Mouse models for dominant dystrophic epidermolysis bullosa carrying common human point mutations recapitulate the human disease.

Heterozygous missense mutations in the human COL7A1 gene - coding for collagen VII - lead to the rare, dominantly inherited skin disorder dominant dystrophic epidermolysis bullosa (DDEB), which is characterised by skin fragility, blistering, scarring and nail dystrophy. To better understand the pathophysiology of DDEB and develop more effective treatments, suitable mouse models for DDEB are required but to date none have existed. We identified the two most common COL7A1 mutations in DDEB patients (p.G2034R and p.G2043R) and used CRISPR-Cas9 to introduce the corresponding mutations into mouse Col7a1 (p.G2028R and p.G2037R). Dominant inheritance of either of these two alleles results in a phenotype that closely resembles that seen in DDEB patients. Specifically, mice carrying these alleles show recurrent blistering that is first observed transiently around the mouth and paws in the early neonatal period and then again around the digits from 5-10 weeks of age. Histologically, the mice show micro-blistering and reduced collagen VII immunostaining. Biochemically, collagen VII from these mice displays reduced thermal stability, which we also observed to be the case for DDEB patients carrying the analogous mutations. Unlike previous rodent models of epidermolysis bullosa, which frequently show early lethality and severe disease, these mouse models, which to our knowledge are the first for DDEB, show no reduction in growth and survival, and - together with a relatively mild phenotype - represent a practically and ethically tractable tool for better understanding and treating epidermolysis bullosa. This article has an associated First Person interview with the first author of the paper.

SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm.

dsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that Sidt1 expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to Sidt2-/- mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to Sidt2-/- mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

A novel rule-based algorithm for assigning myocardial fiber orientation to computational heart models.

Electrical waves traveling throughout the myocardium elicit muscle contractions responsible for pumping blood throughout the body. The shape and direction of these waves depend on the spatial arrangement of ventricular myocytes, termed fiber orientation. In computational studies simulating electrical wave propagation or mechanical contraction in the heart, accurately representing fiber orientation is critical so that model predictions corroborate with experimental data. Typically, fiber orientation is assigned to heart models based on Diffusion Tensor Imaging (DTI) data, yet few alternative methodologies exist if DTI data is noisy or absent. Here we present a novel Laplace-Dirichlet Rule-Based (LDRB) algorithm to perform this task with speed, precision, and high usability. We demonstrate the application of the LDRB algorithm in an image-based computational model of the canine ventricles. Simulations of electrical activation in this model are compared to those in the same geometrical model but with DTI-derived fiber orientation. The results demonstrate that activation patterns from simulations with LDRB and DTI-derived fiber orientations are nearly indistinguishable, with relative differences ≤6%, absolute mean differences in activation times ≤3.15 ms, and positive correlations ≥0.99. These results convincingly show that the LDRB algorithm is a robust alternative to DTI for assigning fiber orientation to computational heart models.

Antibody-based sensors for heavy metal ions.

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA 3000. Four different monoclonal antibodies specific for complexes of EDTA-Cd(II), DTPA-Co(II), 2,9-dicarboxyl-1,10-phenanthroline-U(VI), or cyclohexyl-DTPA-Pb(II) were incubated with the appropriate soluble metal-chelate complex. In the microwell assay format, the immobilized version of the metal-chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10-1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal-chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25+/-11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81-7.77% and 3.62-14.16% for within run and between run precision, respectively.

Binding properties of a monoclonal antibody directed toward lead-chelate complexes.

A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution.

Automated kinetic exclusion assays to quantify protein binding interactions in homogeneous solution.

A method was developed for the quantification of protein-ligand interactions in which the free protein present in homogeneous reaction mixtures was separated and quantified using a KinExA immunoassay instrument. Separation was achieved by rapid percolation of the reaction mixture over a column of microbeads whose surfaces were coated with an immobilized form of the ligand. The protein thus captured was quantified using a fluorescently labeled anti-protein antibody. The features of this new method were illustrated using a model system in which each of the principal reagents was covalently labeled with a different fluorescent molecule: mouse monoclonal anti-biotin primary antibody (fluorescein), biotin (B-phycoerythrin), and goat anti-mouse polyclonal secondary antibody (indodicarbocyanin). Values for the equilibrium and kinetic rate constants for the binding between the anti-biotin antibody and biotin conjugated with B-phycoerythrin were determined and shown to be independent of whether the fluorescent label was located on the primary or secondary antibody. Equilibrium binding experiments conducted with (F(AB))(2) and corresponding F(AB) fragments showed that the valency of the binding protein had no influence on the value of the dissociation constant. The values of the equilibrium and rate constants obtained by this new method are those for the binding reaction in homogeneous solution; the immobilized ligand is only a tool exploited for the separation and quantification of the free protein.

Solid-phase synthesis and characterization of carcinoembryonic antigen (CEA) domains.

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycoprotein expressed on tumors of the colon, breast, ovary, and lung, has seven predicted immunoglobulin-like domains (N-A1-B1-A2-B2-A3-B3), most of which are recognized by distinct monoclonal antibodies. To study the individual domains, we have prepared several of the domains (N, A3, B3, and A3-B3) by solid-phase peptide synthesis. The syntheses were performed by the Fmoc method using single couplings, elevated temperatures for both the coupling and deblocking reactions, and a flexible solvent system for the coupling reactions. The syntheses were accomplished on an in-house built synthesizer which allowed for temperature control and flexible solvent control during the course of the coupling reactions. Due to the large size of the peptides (84-184 residues), it was anticipated that the overall purity of the final product would not exceed 60% even for an average coupling yield of 99.5%. Therefore, several of the peptides were synthesized with a His6 "tail" at the amino terminus, allowing for purification on a Ni-NTA chelate column. For the most part, the purified peptides exhibited single sharp peaks by RP-HPLC, migrated at their expected molecular weights by gel permeation chromatography, gave correct masses by electrospray ionization or matrix-assisted laser desorption ionization time of flight mass spectrometry, gave the expected amino acid analyses, N-terminal sequences, and tryptic maps, and bound their appropriate monoclonal antibodies. The N-domain was extremely hydrophobic, requiring 6M guanidinium hydrochloride for solubilization, the A3 domain was soluble in dilute acid, and the B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) are reported, along with characterization of the 178 amino acid two-domain peptide, A3-B3. Although there is no evidence for proper folding of these domains by NMR, their ability to bind monoclonal antibodies with high affinity suggests that this is a plausible approach for producing individual domains of CEA.

Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes.

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExATM immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

Multiple wavelength anomalous diffraction (MAD) crystal structure of rusticyanin: a highly oxidizing cupredoxin with extreme acid stability.

The X-ray crystal structure of the oxidized form of the extremely stable and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans has been determined by the method of multiwavelength anomalous diffraction (MAD) and refined to 1.9 A resolution. Like other cupredoxins, rusticyanin is a copper-containing metalloprotein, which is composed of a core beta-sandwich fold. In rusticyanin the beta-sandwich is composed of a six- and a seven-stranded beta-sheet. Also like other cupredoxins, the copper ion is coordinated by a cluster of four conserved residues (His85, Cys138, His143, Met148) arranged in a distorted tetrahedron. Rusticyanin has a redox potential of 680 mV, roughly twice that of any other cupredoxin, and it is optimally active at pH values < or = 2. By comparison with other cupredoxins, the three-dimensional structure of rusticyanin reveals several possible sources of the chemical differences, including more ordered secondary structure and more intersheet connectivity than other cupredoxins. The acid stability and redox potential of rusticyanin may also be enhanced over other cupredoxins by a more extensive internal hydrogen bonding network and by more extensive hydrophobic interactions surrounding the copper binding site. Finally, reduction in the number of charged residues surrounding the active site may also make a major contribution to acid stability. We propose that the resulting rigid copper binding site, which is constrained by the surrounding hydrophobic environment, structurally and electronically favours Cu(I). We propose that the two extreme chemical properties of rusticyanin are interrelated; the same unique structural features that enhance acid stability also lead to elevated redox potential.

NMR solution structure of Cu(I) rusticyanin from Thiobacillus ferrooxidans: structural basis for the extreme acid stability and redox potential.

The solution structure of the Cu(I) form of the rusticyanin from Thiobacillus ferrooxidans has been calculated from a total of 1979 distance and dihedral angle constraints derived from 1H, 13C and 15N NMR spectra. The structures reveal two beta-sheets, one of six strands and one of seven strands that are tightly packed in a beta-barrel or beta-sandwich arrangement, and a short helix that extends on the outside of one of the sheets to form a second hydrophobic core. The copper coordination sphere is composed of the standard type I ligands (His2CysMet) in a distorted tetrahedral arrangement. The copper-binding site is located within a hydrophobic region at one end of the molecule, surrounded by a number of aromatic rings and hydrophobic residues. This configuration probably contributes to the acid stability of the copper site, since close association of the aromatic rings with the histidine ligands would sterically hinder their dissociation from the copper. An electrostatic analysis based on a comparison of the structures of rusticyanin and French bean plastocyanin shows that factors determining the high redox potential of rusticyanin include contributions from charged side-chains and from the disposition of backbone peptide dipoles, particularly in the 81 to 86 region of the sequence and the ligand cysteine residue. These interactions should also contribute to the acid stability by inhibiting protonation of His143.

Gene synthesis, high-level expression, and mutagenesis of Thiobacillus ferrooxidans rusticyanin: His 85 is a ligand to the blue copper center.

An artificial gene of the blue copper protein rusticyanin from Thiobacillus ferrooxidans was constructed from eight overlapping oligonucleotides in a recursive "one-pot" polymerase chain reaction. The gene was placed behind the T7/lacOR promoter of pET24a and expressed in Escherichia coli as a soluble protein. A purification scheme involving a pH titration step, cation-exchange chromatography, and reverse-phase HPLC separation provided yields of the apoprotein ranging from 70 to 100 mg/L of cell culture; reconstitution with Cu(II) is quantitative at pH 3.4-5.5. The redox reactions and the electronic absorption and EPR spectra of the recombinant Cu(II)-rusticyanin and NMR spectra of the reduced holoprotein are indistinguishable from those of the protein derived from T. ferrooxidans. Rusticyanin possesses the phylogenetically conserved carboxy-terminal loop of three copper ligands (Cys 138, His 143, and Met 148), but the identity of the fourth ligand was not clear from sequence homology to other blue copper proteins. To address this question directly, we have prepared two site-specific mutants where two of the proposed ligands, Asp 73 and His 85, have been replaced with alanine. The Asp73Ala mutant retained the electronic properties of the wild-type blue copper center (absorption maxima at 452, 597, and 750 nm), whereas the His85Ala variant gave rise to a green type 1 copper protein (absorption maxima at 455 and 618 nm).(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear magnetic resonance 15N and 1H resonance assignments and global fold of rusticyanin. Insights into the ligation and acid stability of the blue copper site.

Nuclear magnetic resonance assignments are reported at pH approximately 3 for a type 1 ("blue") copper protein, rusticyanin, obtained from the acidophilic organism Thiobacillus ferrooxidans. A combination of homonuclear proton and heteronuclear 15N-edited NMR spectra has been used to assign most of the 1H and 15N resonances of reduced rusticyanin. The copper-binding site is shown by analogy with other blue copper proteins to contain the side-chains of Cys138, His143 and Met148 at the C-terminal end of the sequence and a fourth ligand that is most likely a histidine, His85, consistent with the constitution of other type 1 copper sites. The global fold of the molecule is a compact beta-barrel or beta-sandwich, which contains a high proportion of beta-sheet secondary structure and a hydrophobic core particularly rich in aromatic residues. The copper-binding active site is surrounded by aromatic residues, and many of the resonances of the residues flanking the active site are shifted to unusual values, consistent with the effects of ring currents. The protected nature of the copper site is demonstrated by the large number of amide protons that are persistent in this region in 99% 2H2O solution at pH 3.4. We suggest that the unusual acid stability, both of the protein itself and of the blue copper active site, is a direct result of the protected and highly hydrophobic nature of the active site sequence and contacting loops and the high proportion of secondary structure in the protein.

Solubilization of Minerals by Bacteria: Electrophoretic Mobility of Thiobacillus ferrooxidans in the Presence of Iron, Pyrite, and Sulfur.

Thiobacillus ferroxidans is an obligate acidophile that respires aerobically on pyrite, elemental sulfur, or soluble ferrous ions. The electrophoretic mobility of the bacterium was determined by laser Doppler velocimetry under physiological conditions. When grown on pyrite or ferrous ions, washed cells were negatively charged at pH 2.0. The density of the negative charge depended on whether the conjugate base was sulfate, perchlorate, chloride, or nitrate. The addition of ferric ions shifted the net charge on the surface asymptotically to a positive value. When grown on elemental sulfur, washed cells were close to their isoelectric point at pH 2.0. Both pyrite and colloidal sulfur were negatively charged under the same conditions. The electrical double layer around the bacterial cells under physiological conditions exerted minimal electrostatic repulsion in possible interactions between the cell and either of its charged insoluble substrates. When Thiobacillus ferrooxidans was mixed with either pyrite or colloidal sulfur at pH 2.0, the mobility spectra of the free components disappeared with time to be replaced with a new colloidal particle whose electrophoretic properties were intermediate between those of the starting components. This new particle had the charge and size properties anticipated for a complex between the bacterium and its insoluble substrates. The utility of such measurements for the study of the interactions of chemolithotrophic bacteria with their insoluble substrates is discussed.

Respiratory enzymes of Thiobacillus ferrooxidans. Kinetic properties of an acid-stable iron:rusticyanin oxidoreductase.

Rusticyanin is an acid-stable, soluble blue copper protein found in abundance in the periplasmic space of Thiobacillus ferrooxidans, an acidophilic bacterium capable of growing autotrophically on soluble ferrous sulfate. An acid-stable iron:rusticyanin oxidoreductase activity was partially purified from cell-free extracts of T. ferrooxidans. The enzyme-catalyzed, iron-dependent reduction of the rusticyanin exhibited three kinetic properties characteristic of aerobic iron oxidation by whole cells. (i) A survey of 14 different anions indicated that catalysis by the oxidoreductase occurred only in the presence of sulfate or selenate, an anion specificity identical to that of whole cells. (ii) Saturation with both sulfatoiron(II) and the catalyst produced a concentration-independent rate constant of 3 s-1 for the reduction of the rusticyanin, which is an electron transfer reaction sufficiently rapid to account for the flux of electrons through the iron respiratory chain. (iii) Values for the enzyme-catalyzed pseudo-first-order rate constants for the reduction of the rusticyanin showed a hyperbolic dependence on the concentration of sulfatoiron(II) with a half-maximal effect at 300 microM, a value similar to the apparent KM for iron shown by whole cells. On the basis of these favorable comparisons between the behavior patterns of isolated biomolecules and those of whole cells, this iron:rusticyanin oxidoreductase is postulated to be the primary cellular oxidant of ferrous ions in the iron respiratory electron transport chain of T. ferrooxidans.

Enhanced yields of iron-oxidizing bacteria by in situ electrochemical reduction of soluble iron in the growth medium.

An electrochemical apparatus for culturing chemolithotrophic bacteria that respire aerobically on ferrous ions is described. Enhanced yields of the bacteria were achieved by the in situ electrochemical reduction of soluble iron in the growth medium. When subjected to a direct current of 30 A for 60 days, a 45-liter culture of Thiobacillus ferrooxidans grew from 6 x 10 to 9.5 x 10 cells per ml. Growth of the bacterium within the electrolytic bioreactor was linear with time. A final cell density corresponding to 4.7 g of wet cell paste per liter was achieved, and a total of 320 g of wet cell paste was harvested from one culture. The apparatus was designed to deliver protons concomitantly with electrons; therefore, the pH of the culture remained stable at 1.6 +/- 0.1 for the duration of growth. This laboratory-scale apparatus may be readily adapted to pilot or production scale. It is thus anticipated that abundant numbers of iron-oxidizing bacteria may be obtained for both fundamental and applied studies.

Sensitivity of Iron-Oxidizing Bacteria, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans, to Bisulfite Ion.

When grown on iron-salt medium supplemented with the bisulfite ion, Leptospirillum ferrooxidans was much more sensitive to the ion than was Thiobacillus ferrooxidans. The causes of the sensitivity of L. ferrooxidans to the bisulfite ion were studied. The bisulfite ion completely inhibited the iron-oxidizing activities of L. ferrooxidans and T. ferrooxidans at 0.02 and 0.2 mM, respectively. A trapping reagent for the bisulfite ion, formaldehyde, completely reversed the inhibition. The treatment of intact cells with 1.0 mM bisulfite ion for 1 h and washing the bisulfite ion from the cells had no harmful effects on the iron-oxidizing activity of T. ferrooxidans. However, the treatment of L. ferrooxidans with 0.1 mM bisulfite ion for 1 h completely destroyed the iron-oxidizing activity. T. ferrooxidans had sulfite:ferric ion oxidoreductase activity. In contrast, a quite low level of sulfite:ferric ion oxidoreductase activity was found in L. ferrooxidans, suggesting that it is much more difficult for L. ferrooxidans to oxidize the bisulfite ion to the less harmful sulfate than it is for T. ferrooxidans. These results suggest that the sensitivity of L. ferrooxidans to the bisulfite ion is due to a lack of an active sulfite:ferric ion oxidoreductase and the sensitivity of its iron oxidase to bisulfite ion.

Enzyme immunoassay to determine heavy metals using antibodies to specific metal-EDTA complexes: optimization and validation of an immunoassay for soluble indium.

An immunoassay that measures soluble indium at concentrations from 0.005 ppb to 320 ppm is described. The assay utilized a monoclonal antibody that binds specifically to indium-EDTA complexes in an antigen-inhibition format. The sensitivity of the assay could be modulated by changing the nature of the soluble inhibiting antigen. The range of the assay was from 0.6 to 320 ppm, 0.1 to 120 ppm, or 0.005 to 2000 ppb when indium-EDTA, indium-(p-nitrobenzyl)-EDTA, or indium-EDTA-bovine serum albumin, respectively was used as the soluble inhibiting antigen. The assay reliably monitored indium concentration in the presence of a 100-fold excess of manganese, magnesium, or copper ions and the quantitation of indium by immunoassay correlated closely with the values obtained using atomic absorption spectroscopy. This technology could be employed in immunoassays for other metals that are priority pollutants.

Enzymes of aerobic respiration on iron.

Bacteria capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Comparative spectroscopic analyses revealed that phylogenetically distinct organisms expressed copious quantities of spectrally distinct redox-active biomolecules during autotrophic growth on soluble iron. Thiobacillus ferroxidans, Leptospirillum ferrooxidans, Sulfobacillus thermosulfidooxidans, and Metallosphaera sedula possessed iron respiratory chains dominated by a blue copper protein, a novel red cytochrome, a novel yellow protein, and a novel yellow cytochrome, respectively. Further investigation of each type of respiratory chain will be necessary to deduce the advantages and disadvantages of each.

Crystallization and preliminary X-ray crystallographic studies of rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a 16.5 kDa type I blue copper protein isolated from Thiobacillus ferrooxidans. This organism can grow on Fe2+ as its sole energy source. Rusticyanin is thought to be a principal component in the iron respiratory electron transport chain of T. ferrooxidans. As a component of the periplasmic space of an acidophilic bacterium, rusticyanin is remarkably stable at acidic pH. It is redox-active down to pH 0.2. Crystals of rusticyanin have been grown from solutions of PEG 8000 by the hanging-drop vapor diffusion method. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions a = 32.36 A, b = 60.37 A, c = 74.60 A. The crystals diffract to 2.0 A resolution and they are stable in the X-ray beam for at least two days.