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WO1998053849A1 - Metalloproteines synthetiques et procede de preparation correspondant - Google Patents

Metalloproteines synthetiques et procede de preparation correspondant Download PDF

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Publication number
WO1998053849A1
WO1998053849A1 PCT/US1998/011228 US9811228W WO9853849A1 WO 1998053849 A1 WO1998053849 A1 WO 1998053849A1 US 9811228 W US9811228 W US 9811228W WO 9853849 A1 WO9853849 A1 WO 9853849A1
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Prior art keywords
protein
binding site
designed
apoprotein
metal
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PCT/US1998/011228
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English (en)
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John P. Caradonna
Ann L. Pinto
Homme W. Hellinga
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Yale University
Duke University
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Priority to AU77158/98A priority Critical patent/AU7715898A/en
Publication of WO1998053849A1 publication Critical patent/WO1998053849A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention is in the field of synthetic metalloproteins, and relates to compositions having the ability to undergo electron or atom transfer reactions.
  • the invention relates to proteins which are synthetically modified to incorporate a redox-active metal site.
  • the invention further relates to a method for preparing such compositions, which are useful as catalysts in a broad range of applications, including, but not limited to, hydrocarbon oxidation and superoxide dismutation reactions.
  • Metalloproteins offer a particularly interesting target for the design of function, because the biological chemistry of metals is extraordinarily rich. Protein design methodologies (Bryson, J. W., et al, (1995) Science 270, 935-41; Desjarlais, J. R. & Handel, T. M. (1995) Curr. Opin. Biotechnol. 6, 460-6; Barrick, D. (1995) Cwr. Opin. Biotechnol. 6, 411-8) exist to incorporate transition metal ions into proteins (Barrick, supra; Higaki, J. N., Fletterick, R. J. & Craik, C. S. (1992) Trends Biochem. Sci. 17, 100- 4; Matthews, D. J.
  • the present invention shows how to create a true active site which catalyzes the inner sphere transfer of electrons both to and from small molecule substrates.
  • an open coordination position or one occupied by a labile ligand such as H 2 O, is required, but the site must be sufficiently buried to prevent protein dimerization via a metal bridge.
  • Introduction of a cavity for anion binding as well as other factors relating to the attraction and transport of the anion to the active site all must be considered as well.
  • metalloprotein designs have focused primarily on the first two factors (Bryson, J. W., supra; Higaki, J. N., supra; Matthews, D. J., supra; Regan, L., supra; Tawfik, D. S., supra; Hellinga, H. W. (1996) Curr. Opin. Biotechnol, 7, 437-441; Hellinga, H. W. (1996) in Protein Engineering: Principles and Practice, eds.
  • Fe S 4 clusters are among the most common electron transfer centers found in biology (Johnson, M. K. (1994) in Iron-Sulfur Proteins, ed. King, R. B. (John Wiley & Sons, Oxford), Vol. 4, pp. 1896-1915). These clusters act as either simple soluble electron-transfer agents, membrane-bound components of electron-transfer chains, or parts of the electron reservoir found in complex metalloenzymes in plants, animals and bacteria. In addition to participating in electron transport systems and in the metabolism of carbon, oxygen, hydrogen, sulfur and nitrogen, these centers also exhibit gene regulatory, catalytic and structural functions, and have been found as part of a morphogenetic protein (Johnson, M. K., supra).
  • the present inventors demonstrate herein methods having sufficient predictive power to redesign the hydrophobic interior of a protein (Trx) and thereby introduce an iron center that can catalyze the dismutation of superoxide anion.
  • the present inventors also provide a method of preparing a HiPIP cluster constructed by placing a cuboidal ⁇ Fe S 4 ⁇ cluster in the hydrophobic interior of the protein thioredoxin (Trx-Fe 4 S 4 ).
  • one object of the present invention is to provide a method of preparing a functional metalloprotein containing at least one metal binding site, said site consisting of at least one metal ion and ligands associated therewith and having a calculated volume equal to the sums of the volumes of the metal ion(s) and all of the ligands, wherein said ligands are, in a first coordination sphere, atoms bonded directly to the metal ion(s) and in a second coordination sphere, all atoms bonded directly and indirectly to the atoms of the first coordination sphere between the metal ion(s) and the backbone of the protein, which comprises:
  • step (C) preparing a nucleotide sequence corresponding to the set of sequence mutations determined in step (B)(6) for the designed apoprotein;
  • step (D) transfecting a host organism with the nucleotide sequence of the designed apoprotein;
  • Another object of the invention is to provide a functional metalloprotein containing at least one metal binding site, said site consisting of at least one metal ion and ligands associated therewith and having a calculated volume equal to the sums of the volumes of the metal ion(s) and all of the ligands, wherein said ligands are, in a first coordination sphere, atoms bonded directly to the metal ion and, in a second coordination sphere, all the metal ion(s) and the backbone of the protein, which comprises:
  • volume change resulting from each set of primary sequence mutations (5) calculating the volume change resulting from each set of primary sequence mutations; and (6) selecting a set of primary sequence mutations wherein the volume change corresponding thereto is not more than between about 90% and 110% of the volume of the metal binding site and wherein the tertiary structure of the resulting metalloprotein containing such set of mutations is overpacked to an extent of less than about 10% in mean atomic displacement, thereby providing the designed apoprotein;
  • step (C) preparing a nucleotide sequence corresponding to the set of sequence mutations determined in step (B)(6) for the designed apoprotein;
  • a further particular object of the present invention is to provide a synthetic superoxide dismutase containing at least one metal binding site, which superoxide dismutase is useful in the treatment of a range of conditions, including, but not limited to, ischemia and reperfusion-type tissue damage.
  • Another object of the invention is to provide a method of treating a subject suffering from ischemia which comprises administering to the subject a functional metalloprotein prepared from a host protein containing a metal binding site wherein the protein has superoxide dismutase activity in a therapeutically effective amount.
  • a further application of the present invention is a method of treating a subject suffering from reperfusion type tissue damage which comprises administering to the subject a functional metalloprotein having superoxide dismutase activity.
  • Figure 1 shows the location of the designed ⁇ Fe3+ ⁇ Trx-SOD metal-binding site formed by Leu7His, Phe27Asp, Ile ⁇ OHis, and Asn63His.
  • Other mutations include His ⁇ Asn (eliminates potential alternative metal binding residue), Cys32Ser and Cys35Ser (prevents disulfide formation), Leu58Ala and Thr66Ala (rebuilds complementary surface) and Asp26Ala (increases global stability of host protein).
  • Figure 2 illustrates a 20% SDS gel of purified apo Trx-SOD. Lanes: 1, 28 ⁇ g of purified Trx-SOD; 5, 0.28 ⁇ g of purified Trx-SOD; 6, 0.78 ⁇ g of purified Trx-SOD; 2 and 3, molecular weight markers as indicated; 4, 1.8 ⁇ g of E. coli Fe SOD (Sigma).
  • Figure 3 provides a UV/vis spectrum of apo (solid) and reconstituted (dashed) Trx-SOD.
  • the spectra of Trx-SOD (1.1 x 10 "4 M) in 20 mM Hepes, pH 8.0, 25 mM NaCI, 21° C before and after iron reconstitution with ferrous ammonium sulfate in air are shown.
  • Figure 4 illustrates anion binding by a functional metalloprotein.
  • the UV/vis spectrum of ⁇ Fe 3+ ⁇ Trx-SOD (1.1 x 10 "4 M) in 20 mM Hepes, pH 8.0, 25 mM NaCI, 21 °C (solid line) was recorded. The sample was then adjusted to 100 mM sodium azide (dashed line, Figure 4A) or 100 mM sodium fluoride (dashed line, Figure 4B). Dotted lines represent difference spectra in both Figures.
  • Figure 5 provides an assay of ⁇ Fe ⁇ Trx-SOD for SOD activity. Each point represents the average of three assays; the correlation coefficient of the least squares fit is greater than 0.99.
  • Figure 6 shows iron-limiting reconstitution and SOD activity.
  • Apo Trx-SOD was reconstituted in air to the following Fe:protein ratios: 0, 0.25, 0.50, 0.75, 0.95.
  • the samples were then assayed for SOD activity (o) and the optical spectra ( # ) of the samples (20 ⁇ l diluted to 1 ml) were recorded. Each point represents the average of three assays; the correlation coefficient of the least squares fit is greater than 0.99.
  • Figure 7 illustrates ribbon and space-filling representations of the designed cuboidal iron- sulfur protein Trx-Fe S 4 . Iron is cyan, sulfur yellow.
  • FIG. 1 (A) Ribbon diagram showing the location of all mutations used in the construction of Trx-Fe 4 S 4 and the region of the Trx host protein implicated in phage assembly.
  • the cuboidal ⁇ Fe 4 S ⁇ cluster binding residues (Leu24Cys, Leu42Cys, Val55Cys, and Leu99Cys) are buried between the central ⁇ -sheet and two ⁇ -helices.
  • Cys32Ser and Cys35Ser mutations were made to remove the native Trx disulfide bond, thereby eliminating any interaction from cysteine residues that are not part of the designed site.
  • Figure 8 provides an optical spectrum of apo Trx-Fe 4 S 4 (trace A), holo Trx-Fe S (trace B) in 15 mM MOPS, 100 mM NaCI, pH 7.4, 5 °C, and the synthetic cluster ⁇ Fe 4 S 4 (S- EtOH) 4 ⁇ (Me 4 N) 2 (trace C) in 10 mM CHES, 10 mM ⁇ ME, pH 8.5.
  • Figure 9 shows the stability of the free synthetic cluster ⁇ Fe 4 S 4 (S-EtOH) 4 ⁇ (Me 4 N) 2 (D) in the presence of low levels (0.4 mM) of stabilizing exogenous ⁇ ME and reconstituted holo Trx-Fe 4 S ( ⁇ ) in the absence of stabilizing exogenous ⁇ ME.
  • Solution conditions 10 mM MOPS, 100 mM NaCI, pH 7.4, 5 °C.
  • Figure 10 provides an EPR spectrum of holo Trx-Fe 4 S 4 in 15 mM MOPS, 100 mM NaCI, pH 7.4, protein concentration 70 ⁇ 5 ⁇ M.
  • Trace B holo Trx-Fe 4 S 4 oxidized with 10 equivalents of K 3 (Fe(CN) 6 ⁇ .
  • Trace A untreated (resting state) holo Trx-Fe 4 S 4 .
  • Figure 11 shows a set of consensus sequences found in proteins containing cuboidal ⁇ Fe4S4 ⁇ clusters.
  • the present invention provides a rational design approach to construct a synthetic metalloprotein containing a metal center within a protein matrix of known structure. Accordingly, the present invention provides a method of preparing a functional metalloprotein containing at least one metal binding site, said site consisting of at least one metal ion and ligands associated therewith and having a calculated volume equal to the sums of the volumes of the metal ion(s) and all of the ligands, wherein said ligands are, in a first coordination sphere, atoms bonded directly to the metal ion and in a second coordination sphere, all atoms bonded directly and indirectly to the atoms of the first coordination sphere between the metal ion(s) and the backbone of the protein, which comprises:
  • A selecting a host protein having a tertiary structure characterized with a resolution of less than or equal to about 3.0 A; (B) determining a set of primary sequence mutations in the host protein which collectively form the first and second coordination spheres of the metal binding site and result in overpacking of the tertiary structure to an extent of less than about 10% in mean atomic displacement, to provide a designed apoprotein, wherein said determining comprises:
  • step (C) preparing a nucleotide sequence corresponding to the set of sequence mutations determined in step (B)(6) for the designed apoprotein;
  • the invention provides a method as dislcosed above wherein the determining step comprises:
  • identifying residues to be repacked comprising: a) determining whether the residues make steric contact with any portion of the first coordination sphere, wherein van der Waals' radii of atoms comprising the residues and the first coordination sphere overlap, such that if said radii overlap, the residues are repacked; and b) determining whether a replaced residue creates a void by calculating a probe-accessible surface, wherein if a void is created, the replaced residue is repacked;
  • the invention provides a method wherein the functional metalloprotein contains one metal binding site.
  • the method is applicable in preparing functional metalloproteins with two, three or more metal binding (active) sites.
  • the method may be applied to prepare proteins wherein the metal binding site comprises a cuboidal iron-sulfur cluster.
  • the method may be used to prepare metalloproteins wherein the metal binding site comprises a mononuclear iron(II III)-histidine.
  • the required primary sequence mutations may be determined using any molecular modeling protocol adapted to the purpose, for example, DEZYMER, PROPAK and METALSEARCH. Most commonly, the modeling method used in conjunction with the invention to determine the primary sequence mutations is any method based on the DEZYMER algorithm, as further described herein.
  • a three-dimensional atomic coordinate array In order to use a modeling protocol, detailed structural information is required to provide a three-dimensional atomic coordinate array. Where crystals of the host protein are available, crystallographic data derived from X-ray crystallography, electron diffraction or neutron diffraction may be used. Coordinate arrays for protein crystals are readily obtained from public data banks such as the Brookhaven Protein Databank. In cases where no crystals are available, or diffraction data are insufficient, and only a solution- phase structure determination is feasible, a three-dimensional atomic coordinate array may be derived by nuclear magnetic resonance spectroscopy.
  • the method of the present invention may be applied to a wide range of host proteins.
  • functional metalloproteins may be designed and constructed in accord with the invention wherein the host protein is any physiological plasma protein, a thioredoxin or human serum albumin.
  • Other proteins include catalytic antibodies, non- human serum albumins, such as bovine serum albumin, collagen, synthetic oligopeptides and polypeptides.
  • the present invention also provides a functional metalloprotein containing at least one metal binding site, said site consisting of at least one metal ion and ligands associated therewith and having a calculated volume equal to the sums of the volumes of the metal ion(s) and all of the ligands, wherein said ligands are, in a first coordination sphere, atoms bonded directly to the metal ion and in a second coordination sphere, all atoms bonded directly and indirectly to the atoms of the first coordination sphere between the metal ion(s) and the backbone of the protein, which comprises:
  • step (C ) preparing a nucleotide sequence corresponding to the set of sequence mutations determined in step (B)(6) for the designed apoprotein;
  • the invention provides a functional metalloprotein as disclosed above wherein the determining step comprises: (1) representing the structure of the host protein as a three-dimensional atomic coordinate array, wherein side chains are removed leaving the protein backbone;
  • the invention provides a functional metalloprotein wherein the protein contains one metal binding site.
  • functional metalloproteins with two, three or more metal binding (active) sites are also available.
  • One example of functional metalloprotein within the scope of the invention is a protein wherein the metal binding site comprises a cuboidal iron-sulfur cluster.
  • Another example is a functional metalloprotein wherein the metal binding site comprises a mononuclear iron(II/III)-histidine.
  • the functional metalloprotein provided by the invention may be prepared from a host protein which is a physiological plasma protein, a thioredoxin or a human serum albumin.
  • the primary sequence mutations required in the process to prepare the functional metalloproteins may be determined using any molecular modeling protocol adapted to the purpose, such as the DEZYMER, PROPAK and METALSEARCH programs.
  • the modeling method preferably used to determine the primary sequence mutations is any method based on the DEZYMER algorithm.
  • the present invention further provides a synthetic superoxide dismutase containing at least one metal binding site, said site consisting of at least one metal ion and ligands associated therewith and having a calculated volume equal to the sums of the volumes of the metal ion(s) and all of the ligands, wherein said ligands are, in a first coordination sphere, atoms bonded directly to the metal ion and in a second coordination sphere, all atoms bonded directly and indirectly to the atoms of the first coordination sphere between the metal ion(s) and the backbone of the synthetic superoxide dismutase, said synthetic superoxide dismutase being prepared by:
  • the synthetic superoxide dismutase disclosed above is provided wherein the determining step comprises:
  • identifying residues to be repacked comprising: a) determining whether the residues make steric contact with any portion of the first coordination sphere, wherein van der Waals' radii of atoms comprising the residues and the first coordination sphere overlap, such that if said radii overlap, the residues are repacked; and b) determining whether a replaced residue creates a void by calculating a probe-accessible surface, wherein if a void is created, the replaced residue is repacked;
  • the invention provides a synthetic superoxide dismutase wherein said superoxide dismutase contains one metal ion binding site.
  • the synthetic superoxide dismutase may contain two or more metal ion binding sites.
  • the primary sequence mutations necessary in the design of the superoxide dismutase are determined using any suitable modeling protocol, for example, DEZYMER, PROPAK and METALSEARCH, but preferably using the DEZYMER algorithm, as described below.
  • the structure of the host protein is obtained as a three-dimensional atomic coordinate array from X-ray crystallography, electron diffraction or neutron diffraction where crystals are available for the host protein, or from nuclear magnetic resonance spectroscopy where no crystals have bene obtained.
  • Construction of the synthetic superoxide dismutase may be accomplished by selecting any of a variety of host proteins, such as thioredoxin or human serum albumin. Selection of other suitable host proteins in accord with criteria set forth herein would be within the level of skill of the ordinary worker.
  • the present invention also provides a synthetic monooxidase containing at least one metal binding site, said site consisting of at least one metal ion and ligands associated therewith and having a calculated volume equal to the sums of the volumes of the metal ion(s) and all of the ligands, wherein said ligands are, in a first coordination sphere, atoms bonded directly to the metal ion and, in a second coordination sphere, all atoms bonded directly and indirectly to the atoms of the first coordination sphere between the metal ion(s) and the backbone of the synthetic monooxygenase said synthetic monooxygenase being prepared by:
  • step (C) preparing a nucleotide sequence corresponding to the set of sequence mutations determined in step (B)(6) for the designed monooxygenase apoprotein;
  • the synthetic monooxidase is made as disclosed above, wherein the determining step comprises:
  • identifying residues to be repacked comprising: a) determining whether the residues make steric contact with any portion of the first coordination sphere, wherein van der Waals' radii of atoms comprising the residues and the first coordination sphere overlap, such that if said radii overlap, the residues are repacked; and b) determining whether a replaced residue creates a void by calculating a probe-accessible surface, wherein if a void is created, the replaced residue is repacked; (3) calculating volume elements in the host protein to be filled excluding the backbone of the protein equal to between 90%> and 110% of the volume of the metal binding site, said volume elements comprising a sum of replaced residue volumes; (4) executing a repacking algorithm, wherein the primary sequence and residue rotamers are varied, such that sequence mutations are selected wherein the volume change corresponding thereto is not more than between about 90% and 110% of the volume of the metal binding site and wherein the tertiary structure of the
  • the invention provides a synthetic monooxidase prepared as disclosed above wherein the monooxidase contains one metal ion binding site
  • the monooxidase contains one metal ion binding site
  • An example is one containing a dioxo-Mo binding site
  • other synthetic monooxygenases containing more than one metal ion binding site are within the scope of the present invention
  • Preparation of the synthetic monooxidase requires initial consideration of primary sequence mutations which are determined by molecular modeling Any of a variety of modeling protocols are available to assist in deteriming the particular primary sequence mutations Thus, primary sequence mutations are determined using an algorithm selected from the group consisting of DEZYMER, PROPAK and METALSEARCH However, a preferred protocol for modeling uses the DEZYMER algorithm Coordinate data are needed to apply a modeling protocol to determine the primary sequence mutations Structure data of the host protein for use in the design of the synthetic monooxidase is obtained as a three-dimensional atomic coordinate array either from X-ray crystallography, electron diffraction or neutron diffraction, if crystals are available, or from nuclear magnetic resonance spectroscopy, if no crystals are available
  • Suitable host proteins useful in the construction of the synthetic monooxidase include, but are not limited to, thioredoxin or human serum albumin
  • the functional metalloprotein is selected from the group consisting of ferredoxin, human serum albumin, a catalytic antibody, a tyrosinase and a superoxide dismutase
  • the present invention also provides a method of treating a subject suffering from ischemia which comprises administering to the subject a functional metalloprotein prepared from a host protein containing a metal binding site wherein the protein has superoxide dismutase activity in a therapeutically effective amount.
  • the functional metalloprotein may be prepared from a wide range of host proteins, oligopeptides or polypeptides compatible with a medicinal or clinical application.
  • a preferred host protein is a human serum albumin.
  • the metal ion binding site comprises Fe(II/III) and a histidine ligand.
  • the present invention also provides a method of treating a subject suffering from reperfusion type tissue damage which comprises administering to the subject a functional metalloprotein prepared from a host protein containing a metal binding site wherein the protein has superoxide dismutase activity in a therapeutically effective amount.
  • the host protein may be any protein, oligopeptide or polypeptide suited to medicinal application.
  • the host protein is a human serum albumin.
  • the metal ion binding site may comprise any metal ion compatible with a medicinal application, but favorably comprises Fe(II/III) and a histidine ligand.
  • the rational protein design algorithm As applied in the present invention, the rational protein design algorithm
  • DEZYMER Hellinga, H. W. & Richards, F. M. (1991) J. Mol. Biol. Ill, 763-786) is used to search the three-dimensional structure of the host protein (e.g., Trx) to identify locations where it is predicted to be geometrically possible to introduce mutations to form the correct primary coordination sphere. Mutations are chosen to satisfy both the intended metal binding geometry, and the steric requirements of the protein fold. Once a coordination sphere has been positioned, further mutations may be introduced to retain steric compatibility between the metal binding site and the surrounding protein. All the substitutions are therefore structurally conservative in nature, the intent being to maintain the original fold and stability of the host protein.
  • Trx the host protein
  • the DEZYMER algorithm makes predictions based on strict geometric principles without explicit consideration of binding thermodynamics or protein dynamics.
  • the rational design approach used here is based on the placement of an active site into the framework of a known protein fold. Active sites are described as geometrical arrangements of functionally important amino acids around a ligand, for example, a transition metal center.
  • the DEZYMER algorithm systematically examines a protein structure to identify backbone positions that are arranged in such a way that appropriate rotamers of the residues in the binding site definition can be placed to satisfy the desired ligand geometry.
  • additional changes may be introduced to ensure steric complementarity of the placed site with the surrounding protein matrix.
  • the synthetc metalloprotein comprising human serum albumin in which an active site having superoxide dismutase activity, herein referred to as HSA-SOD, and related compositions are useful in treating subjects suffering from ischemia or reperfusion tissue damage.
  • HSA-SOD superoxide dismutase activity
  • SOD may be applied to treat ischemia and reperfusion injury in various tissues such as brain (Matsumiya et al., (1991) Stroke, 22, 1193-1200; Kirsch et al. (1993) Pediatr.
  • HSA-SOD synthetic metalloproteins
  • trauma head injury, multiple organ failure, subarachnoid hemorrhage
  • transplantation rejection of transplant, necrosis
  • radiation therapy protection of bone marrow cells, radiation-induced fibrosis and cystosis
  • acute infection and inflammation burn wound healing, esophagitis, thrombosis, and influenza virus infection.
  • Mammals, and specifically humans, suffering from ischemia or reperfusion tissue damage can be treated by administering to the subject an effective amount of an HSA-SOD or a related metalloprotein in the presence of a pharmaceutically acceptable carrier or diluent.
  • the magnitude of the therapeutic dose of the particular metalloprotein will vary with the nature and severity of the condition to be treated and with the particular metalloprotein and its route of administration.
  • Systemic dosages to achieve relief from symptoms of ischemia and reperfusion type tissue damage typically range from 2 mg/kg to 0.0001 mg/kg per day as a single daily dose or divided daily doses.
  • Typical dosages for topical application range from 0.001 to 100%) by weight of the synthetic metalloprotein.
  • Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a synthetic metalloprotein disclosed herein.
  • the synthetic metalloprotein may be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, parenterally, orally, submucosally, by inhalation, transdermally via a slow release patch, rectally, or topically, in an effective dosage range to achieve relief from symptoms of ischemia and reperfusion type tissue damage.
  • Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, lotions, gels, tinctures, sprays, powders, pastes, slow-release transdermal patches, suppositories for application to rectal, vaginal, nasal or oral mucosa.
  • Thickening agents, emollients, and stabilizers can be used to prepare compositions for administration.
  • thickening agents include petrolatum, beeswax, xanthan gum, or polyethylene, humectants such as sorbitol, emollients such as mineral oil, lanolin and its derivatives, or squalene. All patents, applications, articles, publications and test methods mentioned above are hereby incorporated by reference.
  • Trx-SOD was constructed by standard PCR mutagenesis techniques using Pfu DNA polymerase (Stratagene) and the modified gene was inserted into the expression vector pKK-T7E downstream of a T7 promoter and sequenced in full.
  • the pellet was redissolved in a minimum volume of 20 mM Tris-Cl, pH 8, 25 mM NaCI, adjusted to 50 mM 2-mercaptoethanol and 1 mM each o-phenanthroline, TIRON (4,5-dihydroxy-m-benzenedisulfonic acid) and EDTA to remove endogenous metals and dialyzed overnight against 20 mM Tris-Cl, pH 8, 25 mM NaCI.
  • a final purification step takes advantage of the unusual ability of the Trx host protein to thermally denature and renature reversibly; aliquots of purified apo protein were heated to 90 C for 20 min., spun in a microcentrifuge to remove any precipitated material and rechromatographed on a Superdex S75 column in 20 mM HEPES-Cl, pH 8, 25 mM NaCI where only the properly folded Trx-SOD is isolated.
  • Reconstitution also performed under anaerobic conditions to assess the level of iron binding.
  • Iron binding stoichiometry was measured by varying the initial ferrous ammonium sulfate-to-Trx-SOD ratio (0.1 to 10). After extended incubation (5 hours) in 20 mM Hepes, pH 8.0, 25 mM NaCI, 21 C under strict anaerobic conditions in an inert atmosphere (argon) box, the reconstitution samples were dialyzed overnight against 20 mM Tris-Cl, pH 8, 25 mM NaCI (no EDTA). No oxidation of the iron was observed as judged by the absence of any visible chromophore. After air oxidation (1 hour), Fe to Trx- SOD ratios were measured by atomic absorption and UV spectrophotometric methods; oxidation, however, was complete in minutes as judged by color development.
  • EXAMPLE 5 Activity Measurements The superoxide dismutase activity of holo Trx-SOD was determined by a standard indirect colorimetric assay that measures the level of inhibition of superoxide induced reduction of colorless nitro blue tetrazolium (NBT, Sigma) dye to its oxidized blue formazan form (Flohe, L. & Otting, F. (1984) Methods Enzymol. 105, 93- 104).
  • NBT colorless nitro blue tetrazolium
  • One unit of SOD activity is defined as that amount of enzyme that inhibits the rate of NBT reduction, under the specified conditions, by 50%. Several dilutions of one enzyme solution were investigated in order to accurately extrapolate to the 50%> inhibition level.
  • the iron SOD site definition derived from the X-ray crystal structures of iron SOD from E. coli (Stallings, W. C, et al, (1983) Proc. Nat'lAcad. Sci. USA 80, 3884-8) and Pseudomonas ovalis (Stoddard, B. L., et al, (1990) Biochemistry 29, 8885-93), consisted of a trigonal bipyramidal iron center with axial histidine and exogenous ligand binding sites (for superoxide or other anions) and equatorial (N-His) 2 (O-Asp) ligands.
  • a bound superoxide ligand in the axial position was added to the site definition, based upon the idealized geometry of a Fe-O ⁇ adduct (Hill, A. A. O. & Tew, D. G. (1987) in Dioxygen, Superoxide and Peroxide as Ligands, eds. Wilkinson, G., Gillard, R. D. & McCleverty, J. A. (Pergamon Press, Oxford), Vol. 2, pp. 315-333).
  • the DEZYMER algorithm generated six solutions for the Fe SOD active site definition; the present inventors constructed and characterized one solution (Trx-SOD): Leu7His, Phe27Asp, Ile ⁇ OHis, Asn63His ( Figure 1).
  • This site was initially selected due to the relatively short axial distance between the iron center and the protein surface ( ⁇ 9A), the favorable orientation of the "open” coordination site which is aligned towards the protein surface and not directed towards the protein interior, and the lack of any protein main chain atoms sterically hindering access to the iron site.
  • Trx a relatively small (108 amino acid residues) monomeric protein that contains a single redox active disulfide bond (Cys32, Cys35) (Holmgren, A. (1985) in Ann. Rev. Biochem. 54, 237-271), was chosen (Hellinga, H. W., Caradonna, J. P. & Richards, F. M. (1991) J Mol Biol 222, 787-803) as a scaffold protein since the gene for Trx has been cloned, sequenced, and expressed at high levels (Wallace, B. j. & Kushner, S. R. (1984) Gene 32, 399-401).
  • Trx is a stable protein (oxidized Trx T m : 86 C, reduced Trx T m : 75 C) (Holmgren, A. (1985) in Ann. Rev. Biochem. 54, 237-271) that accommodates conservative as well as some non-conservative mutations (Wynn, R. & Richards, F. M. (1993) Protein Sci. 2, 395-403).
  • Trx is a stable protein (oxidized Trx T m : 86 C, reduced Trx T m : 75 C) (Holmgren, A. (1985) in Ann. Rev. Biochem. 54, 237-271) that accommodates conservative as well as some non-conservative mutations (Wynn, R. & Richards, F. M. (1993) Protein Sci. 2, 395-403).
  • X-ray structure Karlinsky, S. K., LeMaster, D. M. & Eklund, H. (1990) J. Biol. Chem.
  • Trx-SOD Trx-SOD
  • His ⁇ Asn to prevent competition between the designed His7 site and the naturally occurring His6 residue, the only histidine residue in host thioredoxin
  • Leu58Ala Thr66Ala to improve packing around the designed site owing to differences in volume between the naturally occurring residues of Trx and those of the designed Fe site
  • Asp26Ala to improve the global stability of the protein (the Asp26Ala mutant of thioredoxin is 3 kcal stabilized as compared to wild- type) (Langsetmo, K., et al. (1991) Biochemistry 30, 7603-7609; Langsetmo, K., et al (1991) Biochemistry 30, 7609-7614).
  • Fe (a ) has relatively slow exchange kinetics and thus it is possible, if oxidation is rapid relative to complete reconstitution, that reconstitution in air does not yield the thermodynamically favored metal binding site. To address this issue, extended incubation
  • thermodynamically favored iron site Fe /anaerobic conditions
  • SODs are usually assayed indirectly owing to the extreme instability of the superoxide ion in aqueous solution
  • a common assay method utilizes xanthine oxidase and xanthine to generate the superoxide ion which then reduces nitroblue tetrazolium (NBT), a reaction which can be followed spectrophotometrically, inhibition of this reduction is a measure of SOD activity (Flohe, L & Otting, F (1984) Methods Enzymol 105, 93-104)
  • NBT nitroblue tetrazolium
  • Trx-SOD Trx-SOD preparations
  • the activity of Trx-SOD is about 10 " times that of native E. coli Fe SOD; that is, the E. coli enzyme would need to be present at levels of 0.01% to account for the observed activity.
  • heat-treatment of E. coli Fe SOD reduces its activity by at least 1000-fold (a lower limit)
  • the contaminant would have to comprise 10% of present preparations to account for the observed activity after heat-treatment. Examination of Fig. 2 excludes this possibility.
  • the parallel between activity and the intensity of the His-to-Fe LMCT band indicates tha it is iron in the designed histidine rich site that is catalytically active.
  • Trx-Fe 4 S 4 was constructed by Kunkel mutagenesis techniques and the modified gene was inserted into the expression vector pKK-T7E (Kriwacki, R. W., et al, (1992) Proc. Nat 'lAcad. Sci. USA 89, 9759-9763) downstream of a T7 promoter and sequenced in full. Expression of Trx-Fe S4 was achieved in E. coli BL21(DE3). pTrx-Fe S /BL21 was grown in 2xYT, 50 ⁇ g/ml Ampicillin, at 25 C, and induced with DPTG; cells were harvested 4 hours post induction.
  • EXAMPLE 10 Purification of apo Trx-Fe 4 S 4 .
  • the harvested cells from 10 liters of culture were resuspended in 140 ml 20 mM Tris-Cl, pH 7.5, 100 mM NaCI, 10 mM EDTA and lysed by sonication.
  • the cleared lysate was brought to 0.25%> PEI, centrifuged, and then brought to 45%) saturation with solid ammonium sulfate and clarified. The supernatant was then brought to 90% saturation with solid ammonium sulfate.
  • the pellet was dissolved in 50 ml of 50 mM Tris-Cl, pH 8.1, 20 mM NaCI, and dialyzed against the same buffer for 4 hours.
  • the volume was reduced by dialysis against solid PEG 8000 at room temperature for 3 hours, followed by dialysis against 50 mM Tris-Cl, pH 8.1, 20 mM NaCI for 6 hours.
  • the sample was then applied in approximately 60 1.2 ml portions onto a 16 mm x 60 cm S75 gel filtration column and eluted with 50 mM Tris-CL, pH 8.1, 20 mM NaCI, 4 C. Peak fractions were then combined and loaded onto a 25 mm x 7 cm DEAE- cellulose column, washed with five column volumes of 50 mM Tris-Cl, pH 8.1, 20 mM NaCI and eluted with a 20-500 mM salt gradient in 20 mM Tris-Cl, pH 8.1. The isolated protein was concentrated four-fold in an ultrafiltration cell and an equal volume of glycerol was added.
  • EXAMPLE 11 Cluster Reconstitution of Tr -Fe 4 S4. Reconstitution of apo Trx-ye 4 S is performed under strict anaerobic conditions in an inert (argon) atmosphere box using rigorously deoxygenated solutions. Apo Trx-Fe 4 S 4 is passed through a gel filtration column eluted with 15 mM CHES, pH 8.5, to remove glycerol and DTT. The resulting solution is then made 2.0 M urea and 10 mM ⁇ -mercaptoethanol ( ⁇ ME) and allowed to sit for 15 minutes at 25 C. A freshly prepared solution of the synthetic cluster ⁇ Fe S (S- EtOH) 4 ⁇ (Me 4 N) 2 Christou, G. & Carner, C. D.
  • DMSO dimethyl sulfoxide
  • EXAMPLE 12 Measurement of in vivo activity of thioredoxin.
  • An M13mpl9 recombinant phage containing the gene for Trx-Fe S 4 was plated on a bacterial host strain deleted for wild-type thioredoxin (A307, (23, 24)). This strain will not support growth of wild-type M13mpl9 phage, but will support the growth of a M13mpl9 recombinant phage into which an active thioredoxin has been cloned.
  • Activity was scored by comparing phage titers on the ⁇ trxA (A307) strain and its parent trxA strain, K38, of phage stock that had been grown on the permissive strain DH5 ⁇ F': a recombinant phage titer within 1 order of magnitude on both strains (typically 10 to 10 particles/ml) was scored as displaying thioredoxin activity (inactive recombinants give titers of 10 to 10 particles/ml).
  • UV/vis electronic spectra were recorded under strict anaerobic conditions in gas tight optical cuvettes (1 cm pathlength) with a Perkin Elmer Lambda 6 spectrometer.
  • CD spectra were obtained with an AVIV Model 60DS Circular Dichroism Spectropolarimeter using a quartz cell with a 1 mm pathlength at 25 C at a protein concentration of approximately 25 ⁇ M.
  • EPR spectra were collected on a Varian E-line EPR spectrometer operating at a microwave frequency of 9.24 GHz, with 1.5 mW microwave power, 10 G modulation amplitude at 25 K.
  • DEZYMER the rational design algorithm, DEZYMER, is used to introduce a catalytically active iron SOD site into the hydrophobic core of E. coh thioredoxin (Trx), a protein normally devoid of transition metal centers Inspection of the designed site reveals several favorable properties Thioredoxin folds to form a hydrophobic core of ⁇ -pleated sheet flanked on either side by two ⁇ -helices, the structure can be considered as being formed of two domains, ⁇ from residues 1 to 59 and ⁇ from residues 76 to 108 (Katti, S K , LeMaster, D M & Eklund, H (1990) J. Biol Chem.
  • Trx E. coh thioredoxin
  • LMCT ligand-to-Fe charge-transfer
  • Electronic transitions arising from within the ⁇ i-orbital manifold (ligand-field, LF) are all spin- forbidden and are therefore much less intense (e M ⁇ 500). Transitions analogous to those
  • the native E. coli Fe SOD falls into the class of "perfectly evolved" enzymes that operate at the diffusion-controlled limit (Bannister, j. V., Bannister, W. H. & Rotilio, G. (1987) CRC Crit. Rev. Biochem. 22, 111-80). Studies have also shown that copper- manganese- and iron-containing complexes can also catalyze the dismutation of superoxide anion at this same diffusion-controlled limit (Weiss, R H , et al. (1993) J. Biol Chem. 268, 23049-23054, Goldstein, S , Czapski, G & Meyerstein, D (1990) J. Am. Chem. Soc.
  • Trx-SOD enzyme model lacks these conserved secondary active site residues, suggesting that alternative pathways for proton transfer may be operating at slower rates, thereby decreasing the overall rate of superoxide dismutation Unfavorable electrostatic interactions between the Trx-SOD protein surface and
  • the incoming anion may also play a role in diminishing the activity of ⁇ Fe ⁇ Trx-SOD as compared to E. coh FeSOD
  • Favorable electrostatic attraction of the negatively-charged superoxide ion is known to play a role in rapid rate of dismutation by native SODs (Bannister, J. V., supra; Getzoff, E. Dminister et al, (1992) Nature 358, 347-351).
  • a comparison of the surface electrostatic potential of E. coli FeSOD and Trx-SOD reveals that the native enzyme has a strong overall positive charge on the surface near the superoxide coordination position, but that the comparable region in Trx-SOD has a high concentration of negative charge.
  • mutation of surface residues to enhance electrostatic attraction of the negative superoxide ion is expected to boost the overall rate of reaction.
  • the designed enzyme is still highly active, with superoxide dismutation rates on the order of 10 M s .
  • Trx-SOD The activity of Trx-SOD is less than that of aqueous Cu ion and several synthetic model systems whose activities are comparable to the SOD enzyme itself (Valentine, J. S. (1994) in Dioxygen Reactions, eds. Bertini, I., Gray, H. B., Lippard, S. J. & Valentine, J. S. (University Science Books, Mill Valley), pp. 253-314).
  • soluble metal complexes may unpredictably show catalytic dismutase activity ranging from very high to zero, depending on details of the coordination sphere that are not completely defined (Weiss, R. H, et al (1993) J. Biol Chem. 268, 23049-23054; Allen, A. O.
  • Trx-SOD Trx-SOD
  • the functional small molecule SOD mimics will require the completion of both structural characterization and mechanistic/kinetic studies of Trx-SOD, it is reasonable to expect that restricted access to the designed active iron site and the unfavorable electrostatic attraction beween Trx-SOD and the negatively charged superoxide anion are responsible for the lower activity of Trx-SOD versus the small molecule models.
  • Bioinorganic chemistry has traditionally focused its efforts in two directions, the small molecule, synthetic analogue approach (Ibers, J. A. & Holm, R. H. (1980) Science 209, 223-235) and the study of intact metalloproteins.
  • Small molecules permit the investigation of the effects of varying the primary coordination sphere, but cannot provide the elaborate overall environment or long range interactions created by insertion into a protein shell.
  • study of intact metalloproteins does not frequently provide the opportunity to explore the varied contributions of the protein matrix that are often invoked in mechanistic discussions.
  • This protein design methodology allows the incorporation of both approaches into a single line of research; intact proteins can now be manipulated to vary properties of not only the immediate coordination environment but also the adjacent protein matrix while maintaining the overall structural or physiological properties of the chosen host protein.
  • the ⁇ Fe S 4 Cys ⁇ site definition was derived from the X-ray crystal structure of oxidized C. vinosum HiPIP (lHIP)(Carter, C W J , et al, (1972) Proc. Nat'lAcad. Sci.
  • Trx a relatively small (108 amino acid residues) monomeric protein that contains a single redox active disulfide bond (Cys32, Cys35) (Holmgren, A (1985) Annu. Rev. Biochem. 54, 237-271), was chosen (Hellinga, W H , Caradonna, J P & Richards, F M (1991) J. Mol.
  • Trx is a stable protein (oxidized Trx T m 86 °C, reduced Trx T m 75 C) (Holmgren, A , supra) that accommodates conservative as well as some non- conservative mutations (Wynn, R & Richards, F M (1993) Protein Sci. 2, 395-403)
  • Trx is a stable protein (oxidized Trx T m 86 °C, reduced Trx T m 75 C) (Holmgren, A , supra) that accommodates conservative as well as some non- conservative mutations (Wynn, R & Richards, F M (1993) Protein Sci. 2, 395-403)
  • a high-resolution (1 68 A) X-ray structure (Katti, S K , LeMaster, D M & Eklund, H (1990) J. Mol.
  • Trx fold Folding of Designed Protein. Since the designed substitutions are conservative in nature, maintenance of the protein fold is one of the design aims The presence of a near-native Trx fold can be tested in v vo using the absolute requirement of Trx for phage M13 growth (Russel, M & Model, P (1986) J. Biol Chem. 261, 14997-15005, Hellinga, W H , Caradonna, J P & Richards, F M (1991) J. Mol. Biol 222, 787-803, Russel, M & Model, P (1985) Proc. Nat 'lAcad. Sci. U.S.A.
  • Trx is not necessary as the double Cys32Ser, Cys35Ser mutant is capable of catalyzing phage assembly (Russel, M & Model, P (1986) J. B ol Chem. 261, 14997-15005) Mutagenesis studies suggest that the region important for protein- protein interactions in thioredoxin function is formed by residues Gly33, Pro34, Ile75, Pro76, Val91, Gly92 and Ala93 (Eklund, H , et al, (1984) EMBO J.
  • Trx-Fe S 4 supported a normal phage titer when expressed in a non- permissive ⁇ Trx E. coli strain, providing strong evidence that the designed protein adopts a native Trx conformation (Eklund, H , supra) Further evidence for the proper folding of Trx-Fe S 4 is observed in the CD spectrum (195-270 nm) of purified apo Trx-Fe 4 S 4 (inset, Figure 8), which is identical to that of native Trx In addition, both apo Trx-Fe 4 S and native Trx have identical gel filtration retention times (FPLC 16 mm x 60 cm Superdex 75pg, Pharmacia), indicating equivalent Stokes radii The combined phage assay results, CD spectrum, and gel filtration data strongly suggest that the overall aggregate structural elements of Trx are unaltered in the designed Trx-Fe S 4 protein.
  • Trx-Fe 4 S contains no Fe or elemental S. Reconstitution of apo T ⁇ x ⁇ -Pe S was accomplished by exploiting the cooperative and reversible chemical denaturation process of the designed host protein (the midpoint of reduced apo Trx-Fe S 4 unfolding is 2.7 M urea versus 4.6 M urea for reduced Trx) under strict anaerobic conditions in an Ar flushed glove box at 25 C. The fully reduced protein was partially unfolded with 2.0 M urea in the presence of 10 mM mercaptoethanol and high pH (8.5) buffer. A synthetic, preformed tetranuclear iron-sulfur cluster (Hill, C. L., et al, (1977) J. Amer. Chem. Soc.
  • Trx-Fe S 4 was then refolded by 1 : 1 dilution into the same high pH buffer, followed by removal of the remaining urea using gel filtration to exchange into 100 mM NaCI, 15 mM MOPS pH 7.4, 4 C. During this separation step, the cluster remained completely bound to the protein. Reconstituted Trx-Fe S 4 contains 3.9 ⁇ 0.2 moles of iron (atomic absorption spectroscopy) and 3.9 ⁇ 0.2 moles of acid labile sulfide (Beinert, H. (1983) Anal. Biochem.
  • Trx-Fe S 4 a monomer and retains the global structure of Trx and apo Trx-Fe 4 S 4 .
  • EXAMPLE 15 Spectroscopic Properties of holo Trx-Fe 4 S 4 .
  • Figure 8 also shows that the predicted perturbation to the electronic spectrum of the iron-sulfur cluster after its inclusion in the interior of the designed protein relative to the spectrum of the free synthetic cuboidal cluster is observed (Hill, C L , et al, (1977) J. Amer. Chem. Soc.
  • Trx-Fe S 4 is EPR silent, with only minor contributions (0 08 spins/protein) believed to arise from paramagnetic cluster contamination originating from ⁇ Fe 4 S 4 ⁇ cluster fragmentation This resting state property is a direct consequence
  • an EPR-active species is formed whose spectral features are analogous to the ⁇ Fe S 4 (Cys) 4 ⁇ clusters found in oxidized HiPIPs
  • the low intensity signals at g - 2 15, 2 10 and 1 97 not observed in the diamagnetic Trx-Fe 4 S 4 resting state, originate from the ferri cyanide oxidation of the iron- sulfur protein
  • the EPR properties of Trx-Fe S 4 are analogous to those reported for HiPIPs
  • HiPIP ⁇ Fe 4 S 4 (Cys) 4 ⁇ clusters are typically characterized by axial EPR
  • the EPR data in Figure 10 similarly rule out a dominant ⁇ Fe 3 S 4 ⁇ cluster, whose oxidized ground state should contain
  • the present inventors have used the rational design algorithm, DEZYMER, to introduce a cuboidal ⁇ Fe S 4 ⁇ cluster into the hydrophobic interior of E. coli thioredoxin (Trx), a protein normally devoid of transition metal centers. Inspection of the designed site reveals several favorable properties. Thioredoxin folds to form a core of ⁇ -pleated sheet flanked on either side by two ⁇ -helices; the structure can be considered as being formed of two domains, ⁇ from residues 1 to 59 (domain 1) and ⁇ from residues 76 to 108 (domain 2) (Katti, S. K., LeMaster, D. M. & Eklund, H. (1990) J Mol. Biol. 212, 167-184.
  • the DEZYMER solution ( Figure 7), which is located between the central ⁇ -sheet (Cys24, ⁇ -strand 2; Cys55, ⁇ -strand 3) and one ⁇ -helix (Cys42, ⁇ -helix 2; Cys99, ⁇ -helix 4) from each domain, is buried approximately 8-9 A from the surface. All peptide amide NH groups in the vicinity of the designed site participate in secondary structure interactions in this region and are unavailable for hydrogen bonding to the cluster in the model. Finally, the cluster site is buried in the interior of the Trx host and surrounded by a hydrophobic shell generated by Ile41, Ile45, Ala46, Leu53, Leu78, Leu94 and Leul03.
  • the DEZYMER algorithm is based on the premise that the mutations introduced are conservative and maintain the overall protein fold of the host scaffold (inverse folding constraint). This assumption was tested by comparing the in vivo biological properties of Trx-Fe 4 S and wild-type Trx.
  • the cluster binding site was introduced into a region of Trx that is located near the putative protein-protein interaction surface required for the growth of filamentous phages, thereby allowing a test of whether the mutations in Trx-Fe S 4 affect the original functions of Trx.
  • Trx-Fe S 4 exhibited full activity, thereby providing strong evidence that the designed protein adopts a wild-type conformation or at least maintains the essential structural features of the Trx surface that are necessary for this in vivo activity.
  • Trx-Fe 4 S 4 both the CD spectra (apo) and hydrodynamic (apo, holo) properties of Trx-Fe 4 S 4 are equivalent to the Trx host, offering both genetic and physical data suggesting that the overall structure of Trx-Fe S 4 is essentially indistinguishable from Trx.
  • the CD spectrum of holo Trx-Fe 4 S 4 is dominated by signals from the cluster is relatively uninformative regarding the protein fold. Complete structural determinations of apo and holo Trx-Fe 4 S 4 are ultimately required to assess the success of the design.
  • Fds is Cys-X 2 -Cys-X 2 -Cys with a more remote Cys residue supplying the fourth ligating sulf ⁇ r center.
  • the consensus sequence involves two Cys-X 2 -Cys-X -
  • a ferredoxin-like cluster was made from synthetic polypeptides derived from the consensus sequence of natural ferredoxins (Nakamura, A. & Ueyama, N. (1994) in Encyclopedia of Inorganic Chemistry ed. King,
  • the DEZYMER algorithm places no a priori constraint on the choice of the secondary structural environment in which the new place is incorporated, offering the opportunity to test this hypothesis.
  • the fact that the orientation of the amide dipoles in the model of Trx-Fe 4 S are fixed by the folded structure of Trx and not optimized for the presence of the incorporated cluster is consistent with the HiPIP-like redox properties of Trx-Fe 4 S (Stephens, P. J., supra).
  • the geometry of the designed cluster is such that neighboring hydrophobic side-chains can be mutated to introduce hydrogen bonding or charged residues in order to systematically probe the role of these determinants in the electronic properties and redox potential of the cluster.

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Abstract

La présente invention se rapporte à des métalloprotéines synthétiques renfermant au moins un site de liaison constitué d'un ion métallique et des ligands associés. Pour préparer ces métalloprotéines, on utilise un ensemble de coordonnées des structures protéiques de manière à déterminer une série de mutations de séquences primaires dans une protéine hôte présélectionnée, et on construit les métalloprotéines synthétiques par des méthodes de génétique de recombinaison, et d'expression et de purification protéiques. Des applications particulières sont représentées par les superoxydes dismutases et les monooxydases synthétiques. L'invention décrit également un procédé d'oxydation d'un alcane utilisant une métalloprotéine synthétique dans laquelle est installé un site actif de monooxygénase. Parmi les autres utilisations des métalloprotéines, on trouve une méthode permettant de traiter un sujet souffrant d'une lésion tissulaire liée à des perfusions répétées ou d'ischémie.
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CN116174044A (zh) * 2023-02-21 2023-05-30 集美大学 一种蛋白质构架的人工金属酶的新制备方法和用途

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US9625458B2 (en) 2002-10-16 2017-04-18 Duke University Biosensor
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CN116174044A (zh) * 2023-02-21 2023-05-30 集美大学 一种蛋白质构架的人工金属酶的新制备方法和用途

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