Stimulation by cadmium of myohemerythrin-like cells in the gut of the annelid Nereis diversicolor.

Isolated guts of Nereis diversicolor revealed the existence of a cadmium-binding protein, the MPII, belonging to the group of hemerythrins and myohemerythrins. The presence of MPII in the cells of the intestine was demonstrated by immunocytochemistry, using anti-MPII, a monoclonal antibody. In addition, using in situ hybridization and northern blotting, it was shown that MPII-cells are the site of synthesis of this molecule. Exposure of the worms to cadmium led to the cellular activation process of MPII-cells (i.e. transformation of the nucleolus, development of the endoplasmic reticulum and the Golgi apparatus), although MPII mRNA transcript levels were unchanged. Enzyme-linked immunosorbent assay (ELISA) of gut extracts revealed that MPII levels were increased after exposure to Cd, so it appears that this protein is synthesized as a response to Cd exposure without any new synthesis of mRNA. This mechanism of regulation is quite similar to that reported in the case of mammalian ferritin and may be involved in the regulation of Cd levels in this worm.

Visible reflectance hyperspectral imaging: characterization of a noninvasive, in vivo system for determining tissue perfusion.

We characterize a visible reflectance hyperspectral imaging system for noninvasive, in vivo, quantitative analysis of human tissue in a clinical environment. The subject area is illuminated with a quartz-tungsten-halogen light source, and the reflected light is spectrally discriminated by a liquid crystal tunable filter (LCTF) and imaged onto a silicon charge-coupled device detector. The LCTF is continuously tunable within its useful visible spectral range (525-725 nm) with an average spectral full width at half-height bandwidth of 0.38 nm and an average transmittance of 10.0%. A standard resolution target placed 5.5 ft from the system results in a field of view with a 17-cm diameter and an optimal spatial resolution of 0.45 mm. The measured reflectance spectra are quantified in terms of apparent absorbance and formatted as a hyperspectral image cube. As a clinical example, we examine a model of vascular dysfunction involving both ischemia and reactive hyperemia during tissue reperfusion. In this model, spectral images, based upon oxyhemoglobin and deoxyhemoblobin signals in the 525-645-nm region, are deconvoluted using a multivariate least-squares regression analysis to visualize the spatial distribution of the percentages of oxyhemoglobin and deoxyhemoglobin in specific skin tissue areas.

Cloning and expression analysis of a cDNA that encodes a leech hemerythrin.

We report the cDNA sequence of a leech hemerythrin. A cDNA was isolated from a Theromyzon tessulatum cDNA library and encodes a 120 amino acid protein of about 14 kDa. The predicted protein contains the hemerythrin signature sequence and the iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin. The protein displayed the highest identity to myohemerythrin, a non-heme iron-binding protein described in sipunculids. Expression analysis indicated that the mRNA is widely expressed in leech and is stage specific in appearance, being absent after the two first blood meals, appearing after the last blood meal during the period preceding oogenesis and disappearing after egg laying.

Mechanism of photosynthetic water oxidation: combining biophysical studies of photosystem II with inorganic model chemistry.

A mechanism for photosynthetic water oxidation is proposed based on a structural model of the oxygen-evolving complex (OEC) and its placement into the modeled structure of the D1/D2 core of photosystem II. The structural model of the OEC satisfies many of the geometrical constraints imposed by spectroscopic and biophysical results. The model includes the tetranuclear manganese cluster, calcium, chloride, tyrosine Z, H190, D170, H332 and H337 of the D1 polypeptide and is patterned after the reversible O2-binding diferric site in oxyhemerythrin. The mechanism for water oxidation readily follows from the structural model. Concerted proton-coupled electron transfer in the S2-->S3 and S3-->S4 transitions forms a terminal Mn(V)=O moiety. Nucleophilic attack on this electron-deficient Mn(V)=O by a calcium-bound water molecule results in a Mn(III)-OOH species, similar to the ferric hydroperoxide in oxyhemerythrin. Dioxygen is released in a manner analogous to that in oxyhemerythrin, concomitant with reduction of manganese and protonation of a mu-oxo bridge.

Molecular cloning and characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri.

An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies

Oxymyohemerythrin: discriminating between O2 release and autoxidation.

Myohemerythrin (Mhr) is a non-heme iron O2 carrier (with two irons in the active site) that is typically found in the retractor muscle of marine 'peanut' worms. OxyMhr may either release O2, or undergo an autoxidation reaction in which hydrogen peroxide is released and diferric metMhr is produced. The autoxidation reaction can also be promoted by the addition of certain anions to Mhr solutions. This work, using recombinant Themiste zostericola Mhrs, contrasts the results of environmental effects on these reactions. For the O2 release reaction, deltaVdouble dagger(21.5 degrees C) = +28+/-3 cm3 mol(-1), deltaHdouble dagger(1 atm) = +22+/-1 kcal mol(-1), and deltaSdouble dagger(1 atm) = +28+/-4 eu. The autoxidation reaction (pH 8.0, 21.5 degrees C, 1 atm) displays different kinetic parameters: deltaVdouble dagger = -8+/-2 cm3 mol(-1), deltaHdouble dagger = +24.1+/-0.7 kcal mol(-1), and deltaSdouble dagger = +1+/-1 eu. Autoxidation in the presence of sodium azide is orders of magnitude faster than solvolytic autoxidation. The deltaVdouble dagger parameters for azide anation and azide-assisted autoxidation reaction are +15+/-2 and +59+/-2 cm3 mol(-1), respectively, indicating that the rate-limiting steps for the Mhr autoxidation and anation reactions (including O2 uptake) are not associated with ligand binding to the Fe2 center. The L103V and L103N oxyMhr mutants autoxidize approximately 10(3)-10(5) times faster than the wild-type protein, emphasizing the importance of leucine-103, which may function as a protein 'gate' in stabilizing bound dioxygen.

The O(2) binding pocket of myohemerythrin: role of a conserved leucine.

A conserved O(2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), namely, L104, was mutated to several other residues, and the effects on O(2) association and dissociation rates, O(2) affinity, and autoxidation were examined. The L104V, -F, and -Y myoHrs formed stable O(2) adducts whose UV-vis and resonance Raman spectra closely matched those of wild-type oxymyoHr. The L104V mutation produced only minimal effects on either O(2) association or dissociation, whereas the L104F and -Y mutations resulted in 100-300-fold decreases in both O(2) association and dissociation rates. These decreases are attributed to introduction of steric restrictions into the O(2) binding pocket, which are not present in either wild-type or L104V myoHrs. The failure to observe increased O(2) association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not sterically "gate" O(2) entry into or exit from the binding pocket in the rate-determining step(s). L104V myoHr autoxidized approximately 3 times faster than did wild type, whereas L104T autoxidized >10(6) times faster than did wild type. The latter large increase is attributed to increased side chain polarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that restricts water entry into the oxymyoHr binding pocket. Thus, a leucine at position 104 in myoHr appears to have the optimal combination of size and hydrophobicity to facilitate O(2) binding while simultaneously inhibiting autoxidation.

A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech.

The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent K(I) of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the triatoma Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.

Transmembrane and water-soluble helix bundles display reverse patterns of surface roughness.

Amino acid exposure and surface roughness were calculated for 12 helices from three transmembrane alpha-helix bundles and 13 helices from seven water-soluble alpha-helix bundles. Transmembrane helix bundles have relatively rough surfaces exposed to the lipid bilayer hydrocarbon chains and relatively smooth surfaces along helix-helix interfaces. This pattern is the reverse of what occurs in water-soluble helix bundles, where relatively rough surfaces are at the helix-helix interfaces and relatively smooth surfaces are exposed to water. The relatively rough exposed surfaces and buried smooth surfaces of transmembrane helices are likely to contribute to the stability of transmembrane helical bundles in a phospholipid environment.

NADH peroxidase activity of rubrerythrin.

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.

Identification of a gene for a rubrerythrin/nigerythrin-like protein in Spirillum volutans by using amino acid sequence data from mass spectrometry and NH2-terminal sequencing.

A hydrogen peroxide-resistant mutant of the catalase-negative microaerophile, Spirillum volutans, constitutively expresses a 21.5 kDa protein that is undetectable and non-inducible in the wild-type cells. Part of the gene that encodes the protein was cloned using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing. The deduced 158 amino acid polypeptide shows high relatedness to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris. The protein also shows high similarity to putative rubrerythrin proteins found in the anaerobic archeons Archaeoglobus fulgidus, Methanococcus jannaschii and Methanobacterium thermoautotrophicum. This is the first report of this type of protein in an organism that must respire with oxygen. It seems likely that the novel combination of methodologies used in this study could be applied to the rapid cloning of other genes in bacteria for which no genomic library yet exists.

The role of structure in antibody cross-reactivity between peptides and folded proteins.

Peptides have the potential for targeting vaccines against pre-specified epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced fit of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with sub-micromolar affinities. The strong preference for a type II beta-turn predicted for one peptide was confirmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.

A rubrerythrin operon and nigerythrin gene in Desulfovibrio vulgaris (Hildenborough).

Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (Hildenborough). We have sequenced a 3.3-kbp Sal1 fragment of D. vulgaris chromosomal DNA containing the rubrerythrin gene, rbr, identified additional open reading frames (ORFs) adjacent to rbr, and shown that these ORFs are part of a transcriptional unit containing rbr. One ORF, designated fur, lies just upstream of rbr and encodes a 128-amino-acid-residue protein which shows homology to Fur (ferric uptake regulatory) proteins from other purple bacteria. The other ORF, designated rdl, lies just downstream of rbr and encodes a 74-residue protein with significant sequence homology to rubredoxins but with a different number and spacing of cysteine residues. Overexpression of rdl in Escherichia coli yielded a protein, Rdl, which has spectroscopic properties and iron content consistent with one Fe3+(SCys)4 site per polypeptide but is clearly distinct from both rubrerythrin and a related protein, nigerythrin. Northern analysis indicated that fur, rbr, and rdl were each present on a transcript of 1.3 kb; i.e., these three genes are cotranscribed. Because D. vulgaris nigerythrin appears to be closely related to rubrerythrin, and its function is also unknown, we cloned and sequenced the gene encoding nigerythrin, ngr. The amino acid sequence of nigerythrin is 33% identical to that of rubrerythrin, and all residues which furnish iron ligands to both the FeS4 and diiron-oxo sites in rubrerythrin are conserved in nigerythrin. Despite the close resemblance of these two proteins, ngr was found to be no closer than 7 kb to rbr on the D. vulgaris chromosome, and Northern analysis showed that, in contrast to rbr, ngr is not cotranscribed with other genes. Possible redox-linked functions for rubrerythrin and nigerythrin in iron homeostasis are proposed.

Functional role of leucine-103 in myohemerythrin.

Hemerythrins (Hrs) and myohemerythrins (Mhrs) are nonheme iron proteins that function as O2 carriers in four marine invertebrate phyla. Available amino acid sequences and X-ray structures indicate that a conserved leucine, residue 103 in the Themiste zostericola Mhr sequence, occupies a site distal to the Fe-O-Fe center. The side-chain methyl groups of the analogous leucine in Themiste dyscrita oxyHr are in van der Waals contact with bound O2 in the X-ray crystal structure, and this residue may therefore play a role in stabilizing bound dioxygen with respect to autoxidation. In order to test this hypothesis, the gene for T. zostericola Mhr was synthesized and expressed in Escherichia coli. Two mutant Mhrs, L103V and L103N, were also prepared. Optical spectra and kinetics data for these three proteins are presented. Importantly, neither mutant forms a stable oxy adduct; instead, rapid autoxidation results in formation of the corresponding met forms. In addition, the L103N Mhr displays unusually rapid reduction kinetics, suggesting that the amide functionality of Asn-103 destabilizes most bound ligands and additionally promotes rapid semi-metR <==> semi-metO isomerization.

Structures of wild-type chloromet and L103N hydroxomet Themiste zostericola myohemerythrins at 1.8 A resolution.

Myohemerythrin (Mhr) is a nonheme iron oxygen carrier found in the retractor muscles of marine "peanut" worms. The X-ray crystal structures of two recombinant Themiste zostericola Mhrs are reported to a resolution of 1.8 A. Surprisingly, the met wild-type structure (R = 17.8%) was found to contain chloride bound to Fe2, while coordinated hydroxide was found in the met L103N structure (R = 18.3%). An internal water molecule was also found distal to the Fe-O-Fe center of the mutant protein, forming hydrogen bonds with the coordinated hydroxide and the OD1 atom of Asn-103. This finding is consistent with the kinetic and spectroscopic results reported for the L103N mutant Mhr [Raner, G. M., Martins, L. J., & Ellis, W. R., Jr. (1997) Biochemistry 36, 7037-7043]. Possible roles for the side chain of residue 103 (Leu in wild-type Mhr) in gating ligand binding are also discussed.

Primary structure of a myohemerythrin-like cadmium-binding protein, isolated from a terrestrial annelid oligochaete.

Two isoforms of a cadmium-binding protein (Cd-BP 14a and Cd-BP 14b) were isolated from the terrestrial oligochaete annelid, Allolobophora caliginosa. The complete amino acid sequence of the major isoform Cd-BP 14a (molecular mass: 13441 Da; 119 residues) and the amino-terminal sequence (57 residues) of Cd-BP 14b were determined. The sequence of Cd-BP 14a is highly similar to that of myohemerythrins present in marine invertebrates. Furthermore, as myohemerythrins, Cd-BP 14a and Cd-BP 14b bind two atoms of iron and their ultraviolet/visible spectra are typical of non-heme iron-binding proteins. Three substitutions were found in the amino-terminal half of the proteins at positions 19, 21 and 41. The substitutions at positions 19 and 21 are conservative, whereas that at position 41 consists of the replacement of an aspartate residue in isoform a by a lysine residue in isoform b. To our knowledge, it is the first report of a protein belonging to the hemerythrin family in a terrestrial invertebrate.

Contribution of increased length and intact capping sequences to the conformational preference for helix in a 31-residue peptide from the C terminus of myohemerythrin.

In order to examine the effects of chain length on the propensity of short peptides to form helix-like structures in aqueous solution, we have studied a peptide of 31 residues consisting of the C-terminal sequence (residues 88-118) of the four-helix bundle protein myohemerythrin from Themiste zostericola. This peptide, termed MDC, represents the final two elements of secondary structure in the protein, the D-helix and the C-terminal loop sequence, together with a five-residue sequence at the N terminus corresponding to the linker between the C- and D-helices. An N-capping sequence, VDAKNV, immediately precedes the D-helix sequence, and a C-capping sequence, VNHIKGT, corresponding to the alphaL termination motif, occurs at the C-terminal end. The effect of replacement of a cysteine residue in the middle of the sequence with an alanine was explored by the comparison of the MDC peptide and a 16-residue peptide representing the sequence of the D-helix alone, both containing the change Cys99Ala. Significant changes in the NMR and CD spectra were seen for both peptides compared to the wild-type sequence. A comparison of the fluorescence spectra of the wild-type and Cys99Ala peptides indicated that a specific interaction between the side chains of Cys 99 and Trp 102 acts to quench the fluorescence of the tryptophan ring and probably contributes a component that distorts the CD spectrum of the wild-type peptide at approximately 220-235 nm. The effect of an increase in the length of the peptide, with the incorporation of capping sequences derived from the native sequence, was explored by NMR and CD spectroscopy of the 31-residue and 16-residue peptides in aqueous solution and in TFE/water mixtures. Evidence for the formation of a significant population of helical conformers in the region of the MDC peptide corresponding to the D-helix was observed in aqueous solution using CD and NMR spectroscopy. The C-terminal 10 residues of the MDC peptide behave in solution in a manner identical to that of a 10-residue peptide with the same sequence; a highly specific local interaction between an aromatic ring and a glycine amide proton appears to be retained in the longer peptide. Upon addition of trifluoroethanol (TFE), significant shifts are observed in a number of resonances in the NMR spectrum, and both chemical shifts and NOEs provide evidence for a higher population of helix in the D-helix region of the peptide in TFE. However, TFE is unable to promote the propagation of helix beyond the N-cap or alphaL termination motifs, and the specific local interaction observed in the C-terminal sequence is retained in TFE. The CD spectrum in TFE shows an increase in the proportion of helix, to an overall maximum of approximately 55% helix at 50% v/v TFE, corresponding to approximately 100% helix in the D-helix sequence of the peptide, since the N and C termini of the MDC peptide are not helical according to the NMR spectra. The high proportion of helix observed in the D-helix sequence of the longer MDC peptide demonstrates that the presence of intact capping sequences can constrain the peptide conformational ensemble to resemble that seen in the native protein. A compendium of results from this and previous peptide studies has also led to a novel observation, the existence of a correlation between the amide proton chemical shift and temperature coefficient.

Pairwise iterative superposition of distantly related proteins and assessment of the significance of 3-D structural similarity.

A challenge lies in identifying distant protein 3-D structural similarity by rigid-body superposition. The most common measure of structural similarity is r.m.s. distance (r.m.s.d.) between topologically equivalent residues, and most automated methods of protein modelling rely on the assembly of rigid fragments from known 3-D structures. A fast method of improving the definition of a common protein fold by superposition, especially for distant relationships, is described. The definition of topological equivalence by the standard dynamic programming sequence alignment algorithm is extended by refining the entire structure alignment (not just those equivalenced residues within a given cut-off distance) and determining whether the alignment can be continued at the termini. The most appropriate distance-based definition of topological equivalence for a given comparison is identified. Despite the fact that hitherto the distant similarity between the globin fold and colicin A has not been recognized directly by rigid-body superposition, this new approach defines more equivalent residues with a lower r.m.s.d. between them than that obtained by the superposition of equivalences identified by a more elaborate method. A previous distance metric of 3-D structural similarity derived from rigid-body superposition has been extended to the assessment of superpositions where topological equivalences have been determined by methods other than rigid-body ones.

Computational studies of protein folding.

This review describes computational approaches to the determination of protein structure from sequence. The emphasis is on reduced protein models that are sufficiently accurate to represent protein structure at low resolution, yet are computationally efficient enough to allow the extensive search of phase space required to locate the global minimum from an unfolded state. A discussion of both potential functions and algorithmic simulation strategies for such models are presented, along with a number of specific models that have been developed and successfully applied to proteins as large as myoglobin. The results indicate that significant progress is being made in understanding the requirements for computational prediction of protein structure.