RESUMO
Protein solutions can undergo liquid-liquid phase separation (LLPS), where a dispersed phase with a low protein concentration coexists with coacervates with a high protein concentration. We focus on the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein, CPEB4NTD, and its isoform depleted of the Exon4, CPEB4Δ4NTD. They both exhibit LLPS, but in contrast to most systems undergoing LLPS, the single-phase regime preceding LLPS consists mainly of soluble protein clusters. We combine experimental and theoretical approaches to resolve the internal structure of the clusters and the basis for their formation. Dynamic light scattering and atomic force microscopy show that both isoforms exhibit clusters with diameters ranging from 35 to 80 nm. Electron paramagnetic resonance spectroscopy of spin-labeled CPEB4NTD and CPEB4Δ4NTD revealed that these proteins have two distinct dynamical properties in both the clusters and coacervates. Based on the experimental results, we propose a core-shell structure for the clusters, which is supported by the agreement of the dynamic light scattering data on cluster size distribution with a statistical model developed to describe the structure of clusters. This model treats clusters as swollen micelles (microemulsions) where the core and the shell regions comprise different protein conformations, in agreement with the electron paramagnetic resonance detection of two protein populations. The effects of ionic strength and the addition of 1,6-hexanediol were used to probe the interactions responsible for cluster formation. While both CPEB4NTD and CPEB4Δ4NTD showed phase separation with increasing temperature and formed clusters, differences were found in the properties of the clusters and the coacervates. The data also suggested that the coacervates may consist of aggregates of clusters.
Assuntos
Isoformas de Proteínas , Proteínas de Ligação a RNA , Isoformas de Proteínas/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Humanos , Modelos Moleculares , Domínios Proteicos , Separação de FasesRESUMO
Fluorine electron-nuclear double resonance (19F ENDOR) has recently emerged as a valuable tool in structural biology for distance determination between F atoms and a paramagnetic center, either intrinsic or conjugated to a biomolecule via spin labeling. Such measurements allow access to distances too short to be measured by double electron-electron resonance (DEER). To further extend the accessible distance range, we exploit the high-spin properties of Gd(III) and focus on transitions other than the central transition (|-1/2⟩ â |+1/2⟩), that become more populated at high magnetic fields and low temperatures. This increases the spectral resolution up to ca. 7 times, thus raising the long-distance limit of 19F ENDOR almost 2-fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved 19F spectrum in conventional central transition measurements and show quantitative agreement between the experimental spectra and theoretical predictions. We then validate our approach on two proteins labeled with 19F and Gd(III), in which the Gd-F distance is too long to produce a well-resolved 19F ENDOR doublet when measured at the central transition. By focusing on the |-5/2⟩ â |-3/2⟩ and |-7/2⟩ â |-5/2⟩ EPR transitions, a resolution enhancement of 4.5- and 7-fold was obtained, respectively. We also present data analysis strategies to handle contributions of different electron spin manifolds to the ENDOR spectrum. Our new extended 19F ENDOR approach may be applicable to Gd-F distances as large as 20 Å, widening the current ENDOR distance window.
Assuntos
Elétrons , Flúor , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas/química , Marcadores de SpinRESUMO
Half-Integer High Spin (HIHS) systems with zero-field splitting (ZFS) parameters below 1 GHz are generally dominated by the spin |â1/2>â|+1/2 > central transition (CT). Accordingly, most pulsed Electron Paramagnetic Resonance (EPR) experiments are performed at this position for maximum sensitivity. However, in certain cases it can be desirable to detect higher spin transitions away from the CT in such systems. Here, we describe the use of frequency swept Wideband, Uniform Rate, Smooth Truncation (WURST) pulses for transferring spin population from the CT, and other transitions, of Gd(III) to the neighbouring higher spin transition |â3/2>â|â1/2 > at Q- and W-band frequencies. Specifically, we demonstrate this approach to enhance the sensitivity of 1H Mims Electron-Nuclear Double Resonance (ENDOR) measurements on two model Gd(III) aryl substituted 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) complexes, focusing on transitions other than the CT. We show that an enhancement factor greater than 2 is obtained for both complexes at Q- and W-band frequencies by the application of two polarising pulses prior to the ENDOR sequence. This is in agreement with simulations of the spin dynamics of the system during WURST pulse excitation. The technique demonstrated here should allow more sensitive experiments to be measured away from the CT at higher operating temperatures, and be combined with any relevant pulse sequence.
RESUMO
Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron-electron resonance (DEER) technique can track proteins' conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8â nm. Here, we show that GdIII -19 F Mims electron-nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in-cell ENDOR measurements, complemented with room temperature solution and in-cell GdIII -19 F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin-labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in-cell derived GdIII -19 F distances were essentially identical and lie in the 1-1.5â nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19 F regions in the cell.
Assuntos
Elétrons , Gadolínio , Humanos , Espectroscopia de Ressonância de Spin Eletrônica , Gadolínio/química , Proteínas/química , Marcadores de Spin , Ubiquitina , Flúor/químicaRESUMO
The common approach to background removal in double electron-electron resonance (DEER) measurements on frozen solutions with a three-dimensional homogeneous distribution of doubly labeled biomolecules is to fit the background to an exponential decay function. Excluded volume effects or distribution in a dimension lower than three, such as proteins in a membrane, can lead to a stretched exponential decay. In this work, we show that in cases of spin labels with short spin-lattice relaxation time, up to an order of magnitude longer than the DEER trace length, relevant for metal-based spin labels, spin flips that take place during the DEER evolution time affect the background decay shape. This was demonstrated using a series of temperature-dependent DEER measurements on frozen solutions of a nitroxide radical, a Gd(III) complex, Cu(II) ions, and a bis-Gd(III) model complex. As expected, the background decay was exponential for the nitroxide, whereas deviations were noted for Gd(III) and Cu(II). Based on the theoretical approach of Keller et al. (Phys. Chem. Chem. Phys. 21 (2019) 8228-8245), which addresses the effect of spin-lattice relaxation-induced spin flips during the evolution time, we show that the background decay can be fitted to an exponent including a linear and quadratic term in t, which is the position of the pump pulse. Analysis of the data in terms of the probability of spontaneous spin flips induced by spin-lattice relaxation showed that this approach worked well for the high temperature range studied for Gd(III) and Cu(II). At the low temperature range, the spin flips that occured during the DEER evolution time for Gd(III) exceeded the measured spin-lattice relaxation rate and include contributions from spin flips due to another mechanisms, most likely nuclear spin diffusion.
Assuntos
Espectroscopia de Ressonância de Spin EletrônicaRESUMO
Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance spectroscopy (EPR) to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.
Assuntos
Peptídeos , RNA , Espectroscopia de Ressonância de Spin Eletrônica , Peptídeos/química , Marcadores de SpinRESUMO
Knowledge about the structural and dynamic properties of proteins that form membrane-less organelles in cells via liquid-liquid phase separation (LLPS) is required for understanding the process at a molecular level. We used spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the dynamic properties (rotational diffusion) of the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein (CPEB4NTD) across its LLPS transition, which takes place with increasing temperature. We report the coexistence of three spin labeled CPEB4NTD (CPEB4*) populations with distinct dynamic properties representing different conformational spaces, both before and within the LLPS state. Monomeric CPEB4* exhibiting fast motion defines population I and shows low abundance prior to and following LLPS. Populations II and III are part of CPEB4* assemblies where II corresponds to loose conformations with intermediate range motions and population III represents compact conformations with strongly attenuated motions. As the temperature increased the population of component II increased reversibly at the expense of component III, indicating the existence of an III â II equilibrium. We correlated the macroscopic LLPS properties with the III â II exchange process upon varying temperature and CPEB4* and salt concentrations. We hypothesized that weak transient intermolecular interactions facilitated by component II lead to LLPS, with the small assemblies integrated within the droplets. The LLPS transition, however, was not associated with a clear discontinuity in the correlation times and populations of the three components. Importantly, CPEB4NTD exhibits LLPS properties where droplet formation occurs from a preformed microscopic assembly rather than the monomeric protein molecules.
Assuntos
Proteínas , Transição de FaseRESUMO
Proteolysis of Amyloid Precursor Protein, APP, results in the formation of amyloid ß (Aß) peptides, which have been associated with Alzheimer's disease (AD). Recently the failure of therapeutic agents that prohibit Aß aggregation and sequester Cu/Zn in providing symptomatic relief to AD patients has questioned the amyloid and metal ion hypothesis. Alternatively, abnormal heme homeostasis and reduced levels of neurotransmitters in the brain are hallmark features of AD. Heme can bind Aß peptides forming a peroxidase type active site which can oxidatively degrade neurotransmitters like serotonin. To date the reactive species responsible for this activity has not been identified. Using rapid kinetics and freeze quenching, we show that heme bound Aß forms a highly reactive intermediate, compound I. Thus, compound I provides a basis for elucidating the oxidative degradation of neurotransmitters like serotonin, resulting in abnormal neurotransmission, a key pathological feature of AD. Site directed mutants indicate that the Arg5 and Tyr10 residues, unique to human Aß, affect the rates of formation and decay of compound I providing insight into their roles in the oxidative degradation of neurotransmitters. Tyr10 can potentially play a natural protective role against the highly reactive oxidant, compound I, in AD.
RESUMO
A significant abundance of copper (Cu) and iron in amyloid ß (Aß) plaques, and several heme related metabolic disorders are directly correlated with Alzheimer's disease (AD), and these together with co-localization of Aß plaques with heme rich deposits in the brains of AD sufferers indicates a possible association of the said metals with the disease. Recently, the Aß peptides have been found to bind heme and Cu individually as well as simultaneously. Another significant finding relevant to this is the lower levels of nitrite and nitrate found in the brains of patients suffering from AD. In this study, a combination of absorption and electron paramagnetic resonance spectroscopy and kinetic assays have been used to study the interaction of nitrite with the metal bound Aß complexes. The data indicate that heme(III)-Cu(i)-Aß, heme(II)-Cu(i)-Aß, heme(II)-Aß and Cu(i)-Aß can reduce nitrite to nitric oxide (NO), an important biological messenger also related to AD, and thus behave as nitrite reductases. However these complexes reduce nitrite at different rates with heme(III)-Cu(i)-Aß being the fastest following an inner sphere electron transfer mechanism. The rest of the metal-Aß adducts follow an outer sphere electron transfer mechanism during nitrite reduction. Protonation from the Arg5 residue triggering the N-O bond heterolysis in heme(III) bound nitrite with a simultaneous electron transfer from the Cu(i) center to produce NO is the rate determining step, indicating a proton transfer followed by electron transfer (PTET) mechanism.
Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Heme/química , Nitrito Redutases/química , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Humanos , Ferro/química , Cinética , Óxido Nítrico/química , Nitritos/química , Oxirredução , Placa Amiloide/químicaRESUMO
Aß(1-40) peptide is mutated to introduce cysteine residue to allow formation of organized self-assembled monolayers (SAMs) on Au electrodes. Three mutants of this peptide are produced, which vary in the position of the inserted cysteine residue. Fourier transform infrared data on these peptide SAMs show the presence of both α helices and ß sheet in these Aß constructs. These peptide constructs interact with cytochrome c (Cytc), allowing electron transfer between Cytc and the electrode via the Aß peptides. Binding of metals like Zn2+ or Cu2+ induces changes in the morphologies of these assemblies, making them fold, which inhibits their spontaneous interaction with Cytc.
RESUMO
Type 2 diabetes mellitus (T2Dm) is characterized by reduced ß cell mass and amyloid deposits of human islet amyloid polypeptide (hIAPP) or amylin, a 37 amino acid containing peptide around pancreatic ß cells. The interaction of copper (Cu) with amylin and its mutants has been studied in detail using absorption, circular dichroism, electron paramagnetic resonance spectroscopy, and cyclic voltammetry. Cu binds amylin in a 1:1 ratio, and the binding domain lies within the first 19 amino acid residues of the peptide. Depending on the pH of the medium, Cu-amylin shows the formation of five pH-dependent components (component IV at pH 4.0, component III at pH 5.0, component II at pH 6.0, component I at pH 8.0, and another higher pH component above pH 9.0). The terminal amine, His18, and amidates are established as key residues in the peptide that coordinate the Cu center. The physiologically relevant components I and II can generate H2O2, which can possibly account for the enhanced toxicity of amylin in the presence of Cu, causing damage of the ß cells of the pancreas via oxidative stress.
Assuntos
Cobre/química , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Domínio Catalítico , Cobre/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
Heme binds Aß to give a mixture of a mono-histidine bound high spin peroxidase type active site and a bis-histidine bound low spin cytochrome b type active site present in an equilibrium at physiological pH. Of these, the high spin mono-histidine bound complexes produce significant amounts of partially reduced oxygen species (PROS), catalyze the degradation of neurotransmitters and oxidize cytochrome c, with potentially detrimental effects. The presence of excess Aß could lower these effects by creating a low spin bis-histidine cytochrome b type active site which exerts less oxidative stress by producing a much smaller amount of PROS. The presence of Cu(ii) reverses this effect and can convert the benign low spin heme-Aß complex to the detrimental high spin form, even in the presence of excess Aß. Data suggest that the histidine needed to form the bis-histidine site in the low spin heme-Aß complex is likely to be involved in the high affinity Cu binding site in the heme-Cu-Aß complex.
Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Heme/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/química , Histidina/metabolismo , Humanos , Cinética , Teoria Quântica , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Iron (Fe) is the most abundant transition metal ion in the human body and its role, in the form of heme, has been implicated in Alzheimer's disease (AD) and type 2 diabetes mellitus (T2Dm). Heme binds both amyloid beta (Aß) and human islet amyloid polypeptide (hIAPP) to form heme-Aß and heme-hIAPP complexes, respectively, and form reactive oxygen species (ROS) like H2O2, O2Ë-etc., which are known to cause oxidative damage. However the intermediates involved during ROS formation have not yet been isolated. In this study the oxygen bound intermediates of both heme-Aß(1-16) and heme-hIAPP(1-19) have been isolated and characterized using absorption, EPR and resonance Raman (rR) spectroscopy. Fe-O stretches have been found at 575 cm-1 and 577 cm-1 for heme-Aß(1-16) and heme-hIAPP(1-19) respectively. The oxy intermediates are stable at low temperatures. The isolation of the intermediates reveals a mechanistic pathway of ROS generation through the two heme complexes.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Heme/metabolismo , Compostos de Ferro/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , HumanosRESUMO
Heme bound amyloid ß (Aß) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aß, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aß. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aß, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aß complex behaves as CCP.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Citocromo-c Peroxidase/metabolismo , Heme/metabolismo , Peptídeos beta-Amiloides/química , Citocromo-c Peroxidase/química , Heme/química , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Cinética , Análise Espectral RamanRESUMO
Recent evidence has established the colocalization of amyloid-rich plaques and heme-rich deposits in the human cerebral cortex as a common postmortem feature in Alzheimer's disease (AD). The amyloid ß (Aß) peptides have been shown to bind heme, and the resultant heme-Aß complexes can generate toxic partially reduced oxygen species (PROS) and exhibit peroxidase activity. The heme-Aß active site exhibits a concentration-dependent equilibrium between a high-spin mono-His-bound species similar to a peroxidase-type active site and a bis-His-bound six-coordinate low-spin species similar to that of a cytochrome b type active site. The ν(Fe-His) (241 cm(-1)) vibration has been identified in the high-spin heme-Aß active site by resonance Raman spectroscopy. The formation of the low-spin heme-Aß species is promoted by the His14 and noncoordinating second-sphere Arg5 residues. The high-spin state produces more PROS than the low-spin species. Nonbiological constructs modeling different forms of Aß (oligomers, fibrils, etc.) suggest that the detrimental high-spin state is likely to dominate under most physiological conditions.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Citocromos b/metabolismo , Heme/química , Peptídeos/metabolismo , Peroxidases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Redox active iron is utilized in biology for various electron transfer and catalytic reactions essential for life, yet this same chemistry mediates the formation of partially reduced oxygen species (PROS). Oxidative stress derived from the iron accumulated in the amyloid plaques originating from amyloid ß (Aß) peptides and neurofibrillary tangles derived from hyperphosphorylated tau proteins has been implicated in the pathogenesis of Alzheimer's disease (AD). Altered heme homeostasis leading to dysregulation of expression of heme proteins and heme deposits in the amyloid plaques are characteristic of the AD brain. However, the pathogenic significance of heme in neurodegeneration in AD has been unappreciated due to the lack of detailed understanding of the chemistry of the interaction of heme and Aß peptides. As a result, the biochemistry and biophysics of heme complexes of Aß peptides (heme-Aß) remained largely unexplored. In this Account, we discuss the active site environment of heme bound Aß complexes, which involves three amino acid residues unique in mammalian Aß (Arg5, Tyr10, and His13) and missing in Aß from rodents, which do not get affected by AD. The histidine residue binds heme, while the arginine and the tyrosine act as key second sphere residues of the heme-Aß active site that play a crucial role in its reactivity. Generation of PROS, enhanced peroxidase activity, and oxidation of neurotransmitters such as serotonin (5-HT) are all found to be catalyzed by heme-Aß in in vitro assays, and these reactivities can potentially be linked to the observed neuropathologies in AD brain. Association of Cu with heme-Aß leads to the formation of heme-Cu-Aß. The heme-Cu-Aß complex produces a greater amount of PROS than reduced heme-Aß or Cu-Aß alone. Nitric oxide (NO), a signaling molecule, is found to ameliorate the detrimental effects of heme-Aß and Cu bound heme-Aß complexes by detaching heme from the heme-Aß complex and releasing it into the environment solution. Heme-Aß complexes show fast electron transfer with oxidized cytochrome c and rapid heme transfer with apomyoglobin and aponeuroglobin. NO, cytochrome c, and apoglobins can all lead to reduction in PROS generated by reduced heme-Aß. Synthetic analogues of heme, offering a hydrophobic distal environment, have been used to trap oxygen bound intermediates, which provides insight into the mechanism of PROS generation by reduced heme-Aß. Artificial constructs of Aß on nonbiological platforms are used not only to stabilize metastable and physiologically relevant large and small amyloid aggregates but also to monitor the interaction of various drug candidates with heme and Cu bound Aß aggregates, representing a tractable avenue for testing therapeutic agents targeting metals and cofactors in AD.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Heme/metabolismo , Peptídeos beta-Amiloides/química , Heme/química , Humanos , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Serotonina/química , Serotonina/metabolismoRESUMO
Heme-Aß complexes are known to produce toxic partially reduced oxygen species (PROS), catalyze oxidation of neurotransmitters and have been associated with Alzheimer's disease (AD). Neuroglobin (Ngb) play a crucial neuroprotective role against oxidative damage, hypoxic injuries, stroke and apoptosis of neuronal cells. In this study, the interaction of heme-Aß with apoNeuroglobin (apoNgb) has been investigated using a combination of spectroscopic techniques. Absorption and resonance Raman data confirm that apoNgb can uptake heme from heme-Aß and constitute a six-coordinate low-spin ferric heme-active site identical to that of Ngb. ApoNgb can also uptake heme from reduced heme-Aß resulting in the formation of ferrous Ngb. The rate of the heme transfer reaction has been found to be of the order of 10(6) M(-1) s(-1). The reaction is faster for oxidized heme-Aß than the reduced form. The amount of PROS formation by heme-Aß complexes has been found to diminish drastically after reaction with apoNgb. ApoNgb can also sequester ligand-bound heme from heme-Aß, e.g., the CO-bound heme from heme-Aß-CO complex resulting in the formation of Ngb-CO complex. Additionally, ApoNgb can sequester heme from self-assembled monolayer (SAM) of surface-bound heme-Aß formed over Au surface. This heme sequestration by apoNgb from heme-Aß not only diminishes heme-induced toxicity but more significantly it produces Ngb which has well-documented neuroprotective role and can thereby potentially reduce risks associated with AD.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Complexos de Coordenação/metabolismo , Globinas/metabolismo , Heme/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos beta-Amiloides/química , Complexos de Coordenação/química , Globinas/química , Heme/química , Humanos , Peróxido de Hidrogênio/química , Microscopia de Força Atômica , Proteínas do Tecido Nervoso/química , Neuroglobina , OxirreduçãoRESUMO
Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the "hemoglobin (Hb) superfamily." The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His(117) and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His(117) is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.