Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin.

Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.     

Acid and alkaline forms of the higher oxidation state of kangaroo, horse, and sperm whale myoglobin.

Myoglobin(IV), the derivative of myoglobin at the formal oxidation state IV, prepared from kangaroo (Megaleia rufa), horse, or sperm whale myoglobin, when cooled to liquid nitrogen temperature, assumes acid and alkaline forms with different optical spectra. The essential features of the optical spectra of the acid forms are the same as those of leghemoglobin(IV) and are very similar to those of optical spectra of the red higher oxidation states of catalases and peroxidases. This shows that the configuration of the heme iron is the same throughout these compounds. That configuration is believed to be Fe(IV) in a porphyrin environment. The optical spectra of alkaline mammalian myoglobin(IV), like that of alkaline leghemoglobin(IV), resemble those of the alkaline low spin ferric proteins. Kangaroo myoglobin(IV) may be prepared by reaction of ferrous myoglobin with hydrogen peroxide. The acid forms of myoglobin(IV) are conveniently prepared by cooling solutions in borate buffers, initially pH 8.3, to liquid nitrogen temperature. At this temperature borate buffers become acidic.     

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.     

X-ray crystal structure of a recombinant human myoglobin mutant at 2.8 A resolution

We have grown crystals in trigonal space group P3(2)21 of a mutant human myoglobin, aquomet form, in which lysine at position 45 has been replaced by arginine and cysteine at position 110 has been replaced by alanine. Suitable crystals of native recombinant human myoglobin have not been obtained. We have used the molecular replacement method to determine the X-ray crystal structure of the mutant at 2.8 A resolution. At the present stage of refinement, the crystallographic R-value for the model, with tightly restrained stereochemistry, is 0.158 for 5.0 to 2.8 A data. As expected, the overall structure is quite similar to the sperm whale myoglobin structure. Arginine 45 adopts a well-ordered conformation similar to that found in aquomet sperm whale myoglobin.     

A structural domain of the covalent polymer globin chains of artemia. Interpretation of amino acid sequence data

Artemia is unusual in having extracellular hemoglobins of Mr 260,000 comprising two globin chains (Mr 130,000), each of which is a polymer of eight covalently linked domains of about Mr 16,000. The amino acid sequence of one of these domains (E1) has been determined. It has 147 residues and Mr of 17,574 including heme. Sequence alignment revealed 19.0% identity with sperm whale myoglobin, whereas other vertebrate and invertebrate globins had between 13 and 24% identity. However, a much higher percentage of residues has a similar side chain character, suggesting that the domain E1 is very similar to other globins in showing the myoglobin fold. Template model building based on the known three-dimensional structure of myoglobin further supports this conclusion. Conversely, the differences between E1 and other globins are believed to reflect differences in the packing of the domains, first in a covalent polymeric subunit containing eight hemes and subsequently by association of two of these subunits as dimers. These findings provide further evidence for the versatility of the myoglobin fold.     

Comparison of the dynamics of myoglobin in different crystal forms.

Crystals have been grown of "sperm whale" myoglobin produced in Escherichia coli from a synthetic gene and the structure has been solved to 1.9 A resolution. Because of a remaining initiator methionine, this protein crystallizes in a different space group from native sperm whale myoglobin. The three-dimensional structure of the synthetic protein is essentially identical to the native sperm whale protein. However, the crystallographic B-factors for parts of the molecule are quite different in the two crystal forms, and provide a measure of the effect of different packing constraints on the flexibility of the protein. The effect of the packing forces is to reduce the mobility of the protein in the regions of contact and thereby introduce differences in mobilities between the two crystal forms. Discrepancies between mobilities calculated from molecular dynamics simulations and crystallography can be reduced by considering the data from both crystal forms.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether, LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action.     

Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase

Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.     

The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate

The mechanisms by which Hsp40 functions as a molecular chaperone to recognize and bind non-native polypeptides is not understood. We have identified a peptide substrate for Ydj1, a member of the type I Hsp40 from yeast. The structure of the Ydj1 peptide binding fragment and its peptide substrate complex was determined to 2.7 A resolution. The complex structure reveals that Ydj1 peptide binding fragment forms an L-shaped molecule constituted by three domains. The domain I exhibits a similar protein folds as domain III while the domain II contains two Zinc finger motifs. The peptide substrate binds Ydj1 by forming an extra beta strand with domain I of Ydj1. The Leucine residue in the middle of the peptide substrate GWLYEIS inserts its side chain into a hydrophobic pocket formed on the molecular surface of Ydj1 domain I. The Zinc finger motifs located in the Ydj1 domain II are not in the vicinity of peptide substrate binding site.     

Alpha and beta forms of cytochrome c oxidase observed in rat heart myocytes by low temperature Fourier transform infrared spectroscopy

Carbon monoxide bound to myoglobin and cytochrome c oxidase in separated adult rat heart myocytes has been observed with Fourier transform IR spectroscopy at low temperatures. CO complexes of these two proteins can be spectrally separated through temperature manipulation of the relaxation of the photolyzed systems. Photolyzed carboxymyoglobin relaxes very rapidly above 80 K, whereas the CO photolyzed from cytochrome a3 associates with CuB and relaxes very slowly below 140 K. Cytochrome c oxidase is found to be present in two major molecular forms which we designate alpha and beta. Each form contains an a3Fe and its associated CuB which we observe by their CO complexes. The predominant FeCO band, the alpha form of cytochrome oxidase, is similar to that previously seen in beef heart mitochondria, but with a slightly larger activation enthalpy, delta H = 46 kJ/mol. At least one of the beta forms is similar, but two have not been observed in beef heart mitochondria. Upon photolysis of alpha-FeCO, the alpha-CuCO species is formed. This band splits into two at low temperature. Up to half of the FeCO band area of the intact myocytes is distributed among three or more minor species (beta forms). The beta-FeCO bands all appear to be associated with only one beta-CuCO band which does not split at low temperature. After photo-dissociation of CO, the beta forms relax considerably faster than the alpha form, achieving 50% recombination in 10% of the time required for the alpha form. In a tissue slice from an opossum heart exposed to CO, we observed alpha and beta forms of cytochrome oxidase very similar to those in the rat heart myocytes. The cause of the differences between the alpha and beta forms of the enzyme is unknown, but their possible role in the control of respiration is discussed. Carboxymyoglobin contained within intact rat heart myocytes was very similar to sperm whale carboxymyoglobin, but with a much smaller amount of the lower frequency minor component.     

Crystallization and structural analysis of intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri

The intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri (G4-1), which has a raw starch binding domain, has been crystallized. The structure was identified (PDB entry 1GCY) by the molecular replacement method using the structure of its catalytic domain (G4-2). The result showed that the raw starch binding domain is in a disordered state, the corresponding electron densities being almost invisible. Superposition of these two enzyme forms showed evidence for the possible location of the raw starch binding domain (SBD). This crystal is a novel case, in that it forms a regular lattice incorporating flexibly bound SBD in the channel of crystal packing of the catalytic domains.     

A structure for the trimeric MHC class II-associated invariant chain transmembrane domain

The major histocompatibility complex (MHC)-associated invariant chain (Ii) contains a single transmembrane domain that forms trimers. Ii is involved in the assembly of the MHC and antigen presentation, and is thus central to the function of the immune system. Here, we show by attenuated total reflectance, Fourier transform infrared (ATR-FTIR) spectroscopy that the transmembrane domain is alpha-helical and we provide a structural model of the transmembrane domain obtained by a combination of site-specific infrared dichroism and molecular dynamics (MD) simulations. This work resolves the backbone structure of a transmembrane peptide by multiple (13)C=(18)O labelling at ten different residues. A second purely computational approach, based on MD simulations of Ii transmembrane homologous sequences, yields a similar structure that is consistent with our experimental results. The structure presented forms a left-handed coiled coil with an average helix tilt of 13(+/-6) degrees; the residue Gln47 implicated in trimer formation forms strong interhelical contacts, Thr50 points to the inside of the trimeric coil and forms a network of hydrogen bonds.     

Chemical regulation of nitric oxide: a role for intracellular myoglobin?

The detailed chemistry of nitric oxide (*NO) and regulation of this potent signal molecule through interactions with cellular components are complex and not clearly understood. In the vasculature, *NO plays a crucial role in vessel dilation by activating soluble guanylyl cyclase (sGC) in vascular smooth muscle cells (VSMC). *NO is responsible for maintaining coronary blood flow and normal cardiac function. However, *NO is a highly reactive molecule and this reactivity toward a range of alternate substrates may interfere with the activation of its preferred molecular target within VSMC. Interestingly, marked changes to *NO homeostasis are linked to disease progression. Thus, the physiological concentration of *NO is carefully regulated. Myoglobin is a haem-containing protein that is present in relatively high concentration in cardiac and skeletal muscle. Recently, the presence of myoglobin has been confirmed in human smooth muscle. The role of intracellular myoglobin is generally accepted as that of a passive di-oxygen storage protein. However, oxygenated myoglobin readily reacts with *NO to yield higher order N-oxides such as nitrate, while both the ferrous and ferric forms of the protein form a stable complex with *NO. Together, these two reactions effectively eliminate *NO on the physiological time-scale and strongly support the idea that myoglobin plays a role in maintaining *NO homeostasis in tissues that contain the protein. Interestingly, human myoglobin contains a sulfhydryl group and forms an S-nitroso-adduct similar to haemoglobin. In this article we discuss the potential for human myoglobin to actively participate in the regulation of *NO by three distinct mechanisms, namely oxidation, ligand binding, and through formation of biologically active S-nitroso-myoglobin.     

Crystal structure of C-terminal truncated apolipoprotein A-I reveals the assembly of high density lipoprotein (HDL) by dimerization

Apolipoprotein A-I (apoA-I) plays important structural and functional roles in plasma high density lipoprotein (HDL) that is responsible for reverse cholesterol transport. However, a molecular understanding of HDL assembly and function remains enigmatic. The 2.2-? crystal structure of Δ(185-243)apoA-I reported here shows that it forms a half-circle dimer. The backbone of the dimer consists of two elongated antiparallel proline-kinked helices (five AB tandem repeats). The N-terminal domain of each molecule forms a four-helix bundle with the helical C-terminal region of the symmetry-related partner. The central region forms a flexible domain with two antiparallel helices connecting the bundles at each end. The two-domain dimer structure based on helical repeats suggests the role of apoA-I in the formation of discoidal HDL particles. Furthermore, the structure suggests the possible interaction with lecithin-cholesterol acyltransferase and may shed light on the molecular details of the effect of the Milano, Paris, and Fin mutations.     

On the difference in stability between horse and sperm whale myoglobins

The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1 kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both apo and holo forms, and was independent of pH from 5 to 8 and of the presence of sodium chloride. We also show that the substitution of sperm whale myoglobin residues Ala15 and Ala74 to Gly, the residues found at positions 15 and 74 in horse myoglobin, decreased the stability by 1.0 kcal/mol, indicating that helix propensity is an important component of the explanation for the difference in stability between the two proteins.     

Dielectric behavior of water in biological solutions: studies on myoglobin, human low-density lipoprotein, and polyvinylpyrrolidone

The dielectric behavior of the aqueous solutions of three widely differing macromolecules has been investigated: myoglobin, polyvinylpyrrolidone (PVP), and human serum low-density lipoprotein (LDL). It was not possible to interpret unambiguously the dielectric properties of the PVP solution in terms of water structure. The best interpretation of the dielectric data on the myoglobin and LDL solutions was that, in both cases, the macromolecule attracts a layer of water of hydration one or two water molecules in width. For LDL, this corresponds to a hydration factor of only 0.05 g/g, whereas for myoglobin the figure is nearer 0.6 g/g. With myoglobin, part of the water of hydration exhibits its dispersion at frequencies of a few GHz, and the rest disperses at lower frequencies, perhaps as low as 10-12 MHz. The approximate constancy of the width of the hydration shell for two molecules as dissimilar in size as LDL and myoglobin confirms that the proportion of water existing as water of hydration in a biological solution depends critically on the size of the macromolecules as well as on their concentration.     

Equilibrium fluctuations in myoglobin and lysozyme

The angular dependencies of inelastic intensities of Rayleigh scattering of Moessbauer radiation were measured for myoglobin and lysozyme (in the hydration range h = 0.05-0.7). The data were fitted within the framework of model, when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative motions of segments. The best agreement with the experiment at h > 0.05 was obtained when individual motions of small groups together with the cooperative motions of alpha-helices and beta-sheets for lysozyme, and alpha-helices for myoglobin were considered. At further hydration (h = 0.45), mean-square displacements (x2) of both types of motions strongly increase with the increase in hydration degree, while the motions with a large correlation radius (not less than macromolecule radius) remain nearly the same as for h = 0.05. The results of the study of the radial distribution function deduced by Fourier-transform from the diffuse x-ray measurements together with RSMR data allow one to conclude that the water during protein hydration competes with the intramolecular hydrogen bonds, loosens the protein and increases the internal dynamics. Concurrently, water arranges the ordering of macromolecule, which takes the native structure at h = 0.4-0.7. The analysis of auto and cross-correlation functions of bending fluctuations of alpha-helices in the large domain of lysozyme performed by molecular dynamics allows one to come to the final conclusion that it is the difference in the structural organization of myoglobin and lysozyme and not the presence of SS-bonds in lysozyme macromolecule that is responsible for different structural fluctuations in these proteins.     

Human microtubule affinity-regulating kinase 4 is stable at extremes of pH

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of adenosine monophosphate-activated protein kinases, directly associated with cancer and neurodegenerative diseases. Here, we have cloned, expressed, and purified two variants of MARK4 [the kinase domain (MARK4-F2), and kinase domain along with 59 N-terminal residues (MARK4-F1)] and compared their stability at varying pH range. Structural and functional changes were observed by incubating both forms of MARK4 in buffers of different pH. We measured the secondary structure of MARK4 using circular dichroism and tertiary structure by measuring intrinsic fluorescence and absorbance properties along with the size of proteins by dynamic light scattering. We observed that at extremes of pH (below pH 3.5 and above pH 9.0), MARK4 is quite stable. However, a remarkable aggregate formation was observed at intermediate pH (between pH 3.5 and 9.0). To further validate this result, we have modeled both forms of MARK4 and performed molecular dynamics simulation for 15 ns. The spectroscopic observations are in excellent agreement with the findings of molecular dynamics simulation. We also performed ATPase activity at varying pH and found a significant correlation of structure of MARK4 with its enzyme activity. It is interesting to note that both forms of MARK4 are showing a similar pattern of structure changes with reference to pH.