Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Chemical aftertreatment – e.g. – acylation – methylation – etc.
Reexamination Certificate
1999-06-01
2003-11-18
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Chemical aftertreatment, e.g., acylation, methylation, etc.
C530S333000, C436S085000, C436S089000
Reexamination Certificate
active
06649736
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a solution based method for carbon-centered radical mediated protium/heavy hydrogen exchange into the reduced carbon atoms in a molecule of interest. The methods of the present invention can be used to determine which reduced carbon atoms in a molecule are solvent accessible. In particular, the methods of the present invention can be used to determine which carbon atoms in a macromolecule, such as a peptide or protein, are solvent accessible when the macromolecule is in a particular folded state.
2. Background Art
Carbon-centered radical mediated heavy hydrogen labeling of compounds is well known in the art. For example, radiolysis studies have demonstrated that hydroxyl (OH) radical can act as a hydrogen atom abstractor that removes a hydrogen atom from reduced carbon atoms in molecules such as amino acids, peptides, and proteins to form a carbon-centered radical. See e.g. Garrison, 1987,
Chem. Rev.
87:381-398; von Sonntag, 1987, The Chemical Basis of Radiation Biology, Taylor & Francis: London. However, determining which carbon atoms in a molecule react with the hydroxyl radical has been elusive. In the case of DNA, reaction of DNA with a hydrogen atom abstractor results in strand scission. Thus, the site of the reaction between a hydrogen atom abstractor, such as hydroxyl radical, and the DNA can be inferred by studying DNA cleavage patterns. Hertzberg et al.,
Biochemistry
23:3934-3945.
The predominant mode of hydrogen atom abstractor initiated damage of DNA and proteins is removal of a hydrogen atom from a C—H bond of a reduced carbon atom to produce the corresponding carbon-centered radical. von Sonntag, supra. The carbon-centered radical has various chemical fates including: (1) reaction with molecular oxygen, initially forming a hydroperoxyl species that can result in hydroxylation (Fu et al., 1995,
Biochem. J.
311:821-827) and DNA (Breen et al., 1995,
Free Radic. Biol. Med.
18:1033-1077) or protein (Davies,
J. Biol. Chem.
262: 9895-9901) strand scission, (2) recombination of two carbon-centered radicals to form a carbon-carbon crosslink (Karam et al., 1984,
Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
46:715-724; Davies et al., 1987,
J. Biol. Chem.
262:9902-9907; Gajewski et al.,
Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
54:445-449); and (3) chemical repair by H atom donation, such as that mediated by sulfhydryls (Alexander et al., 1955,
Radiobiology Symposium,
pp 49-55, Bacq & Alexander, Academic Press: New York). Thus, the initial site of hydroxyl radical attack on a molecule is often obscured by the multiplicity of resulting products.
In DNA, the abstraction of any ribose hydrogen atom and subsequent oxidation leads to chain scission. Thus, carbon-centered radical mediated assays provide a general method for identifying residues that react with the hydrogen atom abstractor. The ability to randomly initiate cleavage of the DNA backbone by enzymatic or chemical means is the most essential chemical step in DNA footprinting. The sites protected by protein binding are excluded from solvent and consequently, are not susceptible to attack by the hydrogen atom abstractor. The absence of these DNA product fragments in electrophoretic separations identify the DNA nucleotides involved in protein-DNA recognition. The footprint resolution is dependent upon the chemical nature of the DNA cleaving reagent. Base resolution can be achieved by using a small, sterically unhindered molecule that is highly reactive and is nonspecific, indiscriminately cleaving at all base positions of the DNA backbone. Hydroxyl radical generated by &ggr;-radiolysis has been experimentally shown to possess all of these attributes and has been used to identify contacts in protein-DNA complexes at base resolution. Franchet-Beuzig, et al., 1993,
Biochemistry
32:2104-2110.
Despite the success in using carbon-centered radical mediated reactions in DNA footprinting techniques, analogous footprinting techniques to study protein-protein interactions have proven to be unsatisfactory. Small metal chelates have been used to randomly cleave polypeptide chains. The chelate, iron(II)-EDTA, has been utilized in either a tethered or untethered form. The tethered form can be used to map its proximity to neighboring peptide bonds. Rana et al., 1990,
J. Am. Chem.
112:2457-2458. Using untethered iron(II)-EDTA as a nonspecific protein cleaving agent, Heyduk et al. have studied solvent-accessible sites induced by changes in protein conformation upon ligand binding for cAMP receptor protein in the presence and absence of cAMP. Heyduk et al., 1994,
Biochemistry
33:9643-9550. In addition, Greiner and coworkers have used iron(II)-EDTA as a nonspecific protein cleaving agent to map the interactions between the subunits of
E. coli
RNA polymerase. Greiner et al, 1996,
Proc. Natl. Acad. Sci., U.S.A.
93:71-75. In both cases, the peptide fragments were electrophoretically separated and visualized by immunostaining with antibodies specific to the N- and/or C-terminal peptides of the protein. The limitations of this method are (1) iron(II)-EDTA cleavage tends to occur at hypersensitive sites, (2) antibodies for the N- and/or C-termini are required for the proteins of interest, and (3) identification of the sites of protection is usually confined to segments of 10-15 residues in length. Although this protein footprinting methodology permits mapping contact regions of protein domains involved in macromolecular assemblies, the ability of the technique to specifically identify the sites involved in recognition at the amino acid residue level has not been satisfactory.
The failure to achieve single residue resolution in protein footprinting studies despite the success in analogous DNA footprinting studies can also be understood by comparing the reactivity of hydrogen atom abstractors, such as hydroxyl radical, with proteins and DNA. For duplex DNA, hydroxyl radical react with the macromolecule by abstracting a hydrogen atom from solvent-accessible C—H bonds of the deoxyribose ring along the DNA backbone, producing a carbon-centered radical that reacts with O
2
and results in strand scission. Breen et al., 1995,
Free Radic. Biol. Med.
18:1033-1077. Cleavage of globular proteins occurs by a similar mechanism. Stadtman, 1993,
Annu. Rev. Biochem.
62:797-821. Abstraction of a C
&agr;
—H of the protein backbone by hydroxyl radical produces a carbon-centered radical that reacts with O
2
, forming a hydroperoxyl species that leads to protein strand scission. However, the majority of solvent-accessible C—H bonds present on the protein's solvent-accessible surface are not comprised of the backbone (C
&agr;
—H
&agr;
) but those of the side chains. Thus, in protein footprinting the major pathway of hydrogen abstractor reactivity with proteins is not exploited.
The reaction of hydroxyl radical with alkyl C—H bonds is rapid, 10
8
M
−1
s
−1
(Buxton et al., 1988, (
J. Phys. Chem. Ref. Data
17:513-886) a value 10-100 fold less than the diffusion limit. This indicates that a hydrogen atom abstraction occurs on average every 10-100 collisions. This high frequency of reaction prevents hydroxyl radical generated in bulk solution from diffusing into the interior of macromolecular complexes. The success of DNA footprinting with hydroxyl radical demonstrates that formation of macromolecular complexes protects the residues at the molecular interface from reacting with hydroxyl radical. Tullius et al, 1987,
Methods of Enzymology
155:537-558, Wu Ed., Academic Press: New York.
Electron spin resonance studies have also used carbon-centered radical mediated labeling of compounds to study molecules of interest, such as macromolecules. In EPR studies, a hydrogen atom abstractor, such as hydroxyl radical, is used to generate the carbon-centered radical of the molecule of interest. This highly unstable carbon-centered radical is then reacted with a spin trapping agent such as a nitrone. Buettner et al, 1990,
Methods i
Anderson Vernon E.
Goshe Michael B.
Case Western Reserve University
Low Christopher S. F.
Lukton David
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