the catalytic heme pocket and activity loss. Evidences concerning the stabilizing role of the disulfide bridges and the covalently bound heme cofactor are shown and discussed in the context of understanding the structural stability determinants in a relatively large protein.     

Probing the two-domain structure of homodimeric prokaryotic and eukaryotic catalase–peroxidases

Catalase–peroxidases (KatGs) are ancestral bifunctional heme peroxidases found in archaeons, bacteria and lower eukaryotes. In contrast to homologous cytochrome c peroxidase (CcP) and ascorbate peroxidase (APx) homodimeric KatGs have a two-domain monomeric structure with a catalytic N-terminal heme domain and a C-terminal domain of high sequence and structural similarity but without obvious function. Nevertheless, without its C-terminal counterpart the N-terminal domain exhibits neither catalase nor peroxidase activity. Except some hybrid-type proteins all other members of the peroxidase–catalase superfamily lack this C-terminal domain. In order to probe the role of the two-domain monomeric structure for conformational and thermal stability urea and temperature-dependent unfolding experiments were performed by using UV–Vis-, electronic circular dichroism- and fluorescence spectroscopy, as well as differential scanning calorimetry. Recombinant prokaryotic (cyanobacterial KatG from Synechocystis sp. PCC6803) and eukaryotic (fungal KatG from Magnaporthe grisea) were investigated. The obtained data demonstrate that the conformational and thermal stability of bifunctional KatGs is significantly lower compared to homologous monofunctional peroxidases. The N- and C-terminal domains do not unfold independently. Differences between the cyanobacterial and the fungal enzyme are relatively small. Data will be discussed with respect to known structure and function of KatG, CcP and APx.     

Bovine lactoperoxidase - a versatile one- and two-electron catalyst of high structural and thermal stability

Lactoperoxidase (LPO), a member of the peroxidase-cyclooxygenase superfamily, is found in multiple human exocrine secretions and acts as a first line of defense against invading microorganisms by production of antimicrobial oxidants. Because of its ability to efficiently catalyze one- and two-electron oxidation reactions of inorganic and organic compounds, the heme peroxidase is widely used in food biotechnology, cosmetic industry, and diagnostic kits. In order to probe its structural integrity, conformational, and thermal stability, we have undertaken a comprehensive investigation by using complementary biophysical techniques including UV-Vis, circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry (DSC). The oxidoreductase exhibits a high chemical and thermal stability under oxidizing conditions but is significantly destabilized by addition of DTT. Due to its unique ester bonds between the prosthetic group and the protein as well as six intra-chain disulfides, unfolding of the central compact (-helical core occurs concomitantly with denaturation of the heme cavity. The corresponding enthalpic and entropic contributions to the free enthalpy of unfolding are presented. Together with spectroscopic data they will be discussed with respect to the known structure of bovine LPO and homologous myeloperoxidase as well as to its practical application.     

Biosynthesis, processing, and sorting of human myeloperoxidase

Exclusively synthesized by normal neutrophil and monocyte precursor cells, myeloperoxidase (MPO) functions not only in host defense by mediating efficient microbial killing but also can contribute to progressive tissue damage in chronic inflammatory states such as atherosclerosis. The biosynthetic precursor, apoproMPO, is processed slowly in the ER, undergoing cotranslational N-glycosylation, transient interactions with the molecular chaperones calreticulin and calnexin, and heme incorporation to generate enzymatically active proMPO that is competent for export into the Golgi. After exiting the Golgi the propeptide is removed prior to final proteolytic processing in azurophil granules, resulting in formation of a symmetric MPO homodimer linked by a disulfide bond. Some proMPO escapes granule targeting and becomes constitutively secreted to the extracellular environment. Although the precise mechanism is unknown, the pro-segment is required for normal processing and targeting, as propeptide-deleted MPO precursor is either degraded or constitutively secreted. Characterizing the molecular consequences of naturally occurring mutations that cause inherited MPO deficiency provides unique insight into the structural determinants of MPO involved in biosynthesis, processing and targeting.     

The unfolding of the cytochromes c in methanol and acid.

The cytochromes c are a family of hemoproteins that share a number of structural features: a thioether linkage between the protein and the heme, histidine and methionine as the fifth and sixth iron ligands, and a tertiary structure known as the "cytochrome fold." These proteins follow a common mechanism of equilibrium unfolding in methanol and acid, differing only in their reactivity to the denaturing conditions. The reduced cytochromes c exhibit an increased conformational stability which is consistent with the presence of a strengthened iron-methionine linkage in the reduced state.     

Thermal and conformational stability of seed coat soybean peroxidase

Soybean peroxidase (SBP) obtained from the soybean seed coats belongs to class III of the plant peroxidase superfamily. Detailed circular dichroism and steady state fluorescence studies have been carried out to monitor thermal as well as denaturant-induced unfolding of SBP and apo-SBP. Melting of secondary and tertiary structures of SBP occurs with characteristic transition midpoints, T(m), of 86 and 83.5 degrees C, respectively, at neutral pH. Removal of heme resulted in greatly decreased thermal stability of the protein (T(m) = 38 degrees C). The deltaG degrees (H2O) determined from guanidine hydrochloride-induced denaturation at 25 degrees C and at neutral pH is 43.3 kJ mol(-1) for SBP and 9.0 kJ mol(-1) for apo-SBP. Comparison with the reported unfolding data of the homologous enzyme, horseradish peroxidase (HRP-C), showed that SBP exhibits significantly high thermal and conformational stability. We show that this enhanced structural stability of SBP relative to HRP-C arises due to the unique nature of their heme binding. A stronger heme-apoprotein affinity probably due to the interaction between Met37 and the C8 heme vinyl substituent contributes to the unusually high structural stability of SBP.     

Unfolding energetics and conformational stability of DLC8 monomer

To understand the rules governing the protein folding process it is essential to study the stability and unfolding of small monomeric proteins. Here, I present the pH dependent thermal unfolding energetics and conformational stability analysis of monomeric Dynein light chain protein (DLC8) in the pH range 3.5-2.0. DLC8 is the smallest and the most conserved light chain among the light chains of the dynein motor assembly. Thermal unfolding of DLC8 monomer is much complex with the presence of transient intermediates, which is in contrast to the notion that small proteins unfold via simple two-state process. The unfolding seems to be more cooperative at lower pH and the temperature of highest conformational stability (T(s)) is found to be maximum (295.7 K) at pH 2.76. Stability curves have been simulated to understand the thermodynamic parameters that govern the shapes of the experimentally obtained curves. Further, an effort has been made to correlate the observed differences in the denaturation energetics with the protein sequence in order to throw light on the structure-folding paradigm of the DLC8 monomer.     

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.     

Acetonitrile-induced unfolding of porcine pepsin A: A proposal for a critical role of hydration structures in conformational stability

In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.     

The conformation and stability of recombinant-derived granulocyte-macrophage colony stimulating factors

The conformation and stability of recombinant-derived human and murine granulocyte-macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea-gradient polyacrylamide gel electrophoresis and several spectroscopic methods. The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both alpha-helix and beta-sheet structure. The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding. Comparison of the human E. coli-derived (non-glycosylated) and mammalian cell culture-derived (glycosylated) proteins by urea-gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding.     

Heme and pH-dependent stability of an anionic horseradish peroxidase

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N<-->U-->D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.     

Conformational stability of dimeric and monomeric forms of the C-terminal domain of human immunodeficiency virus-1 capsid protein

The unfolding equilibrium of the C-terminal domain of human immunodeficiency virus-1 (HIV-1) capsid protein has been analyzed by circular dichroism and fluorescence spectroscopy. The results for the dimeric, natural domain are consistent with a three-state model (N(2)<-->2I<-->2U). The dimer (N(2)) dissociates and partially unfolds in a coupled cooperative process, into a monomeric intermediate (I) of very low conformational stability. This intermediate, which is the only significantly populated form at low (1 microM) protein concentrations, fully preserves the secondary structure but has lost part of the tertiary (intramonomer) interactions found in the dimer. In a second transition, the intermediate cooperatively unfolds into denatured monomer (U). The latter process is the equivalent of a two-state unfolding transition observed for a monomeric domain in which Trp184 at the dimer interface had been truncated to Ala. A highly conserved, disulfide-bonded cysteine, but not the disulfide bond itself, and three conserved residues within the major homology region of the retroviral capsid are important for the conformational stability of the monomer. All these residues are involved also in the association process, despite being located far away from the dimerization interface. It is proposed that dimerization of the C-terminal domain of the HIV-1 capsid protein involves induced-fit recognition, and the conformational reorganization also improves substantially the low intrinsic stability of each monomeric half.     

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.     

Impact of subunit and oligomeric structure on the thermal and conformational stability of chlorite dismutases

Chlorite dismutases (Cld) are unique heme b containing oxidoreductases that convert chlorite to chloride and dioxygen. Recent phylogenetic and structural analyses demonstrated that these metalloproteins significantly differ in oligomeric and subunit structure. Here we have analyzed two representatives of two phylogenetically separated lineages, namely pentameric Cld from Candidatus "Nitrospira defluvii" and dimeric Cld from Nitrobacter winogradskyi having a similar enzymatic activity at room temperature. By application of a broad set of techniques including differential scanning calorimetry, electronic circular dichroism, UV-vis and fluorescence spectroscopy the temperature-mediated and chemical unfolding of both recombinant proteins were analyzed. Significant differences in thermal and conformational stability are reported. The pentameric enzyme is very stable between pH 3 and 10 (T(m)=92°C at pH 7.0) and active at high temperatures thus being an interesting candidate for bioremediation of chlorite. By contrast the dimeric protein starts to unfold already at 53°C. The observed unfolding pathways are discussed with respect to the known subunit structure and subunit interaction.     

Cold adaptation of enzymes: structural, kinetic and microcalorimetric characterizations of an aminopeptidase from the Arctic psychrophile Colwellia psychrerythraea and of human leukotriene A(4) hydrolase

The relationships between structure, activity, stability and flexibility of a cold-adapted aminopeptidase produced by a psychrophilic marine bacterium have been investigated in comparison with a mesophilic structural and functional human homolog. Differential scanning calorimetry, fluorescence monitoring of thermal- and guanidine hydrochloride-induced unfolding and fluorescence quenching were used to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a low structural stability versus thermal and chemical denaturants and a greater structural permeability to a quenching agent relative to the mesophilic homolog. These findings support the hypothesis that cold-adapted enzymes maintain their activity at low temperatures as a result of increased global or local structural flexibility, which results in low stability. Analysis of the thermodynamic parameters of irreversible thermal unfolding suggests that entropy-driven factors are responsible for the fast unfolding rate of the cold-adapted aminopeptidase. A reduced number of proline residues, a lower degree of hydrophobic residue burial and a decreased surface accessibility of charged residues may be responsible for this effect. On the other hand, the reduction in enthalpy-driven interactions is the primary determinant of the weak conformational stability.     

High-pressure FTIR study of the stability of horseradish peroxidase. Effect of heme substitution,ligand binding,Ca++ removal,and reduction of the disulfide bonds

The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.     

Disruption of the aspartate to heme ester linkage in human myeloperoxidase: impact on ligand binding, redox chemistry, and interconversion of redox intermediates

In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here, monomeric recombinant myeloperoxidase (MPO) and the variants D94V and D94N were produced in Chinese hamster ovary cell lines. Disruption of the Asp(94) to heme ester bond decreased the one-electron reduction potential E'(0) [Fe(III)/Fe(II)] from 1 to -55 mV at pH 7.0 and 25 degrees C, whereas the kinetics of binding of low spin ligands and of compound I formation was unaffected. By contrast, in both variants rates of compound I reduction by chloride and bromide (but not iodide and thiocyanate) were substantially decreased compared with the wild-type protein. Bimolecular rates of compound II (but not compound I) reduction by ascorbate and tyrosine were slightly diminished in D94V and D94N. The presented biochemical and biophysical data suggest that the Asp(94) to heme linkage is no precondition for the autocatalytic formation of the other two covalent links found in MPO. The findings are discussed with respect to the known active site structure of MPO and its complexes with ligands.     

<Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> HmuT: dissecting the roles of conserved residues in heme pocket stabilization

The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV–visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ? HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on π donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT–CO.     

Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies

The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme.     

Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.