METHODS OF UNFOLDING PROTEINS USING METAL COMPLEXES

Abstract: The invention relates to methods of unfolding proteins using metal complexes, particularly cobalt-containing Schiff's base compounds. The proteins can be viral proteins or myoglobin, and the unfolding can be irreversible under physiological conditions. The unfolded proteins exhibit UV/vis absorption spectral changes (see the figure) and a 5 % or greater change in at least one minima or maxima of the circular dichroism spectra of the proteins.

Description: METHODS OF UNFOLDING PROTEINS USING METAL COMPLEXES FIELD OF THE INVENTION The invention relates to methods of unfolding proteins using metal complexes, particularly cobalt-containing Schiff s base compounds. BACKGROUND OF THE INVENTION Partially folded proteins are polypeptides with substantial secondary structure in a largely disordered tertiary structure (Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229; Roder, H. & Colon, W. (1997) Curr. Opin. Struct. Biol. 7, 15-28; P. L. Privalov (1996) J. Mol. Biol. 258, 707-725; Ptitsyn, O. B. (1996) Nat. Struct. Biol. 3, 488-490.). They are often referred to as molten globules ( Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229; Ptitsyn, O. B. (1996) Nat. Struct. Biol. 3, 488-490; Ewbank, j. j., Creighton, T. E.; Hayer-Hartl M. K. & Haiti F. U. (1995) Nat. Struct. Biol. 2, 10-11.). Owing to increased flexibility, hydrophobic amino acid residues may become exposed in a molten globule, leading to a higher membrane affinity (as compared to a fully folded protein), an attribute that could be of importance in biological processes (Ptitsyn, O. B. {1995) Adv. Prot. Chem. 47, 83-229; van der Goot, F. G., Lakey, J. H. & Pattus, F. (1992) Trends Cell Biol. 2, 343-348.). Aggregates formed in vivo from partially folded proteins contribute to a number of important human disease states, such as Alzheimer's disease, other amyloidoses, and the prion diseases (Jaenicke, R. & Seckler, R. (1997) Adv. Prot. Chem. 50, 1-59; Betts, S., Haase-Pettingell, C. & King, J. (1997) Adv. Prot. Chem. 50, 243-264; Fink, A. L. (1998) Folding & Design 3, R9-R23.). In addition, controlled protein unfolding is important for industrial protein manipulation (Mitraki et al., Bio-Technology 7:690 (19989). However, to date, partially folded proteins have been obtained from their more stable folded precursors either under equilibrium conditions in denaturing media, or as shortlived kinetic intermediates in rapid folding experiments (Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229). In addition, with the exception of protein aggregates (Jaenicke, R. & Seckler, R. (1997) Adv. Prot. Chem. 50, 1-59; Betts, S., Haase-Pettingell, C. & King, J. (1997) Adv. Prot. Chem. 50, 243-264; Fink, A. L. (1998) Folding & Design 3, R9-R23), partially unfolded proteins have not been isolated. Some proteins in their natural state may be partially folded. The Co(III)-myoglobin complex is the first example of an isolable partially folded species obtained from a naturally folded precursor. Suitable denaturing media are basically limited (urea, guanidinium-HCl, acids, salts, organic solvents and detergents), and a large excess of denaturant is needed, resulting in nonphysiological and therefore biologically questionable conditions. In addition, the denaturation is generally reversible, as the conditions are chosen to prevent irreversible aggregation of the unfolding proteins. Therefore, it is an object of the invention to provide methods for the unfolding of proteins. SUMMARY OF THE INVENTION In accordance with the objects outlined above, the present invention provides methods of unfolding a protein comprising contacting the protein with a reactive metal complex, wherein said protein exhibits a 5% or greater change in at least one minima or maxima of a circular dichroism (CD) spectra of the protein as a result of binding at least one metal complex to the protein. Preferred embodiments utilize cobalt-containing tetradentate Schiff s base compounds. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : UV/vis absorption spectral changes during the incubation of metMb and 1 in 0.01 M sodium phosphate, at pH 6.5 and 25 °C, with 15 min intervals between measurements. [metMb] 0 = 10 μM, [1] 0 = 0.18 mM. Figures 2A, 2B, 2C and 2D: (A) Soret absorbance as a function of the initial concentration ratios of metMb and 1. (B, C) CD at 222 nm after incubating metMb with (B) 1 or (C) 2. (D) Near-UV CD after incubating metMb with 11 equivalents of 1. All spectra were taken after 96 h at 22°C in 0.01 M sodium phosphate at pH 6.5. [metMb] 0 = 10 μM. Figures 3A and 3B: (A) CD at 222 nm after incubating apoMb (7.5 μM) with 1 and 2. (B) Far-UV CD after incubating metMb and apoMb with 11 equivalents of 1. Conditions as given in the Fig. 2 legend. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the discovery that the addition of metal complexes, particularly Schiff s base chelates of cobalt, to proteins, can result in their binding through the metal of functional moieties of certain accessible amino acids in a protein. This can result in the unfolding of the protein due to the complexing of the functional moiety to the metal complexes, resulting in replacement of some of the protein structure stabilizing interactions by bonding to the metal. Without being bound by theory, the metal complex compounds outlined herein derive their biological activity by the substitution or addition of ligands to the metal complexes. The biological activity of the complexes results from the binding of a new ligand, generally (but not always) in an axial position, most preferably a nitrogen of the side chain of histidine, although as outlined below, other amino acids may be involved. Presumably the amino acid serving as the new ligand of the metal complex is required by the target protein for its structural integrity. This can be due to the involvement of the histidine in structurally important hydrogen bonding, for example, or, as a result of the "bringing together" of two histidines as axial ligands in one metal complex, thus perturbing the structure of the protein. Accordingly, the addition of the metal complexes depicted herein are added to a protein or enzyme, for example, and one or more of the original ligands are replaced by one or more ligands from the protein. This will occur either when the affinity of the protein axial ligand is higher for the metal complex as compared to the original ligand, or when the new axial ligand is present in elevated concentrations such that the equilibrium of ligand binding favors the binding of the new ligand from the protein. This latter possibility may be encouraged by the use of a targeting moiety, as is more fully described below, which increases the presence of the metal complex at or near the surface of the target protein. In a preferred embodiment, the nitrogen atom of an imidazole side chain of the amino acid residue histidine, contained within a target protein, is the new axial ligand. While the examples and disclosure herein particularly describe this histidine embodiment, any "reactive amino acid" may serve as the new ligand. A "reactive amino acid" is one which is capable of binding to the metal compounds of the invention as a new ligand. Thus, while the nitrogen of the imidazole side chain of histidine is particularly preferred, alternative embodiments utilize the nitrogen atom of the aromatic indole side chain of tryptophan, the sulfur atoms of the side chains of cysteine and methionine, nitrogens of the side chains of arginine, lysine, asparagine or glutamine, the oxygen atoms of the side chains of aspartic and glutamic acids and of tyrosine, glutamine, asparagine, serine and threonine, and potentially the π bonds of the aromatic residues of phenylalanine, tyrosine and tryptophan, as well as the protein backbone carbonyl and amino groups, as the moieties which may become axial ligands as outlined above. The availability of these moieties may depend on the pH of the solution containing the protein or enzyme, since in the protonated state, many of these moieties are not good electron donors suitable as ligands. Thus, for example, non-physiological pHs may be used in protein production schemes. Under normal physiological conditions, this binding is effectively irreversible, although, as outlined below, the use of relatively high concentrations of certain reagents can remove the metal complexes from the proteins. Thus, the present invention provides methods of unfolding a target protein comprising contacting the protein with a reactive metal complex. By "unfolding" herein is meant that a protein loses at least a portion of its tertiary structure and/or secondary structure. "Unfolding" in this context can also refer to a conformational change of the protein leading to an alteration in the biological activity. Unfolding can be complete (i.e. an effective denaturation to a generally linear chain of amino acids), or partial. Partial unfolding includes, but is not limited to, a complete loss of tertiary structure with the retention of at least some secondary structure, a partial loss of tertiary structure with no loss of secondary structure, and a partial loss of tertiary structure and a partial loss of secondary structure. In addition, increases in secondary structure, or non-native structures, may be seen. The unfolding can be measured in a number of ways, as will be appreciated by those in the art. Preferred methods include, but are not limited to, changes in absorption spectra, changes in circular dichroism (CD) spectra, changes in fluorescence, changes in nuclear magnetic resonance (NMR) spectra, and changes in small angle X-ray scattering (SAXS) spectra. In a preferred embodiment, unfolding is measured as a change in circular dichroism (CD) spectra, and serves to give a measure of the relative quantities of the secondary structure components of a protein. As for absorption spectra, as is known in the art, native proteins have a characteristic CD spectra, with small changes unique to each particular protein. The shape of the spectra curve, as well as the maxima and minima, provide information about the protein. Thus, for example, peaks present in the 200 to 250 nm wavelength ("far UV") range are generally a "w" shaped spectra with troughs around 222 and 208 being indicative of the presence of α-helical structures, and a "v" shaped spectra with a trough around 217-220 nm being indicative of β-sheet structures. Scans in the "near UV" range, i.e. 250 - 300 nm, give information about tertiary structure. Other parts of the spectrum, as around 410 nm for heme proteins, may yield structural information as well. See description in Freifelder "Physical Biochemistry", 2nd Ed., Freeman, NY 1982, Ch. 16 pp 573-602 for UV CD; and Strickland CRC Critical Reviews in Biochemistry 1974 2:113- 175. For use in protein folding studies see Kelly et al., Biophys. Biochem. Acta 1997 133, 161-185. Thus, in the determination of unfolding by CD, the sample is prepared as is known in the art. For a far UV CD the protein solution is placed in a cuvette and the spectrum is taken. A typical concentration can be 10 μM, in a 1 mm cuvette or 1 μM in a 10 mm cuvette. The concentration needed depends on the light path length through the sample, the protein size and on the solvent properties. A far UV CD is taken similarly, with about a 10 fold higher concentration or pathlength. A spectrum of the buffer alone should be subtracted to obtain the final spectrum. A CD spectra is generally obtained prior to the addition of the metal complexes used in the invention, the complexes are added, and a second spectra is obtained. In general, unfolding is present when changes of at least about 5% of at least one maxima or minima of the spectra are observed, with changes of at least about 10% being preferred, and at least about 25-50 % being particularly preferred, and larger changes also possible. As above, changes in this context includes both increases and decreases. In addition, changes in more than one maxima or minima are preferred. Additionally, unfolding may be measured as a result in changes in the shape of a CD curve. Thus, for example, as outlined in Johnson, Proteins 7:205-214 (1990), the shape of a CD spectra can indicate the percentages of each type of secondary structure: -helix, antiparallel β-sheet, and β-turn. Changes in the shape of the curve, therefore, can show unfolding to a protein containing altered structures and random coils. In a preferred embodiment, unfolding may be measured as a result of changes in fluorescence. There are generally two different types of fluorescence-based assays to measure unfolding. In a preferred embodiment, proteins are specifically altered, generally covalently, with two different fluorochromes (i.e. a fluorescence emitting donor and absorber) to allow fluorescence resonance energy transfer (FRET) as is known in the art (see Freifelder, supra, Ch. 15, pp537-572). That is, the emission spectra of the first fluorochrome overlaps the excitation spectra of the second fluorochrome. Accordingly, if the two fluorochromes are in close enough proximity spatially to allow energy transfer, exciting the first fluorochrome results in a much attenuated fluorescence signal. This may be used to detect unfolding as generally the unfolding of the protein results in a distance increase between any two particular residues; thus, generally, unfolding results in less fluorescence quenching due to the greater distance of the two fluorochromes. Thus, unfolding may be monitored by following the fluorescent signal. As above for CD assays of unfolding, monitoring of unfolding is done by first measuring the fluorescence of the protein in the absence of the metal complex, and then the metal complex is added and the experiment is repeated. In this embodiment, generally a change of at least about 5% of the fluorescence signal is preferred, with at least about 10% being particularly preferred, and at least about 25-50% being especially preferred. As will be appreciated by those in the art, the deficiency of the fluorescence quenching is strongly dependent on the overlap of the emission and absorption spectra of the fluorochromes, and on the distances; thus, a much improved "picture" of unfolding is obtained if the donor and acceptor are moved around the protein and different effects are observed. In a preferred embodiment, the proteins are not modified with fluorescence labels as above, but rather external fluorescent dyes are used whose fluorescence depends on their environment. That is, the dye is substantially non-fluorescent in a certain environment (i.e. a polar environment), but upon a change in medium (i.e. association of the dye with hydrophobic areas of the protein), the dye exhibits a change in fluorescence intensity. Accordingly, upon the unfolding of a protein, hydrophobic areas of the protein are exposed, allowing an increase in fluorescence to be detected. Suitable dyes include, but are not limited to, 8-anilino-l-naphthalenesulphonate (ANS). See for example Englehard et al., Protein Sci. 4:1553 (1996), hereby incorporated by reference in its entirety. Similarly, the reactivity of cysteine side chains as a basis of solvent exposure and thus unfolding has been done; see Ballery et al., Biochemistry 34:1867 (1993), hereby incorporated by reference in its entirety. Alternatively, the accessibility of hydrophilic fluorescence quenchers such as iodide or acrylamide may be done; see Englehard et al., Fod Des 1 :31 (1996), hereby incorporated by reference in its entirety. In this embodiment, as above, monitoring of unfolding is done by first following the fluorescence of the dye with the protein in the absence of the metal complex, and then the metal complex is added and the experiment is repeated. In this embodiment, generally an alteration of at least about 5% of the fluorescence signal is preferred, with at least about 10% being particularly preferred, and at least about 25-50% being especially preferred. As will be appreciated by those in the art, fluorescence monitoring may not be possible with all metal complexes, as some serve as fluorescent quenchers. In a preferred embodiment, unfolding is measured as a change in UV absorption spectra. Some native proteins (for example, heme containing proteins) have a characteristic absorption spectra that contains at least one maxima (λ max ), generally characteristic of the protein. Thus, in a preferred embodiment, a change of the absorbance of the protein at its maximal absorbance λ max of at least about 5%, with at least about 10% being preferred, and at least about 25-50% being particularly preferred. Changes in this case include both increases and decreases in the absorption. In addition, as is known in the art, there are a number of other ways to determine protein unfolding, although quantification of these effects may be difficult. Thus, for example, changes in the NMR spectra of a protein as a result of the addition of the metal complexes of the invention can show unfolding. Similarly, changes in small angle X-ray scattering (SAXS), quasi-electric light scattering, and even X-ray crystallography can be used; for a review of these and other methods, see Plaxco et al., Current Opin. in Structural Biol. 6:630 (1996). In general, movements of atoms, preferably backbone atoms, of greater than 5 A from their position prior to the addition of the metal complex, is indicative of unfolding. By "reactive metal complex" herein is meant a metal complex that is capable of binding to a reactive amino acid, as defined above, and causing unfolding of the protein containing the reactive amino acid. The metal complexes of the invention comprise a metal ion and chelator, as are more fully described below. Without being bound by theory, it appears that the metal complexes useful in the invention have several important characteristics. In general, they are able to bind at least two amino acid side chains on the protein's surface; that is, at least two coordination atoms (generally provided by two ligands) of the metal complex are "substitutionally labile"; that is, these ligands generally have good leaving group properties, and can be replaced, for example by the nitrogen of the histidine side chain or by the sulfur atom of a cysteine side chain. In other types of complexes, such as square planar d 8 complexes, bidentate binding of the protein utilize the apical fifth position, although there is no non-solvent ligand present. Thus, preferred embodiments utilize two or three substitutionally labile positions, such that either two or three side chains may bind to the metal complex. However, it should be noted that in some instances, the metal complexes used in the invention may only bind one side chain, if hydrogen bonds of the side chain are structurally important. In addition to forming at least two coordination bonds with two separate side chains of the target protein, the metal complexes of the invention are preferably small, and in some cases may be relatively hydrophobic, enough to at least partially penetrate into the protein. Without being bound by theory, it appears that the metal complexes penetrate into the hydrophobic interior of a protein to disrupt at least one structurally important residue; once the first unfolding event has occurred, others can then follow, as additional residues become exposed to the solvent containing the metal complex. Thus, as outlined below, preferred chelators that utilize R substitution groups generally utilize small and/or hydrophobic groups. In addition, the chelators may exhibit regiospecific hydrophilicity/hydrophobicity; that is, R groups on one "side" of the chelate may be hydrophobic, and the other "side" may comprise one or more hydrophilic residues (for example, in Structure 1, R 7 , R 8 , RQ and R π are hydrophobic, and at least one of R„ R 2 , R 3 and R !0 is hydrophilic, although other combinations resulting in amphiphathic characteristics are also possible, as will be appreciated by those in the art). This may be particularly desirable since this regiospecific hydrophilicity/hydrophobicity can allow better positioning of the metal complex into or near the surface of a protein or enzyme, as is discussed below. Without being bound by theory, it appears that this regiospecific hydrophilicity/ hydrophobicity can allow the metal complex to more efficiently interact with the protein or enzyme, which generally displays both hydrophobic and hydrophilic regions. As a functional test for the ability of the metal complex to serve as a reactive metal complex, the complex may be added to a test protein, for example myoglobin, and the ability of the metal complex to cause protein unfolding may be measured. Accordingly, as will be appreciated by those in the art, there are a large number of metal ion-chelator pairs that are suitable for use in the methods of the invention. In general, the chelator has a number of coordination sites containing coordination atoms which bind the metal ion, to ensure that not all ligands will dissociate, and, in some cases, to labilize the axial ligands. The number of coordination sites, and thus the structure of the chelator, depends on the metal ion. The choice of the metal ion in turn depends on the identity of the reactive amino acid, with preferred metals being selected from the group consisting of cobalt (either Co(I), Co(II) or Co(III)), copper (including Cu(I) and Cu+2 or Cu(II)), nickel (including Ni+2 or Ni(II)), palladium (including Pd+2 or Pd(II)) and platinium (including Pt+2 or Pt(II)), with silver (Ag) and gold (Au), as well as Rh, Ir, Ru, Fe, Os, Cr, Mn, Zn, Mo, Ru, and Cd, being possible in some embodiments as described herein. Thus, for example, when histidines or other nitrogen containing side chains are to be targeted, cobalt is particularly preferred. When cysteine and methionine residues are to be targeted, suitable metal ions include gold, nickel, palladium, platinum and copper, as these metals have a strong propensity to bind sulfur preferentially to other elements such as oxygen, nitrogen and carbon. Consequently these complexes will preferentially bind to the sulfur atom of a cysteine or methionine residue. As will be appreciated by those in the art, there are a large number of suitable chelators that can be used in the methods of the invention. As outlined above, preferred metal complexes can bind at least two different side chains of the target protein, and thus, in general, when n is the number of coordination sites of the metal ion, the chelator provides n-2, n-3 or n-4 coordination atoms, and the remaining sites are filled by ligands that provide preferably a single coordination atom each, to allow for the substitutional lability of the ligands. There are several classes of metal complexes which find particular use in the invention, as are generally depicted in Structures 1 to 15 depicted below. As will be appreciated by those in the art, these structures are not meant to limit the class of suitable reactive metal complexes, and are intended as illustrative only. These complexes are made as is known in the art; see for example U.S. Patent Nos. U.S. Patent Nos. 4,866,054, 4,735,634, 5,324,879, 4,866,053, 5,210,096, 5,049,557, 5,106,841, and 5,142,076, and U.S.S.N.s 08/358,068, 08/570,761 and 08/571,364, all of which are expressly incorporated herein by reference. In a preferred embodiment, the chelator is a Schiff s base compound. By the term "Schiff s base" herein is meant a substituted imine. Schiff s bases are generally the condensation products of amines and aliphatic aldehydes forming azomethines substituted on the nitrogen atom. Schiff s base compounds can be di-, tri- and tetravalent, with a tetravalent Schiff s base being generally depicted in Structure 1, below. Particularly preferred in this embodiment are cobalt containing complexes, and particularly preferred compounds are outlined in U.S. Patent Nos. 4,866,054, 4,735,634, 5,324,879, 4,866,053, 5,210,096, 5,049,557, 5,106,841, and 5,142,076, and U.S.S.N.s 08/358,068, 08/570,761 and 08/571 ,364, all of which are expressly incorporated herein by reference. Structure 1 Structure 1 depicts the metal as cobalt, but other metals, particularly d 6 metals, can also be used in Structure 1. It should be noted that if Co(II) is used, the axial ligands may not be present, but upon oxidation of the Co(II) to Co(III), two axial ligands will be picked up. In addition, the two oxygen atoms may be replaced with either sulfur or selenium atoms as well, and the nitrogen by phosphorus. In Structure 1, L, and L 2 are axial ligands, also called neutral coordinating ligands herein, and each of the R groups is a substitution group. By "axial ligand" herein is meant a ligand L, or L 2 located at either the fifth or sixth coordination sites, generally depicted in Structure 1 above, in the equatorial plane defined by the chelate ad the metal. Generally, Co(II) compounds have up to four coordination atoms, although it is possible that other molecules (usually the solvent) may be weakly associated in one or both axial ligand positions. Similarly, Co(III) compounds have up to six coordination atoms, of which two are defined herein as axial ligand positions. The complex is synthesized or formulated with two particular axial ligands, and then when the complex is added to a protein, for example, the original axial ligand or ligands are replaced by one or more ligands from a protein. This will occur either when the affinity of the protein axial ligand is higher for the metal complex as compared to the original axial ligand, or when the new axial ligand is present in elevated concentrations such that the equilibrium of axial ligand binding favors the binding of the new axial ligand from the protein. Thus, Co(III) complexes are made with axial ligands that can be substituted with other ligands. Without being bound by theory, when the cobalt is Co(II), such complexes may, under certain circumstances, have a first axial ligand. The Co(II) compounds of the invention are preferably synthesized with no axial ligands. Upon incubation with a protein, certain moieties, such as the nitrogen atom of the imidazole of the side chain of histidine, within the protein can become an axial ligand, resulting in a tightly-bound protein-cobalt compound complex. This occurs when the Co(II) compound, with its four coordinating atoms from the Schiff s base, binds an imidazole moiety, for example, and is oxidized to a Co(III) compound. In one sense, this may be considered a redox reaction, since the Co(II) compound is oxidized to a Co(III) compound upon binding to the protein. Thus, the imidazole axial ligand serves as a fifth coordinating atom, and is tightly bound. The axial ligands or neutral coordination ligands are preferably water soluble groups having weak to intermediate ligand field strength (that is, they are substitutionally labile in that the axial ligands can be replaced by the reactive amino acid side chains, such as the nitrogen atom of the imidazole side chain of histidine). As is known in the art, ligands may be arranged in a spectrochemical series according to the magnitude of the field strength. Accordingly, suitable axial ligands include, but are not limited to, halides, amine groups, water, dimethyl sulfoxide, any bulky ligand, alcohols, alkoxides, thioethers, carbonyl bound compounds, hydrophylic olefines, etc. In addition, when two axial ligands are present, they may be the same or different. In Structure 1, each of the R groups is a substitution group. Suitable R substitution groups include a wide variety of groups, as will be understood by those in the art. Each R group may be independently selected from the group include hydrogen, halides, alkyl groups (including substituted alkyl groups and heteroalkyl groups), aryl groups (including substituted aryl and heteroaryl groups), organic acids, glycols, alcohols, amines, amides, esters, ethers, nitro groups, aldehydes, sulfur containing moieties, phosphorus containing moieties, cyano moieties, and targeting moieties. As will be appreciated by those skilled in the art, some positions (i.e. R 4 and R 5 in Structure 1) designated above may have two R groups attached (R' and R' '), although in a preferred embodiment only a single non-hydrogen R group is attached at these positions. In addition, in some embodiments, two adjacent R groups may be bonded together to form ring structures together with the carbon atoms of the chelator. By "alkyl group" or grammatical equivalents herein is meant a straight or branched chain alkyl group, with straight chain alkyl groups being preferred. If branched, it may be branched at one or more positions, and unless specified, at any position. The alkyl group may range from about 1 to about 30 carbon atoms (Cl -C30), with a preferred embodiment utilizing from about 1 to about 20 carbon atoms (Cl -C20), with about Cl through about C12 to about C15 being preferred, and Cl to C5 being particularly preferred, although in some embodiments the alkyl group may be much larger. Also included within the definition of an alkyl group are cycloalkyl groups such as C5 and C6 rings, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus. Alkyl also includes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and silicone being preferred. Alkyl includes substituted alkyl groups. By "substituted alkyl group" herein is meant an alkyl group further comprising one or more substitution moieties "R", as defined above. By "aryl" or "aromatic" groups or grammatical equivalents herein is meant an aromatic monocyclic or polycyclic hydrocarbon moiety generally containing 5 to 14 carbon atoms (although larger polycyclic rings structures may be made) and any carbocylic ketone or thioketone derivative thereof, wherein the carbon atom with the free valence is a member of an aromatic ring. Aromatic groups include arylene groups and aromatic groups with more than two atoms removed. For the purposes of this application aromatic includes heterocycle. "Heterocycle" or "heteroaryl" means an aromatic group wherein 1 to 5 of the indicated carbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen, sulfur, phosphorus, boron and silicon wherein the atom with the free valence is a member of an aromatic ring, and any heterocyclic ketone and thioketone derivative thereof. Thus, heterocycle includes thienyl, furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl, isoquinolyl, thiazolyl, imidozyl, etc. By "amino groups" or grammatical equivalents herein is meant -NH 2 , -NHR and -NR 2 groups, with R being as defined herein. By "nitro group" herein is meant an -NO 2 group. By "sulfur containing moieties" herein is meant compounds containing sulfur atoms, including but not limited to, thia-, thio- and sulfo- compounds, thiols (-SH and -SR), and sulfides (-RSR-). By "phosphorus containing moieties" herein is meant compounds containing phosphorus, including, but not limited to, phosphines and phosphates. By "silicon containing moieties" herein is meant compounds containing silicon. By "ether" herein is meant an -O-R group. Preferred ethers include alkoxy groups, with - O-(CH 2 ) 2 CH 3 and -O-(CH 2 ) 4 CH 3 being preferred. By "ester" herein is meant a -COOR group. By "halogen" or "halide" herein is meant bromine, iodine, chlorine, or fluorine. By "aldehyde" herein is meant -RCHO groups. By "alcohol" herein is meant -OH groups, and alkyl alcohols -ROH. The alkyl alcohol may be primary, secondary or tertiary, depending on the alkyl group. In a preferred embodiment, the alkyl alcohol is a straight chain primary alkyl alcohol, generally containing at least 3 carbon atoms. Preferred alkyl alcohols include, but are not limited to, n-propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-heptyl alcohol, or n-octyl alcohol. By "amido" herein is meant -RCONH- or RCONR- groups. By "ethylene glycol" or "(poly)ethylene glycol" herein is meant a -(O-CH 2 -CH 2 ) n - group, although each carbon atom of the ethylene group may also be singly or doubly substituted, i.e. -(O-CR 2 -CR 2 ) n -, with R as described above. Ethylene glycol derivatives with other heteroatoms in place of oxygen (i.e. -(N-CH 2 -CH 2 ) n - or -(S-CH 2 -CH 2 ) n -, or with substitution groups) are also preferred. By "organic acid" or grammatical equivalents herein is meant an alkyl group containing one or more carboxyl groups, -COOH, i.e. a carboxylic acid. As defined above, the alkyl group may be substituted or unsubstituted. Cl - C20 alkyl groups may be used with at least one carboxyl group attached to any one of the alkyl carbons, with Cl - C5 being preferred. In a preferred embodiment, the carboxyl group is attached to the terminal carbon of the alkyl group. Other preferred organic acids include phosphonates and sulfonates. A preferred organic acid is propionic acid. By "alcohol" herein is meant an -OH group. By "alkyl alcohol" herein is meant an alkyl group containing one or more alcohol groups, similar to the alkyl acids. As defined above, the alkyl group may be substituted or unsubstituted. The alkyl alcohol may be primary, secondary or tertiary, depending on the alkyl group. In a preferred embodiment, the alkyl alcohol is a straight chain primary alkyl alcohol, generally containing at least 2 carbon atoms. Preferred alkyl alcohols include, but are not limited to, ethanol, n-propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-heptyl alcohol, or n-octyl alcohol. As for the alkyl acids, preferred alkyl alcohols have an alcohol group attached to the terminal carbon of the alkyl group. By "alkyl thiol" herein is meant an alkyl group containing a thiol (-SH) group at any position, with terminal positions preferred as for acids and alcohols. By "carbonyl oxygen" herein is meant an oxygen double bonded to a carbon atom. By "phosphonyl oxygen" herein is meant an oxygen double bonded to a phosphorus atom. By the term "targeting moiety" herein is meant a functional group that will specifically interact with the target protein, and thus is used to target the metal complex to a particular target protein. That is, the metal complex is covalently linked to a targeting moiety that will bind or associate, preferably specifically, with a target protein. For example, the metal complexes used in the invention may include a polypeptide inhibitor that is known to inhibit a protease, thus effectively increasing the local concentration of the metal complex around the target protein. Suitable targeting moieties include, but are not limited to, polypeptides, nucleic acids, carbohydrates, lipids, hormones including proteinaceous and steroid hormones, growth factors, receptor ligands, antigens and antibodies, and the like. By the term "polypeptide" herein is meant a compound ranging from about 2 to about 15 amino acid residues covalently linked by peptide bonds. Preferred embodiments utilize polypeptides from about 2 to about 8 amino acids, with about 4 to about 6 being the most preferred. Preferably, the amino acids are naturally occurring amino acids in the L- configuration, although amino acid analogs are also useful, as outlined below. Under certain circumstances, the polypeptide may be only a single amino acid residue. Additionally, in some embodiments, the polypeptide may be larger, and may even be a protein, although this is not preferred. In one embodiment, the polypeptide is glycosylated. Also included within the definition of polypeptide are peptidomimetic structures or amino acid analogs. Thus, for example, non-naturally occurring side chains or linkages may be used, for example to prevent or retard in vivo degradations. Alternatively, the amino acid side chains may be in the (R) or D-configuration. Additionally, the amino acids, normally linked via a peptide bond or linkage, i.e. a peptidic carbamoyl group, i.e. -CONH-, may be linked via peptidomimetic bonds. These peptidomimetic bonds include CH 2 -NH-, CO- CH 2 , azapeptide and retroinversion bonds. By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31 :1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al, J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate addition to the metal complex, or to increase the stability and half-life of such molecules in physiological environments. As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids and analogs, and mixtures of different nucleic acid analogs may be made. The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. As used herein, the term "nucleoside" includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside. By "carbohydrate" herein is meant a compound with the general formula C x (H 2 O) y . Monosaccharides, disaccharides, and oligo- or polysaccharides are all included within the definition and comprise polymers of various sugar molecules linked via glycosidic linkages. Particularly preferred carbohydrates are those that comprise all or part of the carbohydrate component of glycosylated proteins, including monomers and oligomers of galactose, mannose, fucose, galactosamine, (particularly N-acetylglucosamine), glucosamine, glucose and sialic acid, and in particular the glycosylation component that allows binding to certain receptors such as cell surface receptors. Other carbohydrates comprise monomers and polymers of glucose, ribose, lactose, raffinose, fructose, and other biologically significant carbohydrates. "Lipid" as used herein includes fats, fatty oils, waxes, phospholipids, glycolipids, terpenes, fatty acids, and glycerides, particularly the triglycerides. Also included within the definition of lipids are the eicosanoids, steroids and sterols, some of which are also hormones, such as prostaglandins, opiates, and cholesterol. Hormones include both steroid hormones and proteinaceous hormones, including, but not limited to, epinephrine, thyroxine, oxytocin, insulin, thyroid-stimulating hormone, calcitonin, chorionic gonadotropin, cortictropin, follicle-stimulating hormone, glucagon, leuteinizing hormone, lipotropin, melanocyte-stimulating hormone, norepinephrine, parathryroid hormone, vasopressin, enkephalins, seratonin, estradiol, progesterone, testosterone, cortisone, and glucocorticoids. Receptor ligands include ligands that bind to receptors such as cell surface receptors, which include hormones, lipids, proteins, glycoproteins, signal transducers, growth factors, cytokines, and others. As noted above, preferred chelators are relatively small and hydrophobic. Accordingly, preferred R substitution groups include hydrogen, small alkyl groups, halides, OH, OH, NHR, CN, COOH, and COO-Na+. When hydrophilic or larger R groups are used, preferred embodiments utilize chelators that have only 1 to 3 of these groups, with 1 being preferred. In addition to Schiff s base chelators, other chelators find use in the present invention, as are shown below: Structure 2 In this embodiment, M is a transition metal ion, A is either nitrogen, phosphorus, sulfur or oxygen, E is oxygen, sulfur, nitrogen, phosphorus or selenium and D is carbon, boron (B) or phosphorus (P). X is either a counter-ion or a neutral coordinating ligand. R, is a substitution group as outlined herein, or may be absent when A is oxygen. R 2 is a substitution group as outlined herein, carbonyl oxygen, phosphonyl oxygen, or -OR when A is boron. R 3 is a substitution group as outlined herein, or -OR when A is boron or phosphorus, or is absent when R 2 is carbonyl oxygen. The other R groups are substitution groups as outlined herein. The choice of A, E, X and M will depend on a variety of factors. Since, in a preferred embodiment, the metal complexes of the invention are neutral, i.e. uncharged, the collective charge of the A, E, X and M moieties preferably equal zero. Thus, as is depicted herein, the choice of A and E will determine whether X is a counter-ion or a ligand. Thus, when A and E are such that they both carry a negative charge (for example when A is oxygen and R, is absent, and E is sulfur, oxygen, or selenium, with R 8 being absent) then X is a neutral ligand. Alternatively, when one or the other of A and E is negatively charged, and the other is neutral, X is a counter-ion. As will be appreciated by those in the art, either A or E should carry a negative charge. Thus, preferred embodiments utilize both A and E with negative charges; A as nitrogen (with R, present) and E as oxygen, sulfur or selenium, with R 8 being absent; or A as oxygen (R, absent) and E as oxygen or nitrogen with R 8 present. Suitable counter-ions include, but are not limited to, halides; -OR; -SR; SO 4 2-, PF 6 -, BF 4 -, Bar4, RCCO, citrate, and -NHR, where R is a substituent group as herein defined, preferably alkyl and aryl. It should be noted that the choice of the counter-ion can influence conformational changes, so chaotropic and kosmotropic anions are included. By "neutral coordinating ligand" herein is meant a neutral molecule capable of donating electrons to a metal to form a metal-ligand complex without a formal change in oxidation state. Suitable neutral coordinating ligands include, but are not limited to, water (H 2 O), dioxane, THF, ether (ROR), thioether (RSR), amine (NR 3 ) and phosphine (PR 3 ), with R being any number of groups but preferably an alkyl group. Preferred embodiments of Structure 2 are shown below in Structures 3 to 7: Structure 3 In Structure 3, E is oxygen, sulfur or selenium, R 3 is hydrogen; and X is a counter-ion. Structure 4 In Structure 4, E is oxygen, sulfur, or selenium and X is a neutral coordinating ligand. Structure 5 In Structure 5, E is oxygen, sulfur, or selenium, and X is a neutral coordinating ligand. Structure 6 In Structure 6, E is oxygen, sulfur, or selenium and X is a neutral coordinating ligand. Structure 7 In Structure 7, E is nitrogen, oxygen or sulfur, and X is a counter-ion. In a preferred embodiment, the metal complexes of the invention have the formula depicted below in Structure 8: Structure 8 In Structure 8, M is a transition metal ion selected from the group consisting of Co, Cu, Ag, Au, Ni, Pd and Pt, and E is oxygen, sulfur, or selenium, with oxygen being preferred. Rq, R 10 , R n , R 12 , R )3 , R )4 , R 15 and R ]6 are each independently a substitution group as defined herein, although R,, and R ]2 together may form a cycloalkyl or aryl group. Similarly, R 15 and R 16 together may form a cycloalkyl or aryl group. In a preferred embodiment, the metal complexes of the invention have the formula depicted below in Structure 9: Structure 9 In Structure 9, M is a transition metal ion with an oxidation state of +1, preferably Cu(+1), Au(+1), or Ag(+1). X is a counter-ion. R 25 , R 26 , R 27 , R 2g , R 29 , R 30 , R 3] , R 32 and R 33 are independently substitution groups, that may, with an adjacent R group forms a cycloalkyl or aryl group. In a preferred embodiment, the metal complexes of the invention have the formula depicted below in Structure 10: Structure 10 In Structure 10, M is a transition metal ion selected from the group consisting of Cu, Ag, Au, Ni, Pd and Pt, with Au+2 being preferred. X is a counter-ion. R 35 , R 36 and R 37 are independently hydrogen, halogen, alkyl, alkyl alcohol, alcohol, alkyl thiol, alkyl acid, alkyl amine, amine, aryl, a targeting moiety, or, together with an adjacent R group forms a cycloalkyl (preferably heterocycloalkyl, with the heteroatom being nitrogen, oxygen, or sulfur) substituted cycloalkyl, aryl, or substituted aryl groups. In a preferred embodiment, at least one R 35 , R 36 , R 37 or the R substituents of the cycloalkyl or aryl group is a targeting moiety, with polypeptides and nucleic acids being preferred. Thus, preferred embodiments include the structures depicted below: Structure 11 In Structure 11 , the R group on the nitrogen atom may be an R group as defined herein or it may be hydrogen. In a preferred embodiment, the metal complexes of the invention have the formula depicted below in Structure 12: Structure 12 In Structure 12, M is a transition metal ion selected from the group consisting of Cu, Ag, Au, Ni, Pd and Pt, with Cu, Ni, Pd and Pt being preferred. X is a counter-ion. R 38 , R 39 , R 40 , R 41 , R 42 and R 43 are independently hydrogen, halogen, alkyl, alkyl alcohol, alcohol, alkyl thiol, alkyl acid, alkyl amine, amine, aryl, or a targeting moiety. In a preferred embodiment, at least one of R 38 to R 43 is a targeting moiety. In a preferred embodiment, the metal complexes of the invention have the formula depicted below in Structure 13: Structure 13 In Structure 13, M is a transition metal ion selected from the group consisting of Cu, Ag, Au, Ni, Pd and Pt, with Cu, Ni, Pd and Pt being preferred. E is oxygen, sulfur or selenium, with oxygen being preferred. Each X is independently a counter-ion. R 44 is hydrogen, halogen, alkyl, alkyl alcohol, alcohol, alkyl thiol, alkyl acid, alkyl amine, amine, aryl, or a targeting moiety. In a preferred embodiment, at least one of R 3 to R 43 is a targeting moiety. In this case, A, B, C and D are independently single or double bonds, with the latter being preferred. The R groups are independently substitution groups, and as above, two adjacent R groups may together form a cycloalkyl or aryl ring. The metal complexes are contacted with the target protein. By "target protein" herein is meant a protein that is to be unfolded using the methods of the invention. Without being bound by theory, it appears that target proteins that may be unfolded using the methods of the invention. The target protein should have reactive amino acids on the surface. As will be appreciated by those in the art, the identity of the target protein will depend on the utility of the present methods. In a preferred embodiment, the methods are used to unfold proteins to alter their biological activity, generally by inhibiting or decreasing their biological activity. In this embodiment, suitable target proteins include, but are not limited to, enzymes (including hydrolases such as proteases (including, but not limited to, serine (including, but not limited to, plasminogen activators and other therapeutically relevant mammalian serine proteases as well as bacterial serine proteases such as subtilisins) aspartyl, metal, acid and cysteine proteases (including, but not limited to cathepsins (including cathepsins B, H, J, L, N, S, K, O, T and C, (cathepsin C is also known as dipeptidyl peptidase I), interleukin converting enzyme (ICE), calcium-activated neutral proteases, calpain I and II); carbohydrases, lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases and phophatases, etc.); immunoglobulins, particularly IgEs, IgGs and IgMs; viral proteins, (including enzymes and coat proteins from: orthomyxoviruses, (e.g. influenza virus), paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variola virus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus), hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus), rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and -II), papovaviruses (e.g. papillomavirus), polyomaviruses, and picornaviruses, and the like); bacteria proteins (including proteins from a wide variety of pathogenic and non-pathogenic prokaryotes of interest including Bacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C.perfringens; Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G. lambliaY. pesiis, Pseudomonas, e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis; Treponema, e.g. T. palladium; and the like); proteinaceous hormones and cytokines (many of which serve as ligands for cellular receptors) such as erythropoietin (EPO), thrombopoietin (TPO), the interleukins (including IL-1 through IL- 17), insulin, insulin-like growth factors (including IGF-1 and -2), epidermal growth factor (EGF), transforming growth factors (including TGF-α and TGF-β), human growth hormone, transferrin, epidermal growth factor (EGF), low density lipoprotein, high density lipoprotein, leptin, VEGF, PDGF, ciliary neurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH), calcitonin, human chorionic gonadotropin, cotrisol, estradiol, follicle stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and leutinzing hormone (LH). In an alternative embodiment, the methods are used to unfold the proteins in order to allow the protein to traverse a membrane more easily, as is generally described in van der Goot et al, Trends in Cell Biology 2, p 343 (1992). The metal complexes are contacted with the target protein under conditions that allow the binding of the metal complex to the protein, and the mixture is allowed to incubate for some period of time. By "contacted" or "added" herein is meant that the solutions containing the two are mixed, with homogeneous solutions being preferred. The salt concentration, buffer composition and concentration, heat, pressure and pH can all be varied. Low pH (i.e. 5.5) generally facilitates the reaction. As shown in the Example, the number of metal complexes bound to any particular protein depends on both the number of reactive amino acids and the concentration of the added metal complex; thus, for example, using less than stochiometric ratios of the metal complex can allow a partial unfolding in some cases. In a preferred embodiment, the metal complexes are added to the protein in the absence of any significant amounts of traditional denaturants, such as high salt concentrations, guanidinium-HCl, detergents, etc. That is, the proteins do not significantly unfold unless the metal complex is present. By "binding" herein is meant the formation of a coordination bond. That is, the metal complex gives up at least one, and preferably two, of its substitutionally labile ligands in favor of binding one or more amino acid side chains from the target protein. The metal complexes bind to the proteins, generally to solvent accessible reactive amino acids. As outlined above, amino acid side chains that are buried within the interior of a folded protein may become exposed as the protein unfolds. As a result of the binding, the protein unfolds, which is measured as outlined above. This may be accompanied by a loss of biological activity, which, as those in the art will appreciate, will be determined as dictated by the identity of the target protein. The following examples serve to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are incorporated by reference in their entirety. EXAMPLES Isolation of a Myoglobin Molten Globule by Selective Co(III)-induced Unfolding The reaction between a cobalt(III) Schiff base complex, [Co(acetylacetonate ethylenediimine)(NH 3 ) 2 ] (1) (Bδttcher, A., Takeuchi, T., Hardcastle, K. I., Meade, T. J., Gray, H. B., Cwikel, D., Kapon, M. & Dori, Z. (1997) Inorg. Chem. 36, 2498-2504; Costa, G., Mestroni, G., Tauzher, G. & Stefani, L. (1966) J Organomet. Chem. 6, 181-187;, and metmyoglobin (metMb) yields a partially folded protein isolated in a biologically relevant medium; while some proteins in their natural state may be partially folded, this Co(III)- myoglobin complex is the first example of an isolatable partially folded species obtained from a naturally folded precursor. Uniquely, some other proteins are not affected by 1 under the same conditions, demonstrating that this novel unfolding method can be selective. Mechanistic studies indicate that the irreversibility and selectivity of unfolding by 1 originate in the strong bond formed preferentially between cobalt and an imidazole nitrogen of a histidine. Our findings open the way for applications based on the unique properties of molten globules as toxin-like prodrugs. Materials: [Co(acacen)L 2 ]Cl (acacen = acetylacetonate ethylenediimine; L = NH 3 , 1; L = imidazole, 2; L = 4-Me-imidazole, 3; L = JV-Me-imidazole, 4; L = 2-Me-imidazole, 5; L = MeNH 2 , 6) complexes were prepared by literature methods (Bottcher, A., Takeuchi, T., Hardcastle, K. I., Meade, T. j., Gray, H. B., Cwikel, D., Kapon, M. & Dori, Z. (1997) Inorg. Chem. 36, 2498-2504. Horse skeletal muscle metMb, apoMb and bovine serum albumin (Sigma) were of the highest purity available, and were used as received. Spectroscopy. UV/vis absorption spectra were acquired on a Hewlett Packard HP 8452 diode array spectrophotometer. CD spectra were measured on an Aviv 62DS spectropolarimeter at 25 °C (1 mm cell for far-UV CD; 10 mm cell for near-UV CD). Measurements were made on the incubation mixtures, without additional treatment, to prevent dissociation of weakly bound Co(III) complexes. Note, however, that removal of free 1 by dialysis did not change the spectrum of the incubation product of metMb with 11 equivalents of 1. The 200 MHz 'H NMR spectrum (hemin region) of metMb (1 mM) with excess 2 ([2] 0 = 12 mM) in 0.05 sodium phosphate buffer (pH 7.0) at 25 °C was measured on a Bruker AM200. Unfolding Experiments. Different amounts from a stock solution of the relevant cobalt complex in 0.01 M sodium phosphate buffer solution at pH 6.5 were added to a metMb or apoMb solution in the same medium. Volume corrections with pure buffer solution (so that a final reaction volume of 20 ml would be reached) were done before addition of the cobalt solution. The final protein concentration was 1 • 10 "5 M (except for determining the concentration dependence of the 222 nm CD signal on the protein concentration). Incubation was for 96 h at 22 °C. Analysis of the products was after dialysis of the free cobalt, unless otherwise mentioned. Dialysis at 4°C was either from bags (3000 D cutoff) for 12 h against pure buffer solutions, or from Centricon concentrators (3000 D, Amicon). Determination of the Cobalt to Protein Binding Ratio. Final protein concentrations were determined by the Folin-Ciocalteu method (Layne, E. (1975) Methods Enzymol. 3, 447-454). [1] after dialysis was determined by graphite furnace atomic absorption of cobalt (duplicate 20 μl samples were examined, using argon as the inert gas; the absorbance was measured at 240.7 nm (0.2 nm slit, 1 s) on a Varian SpectrAA-20, equipped with PSC-56 and GTA-96). Binding ratios are rounded to the nearest whole number. Attempts to Reverse the Unfolding. These experiments were on 10 "5 M solutions of the unfolded protein from which the free unfolding reagent was removed by dialysis. The reaction with excess dithionite was run for 3 h under argon before the UV/vis spectrum of the solution was recorded. The product was exposed to air, and the spectrum was remeasured. The reactions with excess imidazole (25, 200, and 10 5 ) were followed periodically by UV/vis absorption. The reaction with 25 fold excess hemin (solubilized in a minimum volume of a 0.01 M NaOH solution) was allowed to proceed at room temperature for 24 h. The solution was then transferred through a Sephadex G-25 column (1.5»20 cm) to separate the free hemin, and the absorption spectrum was acquired. Ligand Exchange. Reactions of complexes 4 and 5 with free imidazole occur in water. UV/vis absorptions of these reaction mixtures were monitored at 3 or 4 different wavelengths (per experiment) in which the absorbance difference between the starting compound and the expected product (prepared separately) was maximized. The Co(III) complex concentration was 10 μM, and the free ligand was at 25 and 200 fold excess. NMR spectral analysis of the initial and final solution established the identity of the products. Results: Incubation of metMb with 1 leads to broadening, a strong decrease in the molar extinction coefficient and a blue shift of the Soret band in the absorption spectrum from 409 (e ~ 1.7- 10 5 ) to 392 nm (e ~ 5.9» 10 4 M "1 cm 1 ) (Fig. 1). The intraligand π-π* band of 1 shifts from 334 to 340 nm, with no apparent change in extinction coefficient (e 334 = 7100 M" 1 cm" 1 ). A minimum Co(III) to protein ratio of 11 to 1 is required for the full spectroscopic transformation (Fig. 2a). The far UV circular dichroism (CD) shows a 50% decrease in the α-helical secondary structure of metMb (Fig. 2b) (14) that parallels the decrease in the Soret band (Fig. 2a). The loss of the near-UV CD (Fig. 2d) indicates diminished packing around the aromatic amino acids. At an initial 1 :metMb ratio of 11 : 1 , virtually no near-UV CD signal is observed. To verify that the CD signal intensity loss is not due to protein aggregation in experiments conducted with 10 μM metMb or less, the dependence of the CD signal at 222 nm on the metMb concentration was determined (keeping [metMb] :[1] = 10 and other conditions as reported in Fig. 2). A linear correlation was obtained between 1 and 18 μM metMb (data not shown). The reaction is irreversible in the sense that extensive dialysis leaves an average of 6 cobalt complexes associated with the protein, and that the product retains 1 for prolonged periods in solution even in the absence of free 1. External reagents reverse the reaction. Excess dithionite reduces the hemin to ferroheme, and causes cobalt dissociation from the protein (probably by reducing Co(III) to labile Co(II)). Exposure to air regenerates metMb. A large excess of imidazole leads to slow, incomplete recovery of the Soret band. Excess hemin does not affect the product UV/vis spectrum. ApoMb reacts with 1 much faster than metMb, and a smaller 1 to apoMb ratio completes the transformation (about 6:1 L.apoMb (Fig. 3a)). The product is identical with the one obtained from metMb and excess 1 (overlapping far-UV CD (Fig. 3b), with 6 Co(III) complexes binding to the protein). The observation that these products are the same indicates that 1 causes hemin dissociation from the active site of metMb. The dissociated hemin is not separated from the cobalt-myoglobin product by dialysis, as confirmed by iron atomic absoφtion measurements. It probably binds nonspecifically to the unfolded protein, as observed by Hargrove, M. S. & Olson, J. S. (1996) Biochemistry 35, 11310- 11318.^. The similarity of the electronic spectrum of metMb after reaction with 1 with that of free hemin in a polar medium (Cann, J. R. (1964) Biochemistry 3, 714-722) also supports this conclusion. The far-UV CD and UV/vis spectral changes (Figs. 2a,b) are similar to those obtained upon treatment of metMb with acid or low concentration guanidine»HCl (Puett, D. (1973) J. Biol. Chem. 248, 4623-4634; Bismuto, E., Colona, G. & Irace, G. (1983) Biochemistry 22, 4165-4170; Palaniappan, V. & Bocian, D. F. (1994) Biochemistry 33, 14264-14274), where it is well established that there is disruption of the tertiary structure in the B-F folding domain (Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544-1548; Jennings, P. A. & Wright, P. E. (1993) Science 262 892-896; Eliezer, D., Yao, J., Dyson, H. J. & Wright, P. E. (1998) Nat. Struct. Biol. 5, 148-155.;. As the active site is contained within this domain, evidence for unfolding by 1 in this region is our observation of hemin dissociation, and in the correlation between that dissociation (monitored by the Soret absoφtion) and the changes in the protein secondary structure (as indicated by [Θ] 222 ) (Figs. 2a, b). As the near-UV CD bands at λ > 268 nm are due to electronic transitions in tryptophans and tyrosines (Strickland, E. H. (1974) CRC Crit. Rev. Biochem. 2, 113-175; Sirangelo, I., Bismuto, E., Tavassi, S. & Irace, G. (1998) Eur. Biophys. J. 27, 27-31.;, they probe the tertiary structure of horse myoglobin only in the AGH domain, where such residues are found (Evans, S. V. & Brayer, G. D. (1988) J. Biol. Chem. 263, 4263-4268). The disappearance of the near-UV CD features suggests that this domain loses its tertiary fold upon reaction with 1. In contrast, near-UV CD (Gast, K., Damaschun, H., Misselwitz, R., Mϋller-Frohne, M.; Zirwer, D. & Damaschun, G. (1994) Eur. Biophys. J. 23, 297-305; Irace, G., Bismuto, E., Savy, F. & Colona, G. (1986) Arch. Biochem. Biophys. 244, 459- 469; Fink, A. L., Oberg, K. A. & Seshadri, S. (1998) Folding & Design 3, 19-25; and other studies (Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544- 1548; Jennings, P. A. & Wright, P. E. (1993) Science 262 892-896; Eliezer, D., Yao, J., Dyson, H. J. & Wright, P. E. (1998) Nat. Struct. Biol. 5, 148-155; indicate that the AGH domain remains largely intact upon myoglobin unfolding by acid or low concentration guanidine » HCl. The partially unfolded Co(III)-myoglobin can be described as a molten globule ( Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229; Ptitsyn, O. B. (1996) N t. Struct. Biol. 3, 488- 490; Ewbank, J. J., Creighton, T. E.; Hayer-Hartl M. K. & Hartl F. U. (1995) Nat. Struct. Biol. 2, 10-11.), since it retains much secondary structure but very little tertiary structure (Fig. 2d). As all free denaturant can be removed, this is in every sense an isolated, kinetically stable, partially folded protein. [Co(acacen)(imidazole) 2 ] + (2) reacts differently than 1 with metMb. Only one molecule of 2 binds each protein, causing no change in the Soret absoφtion, and only a small change in the far-UV CD (Fig. 2c). The 4-methyl- and N-methyl-imidazole derivatives of 1 (complexes 3 and 4) behave as 2 toward metMb (only 1 cobalt binds), while the 2-methyl- imidazole and methylamine derivatives (complexes 5 and 6) react like 1 (6 cobalts bind to the protein). Nevertheless, 2 unfolds apoMb. The CD unfolding profile at 222 nm is similar to that of 1 (Fig. 3a), although only 3 cobalt complexes are bound to the isolated product. Some of the imidazole liberated by the reaction of metMb with 2 binds at the active site, generating low-spin imidazole-bound hemin in equilibrium with high-spin water-bound hemin. This is evident from the paramagnetic 'H-NMR spectrum of metMb with excess 2, which is a supeφosition of the spectra of native metMb and imidazole-bound metMb (data not shown). Removal of the imidazole by dialysis regenerates a pure high-spin species. The position and intensity of the band at 340 nm confirm that the acacen ligand remains bound to cobalt. The π-π* transition of the uncomplexed ligand is at 323 nm (12). When this product is reacted with excess 2, no low-spin product is observed by 'H NMR, as the cobalt binding site remains occupied after removal of unreacted 2. This evidence strongly suggests that the reaction of metMb with 2 involves cleavage of the cobalt-imidazole bond and replacement of this ligand by a protein side chain with ligating capabilities. The small changes in wavelength (6 nm shift) and extinction coefficient of the π-π* absoφtion band of 1 (Fig. 1) indicate that it binds to a nitrogen donor of the protein. While these values are similar for ammonia, alkylamines, pyridine, and substituted imidazoles as axial ligands (Ηottcher, A., Takeuchi, T., Hardcastle, K. I., Meade, T. J., Gray, H. B., Cwikel, D., Kapon, M. & Dori, Z. (1997) Inorg. Chem. 36, 2498-2504; Costa, G., Mestroni, G., Tauzher, G. & Stefani, L. (1966) J. Organomet. Chem. 6, 181-187), they do change upon replacement of a nitrogen donor by an oxygen donor (Sima, J. (1994) Polish J. Chem. 68, 1689-1697). Moreover, attempts to prepare [Co(acacen)(amino-acid) 2 ] + complexes in which amino acids bind η 1 through a carboxylate oxygen have failed, resulting in binding only to the amino group (Fujii, Y. (1972) Bull. Chem. Soc. Jpn. 45, 3084-3092). Ligand exchange experiments demonstrate that 1 and 2 prefer histidines over lysines. Indeed, two imidazole equivalents replace both ammines of 1 in water at 25 °C, while no reaction is observed between 2 and two equivalents of w-butylamine. Thus we conclude that the protein ligand in the 1-Mb derivative is a histidine imidazole. The kinetic lability of the Co(III) axial ligand is important in determining the reaction product with metMb. This is apparent upon comparisons among 2, 3, 4, and 5. Ligand exchange of 5 with excess imidazole is much faster than the analogous reaction with 4. This is attributed to steric repulsion between the methyl group at the imidazole 2-position and the cis disposed Schiff base. As 2, 3, and 4 bind only to one (or two, if bridging, see below) of the exposed histidines of metMb. As many as 6 surface histidines on metMb can be modified: Konopka, K. & Waskell, L. (1988) Biochim. Biophys. Acta 954, 189-200. Three [Ru(NH 3 ) 5 ] 2+ complexes can be bound to metMb without perturbing the protein structure: Toi, H., La Mar, G. N., Margalit, R., Che, C.-M.& Gray, H. B. (1984) J. Am. Chem. Soc. 106, 6213-6217;, it would appear that ligand exchange on the cobalt center of 2, 3, or 4 is assisted by interactions with the protein. This assistance probably depends on neighboring side chain interactions with the cobalt complex. Protonation of the axial ligand by an acidic residue is a likely possibility. The fact that during unfolding apoMb binds six molecules of 1, but only three of 2, supports this suggestion. Why do 1 and 5 unfold metMb, while other histidine modifiers {2, [Ru(NH 3 ) 5 (H 2 O)] 2+ . Three [Ru(NH 3 ) 5 ] 2+ complexes can be bound to metMb without perturbing the protein structure: Toi, H., La Mar, G. N., Margalit, R., Che, C.-M.& Gray, H. B. (1984) J Am. Chem. Soc. 106, 6213-6217, and [Ru(en) 2 (H 2 O) 2 ] 2+ (en = 7,2-ethylenediamine) (Che, C- M., Margalit, R., Chiang, H.-J. & Gray, H. B. (1987) Inorg. Chim. Ada 135, 33-35) do not? An intriguing explanation is that 1 and 5, which could sequentially dissociate both axial ligands, bind to two trans disposed histidines. Cobalt(III) Schiff-base complexes differ from other d 6 complexes in their higher axial ligand labilities (31). Possible support for this suggestion is the observation that 6 (on average) molecules of 1 bind to unfolded myoglobin. As horse myoglobin has 11 histidines, this number may reflect 5 pairs of cobalt-bridged histidines, plus a histidine that is predominantly bound to a nonbridging cobalt (inteφrotein linking by cobalt is also a possibility). The interaction of Co(III) with one or more surface histidines is a likely initial step in the unfolding of metMb. The relative rates of unfolding by 1 are metMb « apoMb, indicating that hemin dissociation is rate determining. Hence, the parallel changes of the hemin absoφtion at 408 nm (Fig. 2a) and the backbone far-UV CD (Fig. 2b) imply that hemin dissociation precedes unfolding, as suggested for metMb unfolding under equilibrium conditions. The dissociated hemin is not separated from the cobalt-myoglobin product by dialysis, as confirmed by iron atomic absoφtion measurements. It probably binds nonspecifically to the unfolded protein, as observed by Hargrove, M. S. & Olson, J. S. (1996) Biochemistry 35, 11310-11318; Konermann, L., Rosell, F. I., Mauk, A. G. & Douglas, D. J. (1997) Biochemistry 36, 6448-6454. Barrick and Baldwin have proposed that two protons per protein unfold apoMb (Barrick, D. & Baldwin, R. L. (1993) Biochemistry 32, 3790-3796.) by disrupting the structurally important His 24 to His 119 hydrogen bond (Barrick, D., Hughson, F. M. & Baldwin, R. L. (1994) J. Mol. Biol. 237, 588-601;. Like Co(III), Zn 2+ and Cu 2+ associate with the histidines of metMb, probably triggering partial unfolding (Cann, J. R. (1964) Biochemistry 3, 714-722; Hartzell, C. R., Hardman, K. D., Gillespie, J. M. & Gurd, F. R. N. (1967) J Biol. Chem. 242, 47-53). It is reasonable, therefore, to propose that metal cations play a role similar to that of protons in their interactions with apoMb. The unfolding of metMb by 1 is highly selective. Under the same reaction conditions, neither the absoφtion nor the far-UV CD spectra of cytochrome c, azurin, thrombin, and thermolysin (Takeuchi, T., Ph. D. Thesis, California Institute of Technology, 1996; are affected by treatment with 1; and the catalytic activity of carbonic anhydrase is not diminished. Unlike metMb, these proteins do not possess enough accessible metal-ion binding sites, and they probably do not have histidines that are involved in structurally critical hydrogen bonds (unlike apoMb), thus explaining their markedly different behavior towards Co(III). Employing suitably structured metal complexes, it should be possible to unfold proteins rich in other hydrogen-bond-forming amino acids, in addition to those that possess a single hydrogen bond that is critical for tertiary-structure stabilization. Molten globules are superior to folded proteins in their ability to translocate across or insert into membranes (van der Goot, F. G., Lakey, J. H. & Pattus, F. (1992) Trends Cell Biol. 2, 343-348;, as they have increased affinity for hydrophobic surfaces (Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229;. An isolable partially folded protein can, therefore, be a powerful new type of pro-drug, functioning in a manner similar to that suggested for some bacterial toxins (van der Goot, F. G., Lakey, J. H. & Pattus, F. (1992) Trends Cell Biol. 2, 343-348;. Metal-ion-induced unfolding may also be utilized for selective protein precipitation, thereby aiding the formation of solid inclusion bodies, which could lead to improvements in large-scale protein biosynthesis (Betts, S., Haase-Pettingell, C. & King, J. (1997) Adv. Prot. Chem. 50, 243-264;.

Claims: CLAIMS We claim: 1. A method of unfolding a protein comprising contacting said protein with a Schiff s base metal complex, wherein said protein exhibits a 5% or greater change in at least one minima or maxima of the circular dichroism (CD) spectra of said protein as a result of binding at least of said one metal complexes to said protein. 2. A method according to claim 1 wherein said metal complex is a cobalt-containing complex. 3. A method according to claim 2 wherein said cobalt-containing complex is a Co(III) containing complex. 4. A method according to claim 1 wherein said metal complex has the formula: Structure 1 wherein L, and L 2 are axial ligands; and R, -R 8 are substitution groups. 5. A method according to claim 1 wherein said unfolding is irreversible under physiological conditions. 6. A method according to claim 1 wherein said protein is a viral protein. 7. A method according to claim 1 wherein said protein has at least 2 accessible metal-ion binding sites.

Inventors: BLUM OFER, CWIKEL DORY, DORI ZVI, GRAY HARRY B, HAIEK ABED, MEADE THOMAS J, BLUM, OFER, CWIKEL, DORY, DORI, ZVI, GRAY, HARRY, B., HAIEK, ABED, MEADE, THOMAS, J.
Applicants: CALIFORNIA INST OF TECHN, CALIFORNIA INSTITUTE OF TECHNOLOGY
Application year: 1998
Main classification: C07K1
IPC codes: C07K1, C07K14