Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-07-12
2001-01-09
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S501000, C436S517000
Reexamination Certificate
active
06171804
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to methods for determining the relative orientation of the individual components of a macromolecule with respect to the global molecular coordinate frame of the macromolecule. Such methods can be used to determine the structure of multicomponent molecules.
BACKGROUND OF THE INVENTION
Knowledge of the detailed three-dimensional structure of any given macromolecule is critical for optimizing and/or regulating the use of that macromolecule, be it a protein that is malfunctioning in a metabolic pathway, or a synthetic polymer used in microchip technology. Currently, there are two major strategies for determining the detailed three-dimensional structure of a macromoleucle: X-ray crystallography and nuclear magnetic resonance. X-ray crystallographic analysis requires the time-consuming process of preparing high quality crystals, whereas classical NMR three-dimensional analysis is limited to macromolecules that are under 35 kilodaltons [Yu,
Proc. Nat. Acad. Sci
. USA 96:332-334 (1999)]. Furthermore, such methods of high-resolution structure determination are generally applicable to macromolecules formed by tight contacts between the individual, well-structured components of the macromolecule. These methods have more limited applicability in those cases where there are weaker interactions between the component; examples include the relatively transient associations formed in complexes involved in signal transduction, or in transcriptional control. Crystal structures of such complexes might be biased by packing forces comparable to the interdomain interactions, while the precision and accuracy of the conventional NMR structural approaches are necessarily limited by the restricted number of nuclear Overhauser effect (NOE) contacts and by interdomain flexibility rendering the available NOE information uninterpretable.
Recently proposed NMR approaches [Tolman et al.,
Proc. Natl. Acad. Sci
., U.S.A., 92:9279-83 (1995); Bruschweiler et al.,
Science
, 268:886-9 (1995); Broadhurst et al.,
Biochemistry
, 34:16608-17 (1995); Tjandra et al.,
Nat. Struct. Biol
., 4:443-9 (1997); and Tjandra and Bax,
Science
, 278:1111-4] are potentially capable of improving both the accuracy and precision of structure determination in solution and might prove to be the method of choice in those cases when the number of available short-range NOE contacts is limited. These methods are based on ‘long-range’ structural information in the form of inter-nuclear vector constraints with respect to an overall, molecular reference frame. These constraints may arise from correlation with the anisotropic hydrodynamic properties of the molecule [Bruschweiler et al.,
Science
, 268:886-9 (1995); Broadhurst et al,
Biochemistry
, 34:16608-17 (1995); and Tjandra et al,
Nat. Struct. Biol
., 4:443-9 (1997)], or from weak alignment of molecules in solution caused by either their interaction with the magnetic field [Tolman et al.,
Proc. Natl. Acad. Sci
, U.S.A., 92:9279-83 (1995)] or by the liquid crystalline characteristics of the medium [Tjandra and Bax,
Science
, 278:1111-4]. The NMR relaxation approach [Bruschweiler et al.,
Science
, 268:886-9 (1995); Broadhurst et al.,
Biochemistry
, 34:16608-17; and Tjandra et al,
Nat. Struct. Biol
., 4:443-9 (1997)] which takes advantage of the anisotropic character of the overall rotation, is most generally applicable to a wide range of macromolecules in their native milieu. The magnetic alignment method [Tolman et al.,
Proc. Natl. Acad. Sci
., U.S.A., 92:9279-83 (1995) and Tjandra et al.,
Nature Structural Biology
, 4:732 (1997)] requires macromolecules to possess a sufficiently high anisotropy of the magnetic susceptibility, and is not, therefore, widely applicable. The approaches based on weak alignment of macromolecules in liquid crystalline medium may be restricted by possible interactions between the molecule under investigation and the medium. For a list of intractable target proteins by this method using lipid bicelles see footnote 8 in Clore et al.,[
J. Am. Chem. Soc
., 120:10571-2 (1998)], although more recent alignment methods may alleviate this issue [Clore et al.,
J. Am. Chem. Soc
., 120:10571-2 (1998); Hansen et al.,
J. Am. Chem. Soc
., 120:11210-11 (1998); Koenig et al.,
J. Am. Chem. Soc
., 121:1385-6 (1999); and Sass et al.,
J. Am. Chem. Soc
., 121:2047-55 (1999)].
Naturally occurring polymers such as nucleic acids and proteins are macromolecules that have distinct three-dimensional structures. Indeed, the ability of any given protein to carry out its physiological role, regardless of whether it functions as a structural element, a binding partner, and/or a biochemical catalyst, requires that the protein assume a specific conformation. This conformation is dependent on the three-dimensional folding of the protein into specific domains and the orientation of these domains to each other, as well to the corresponding domains of other proteins.
The binding of a ligand to a protein (e.g., a substrate to an enzyme), generally results in a local alteration of the three-dimensional structure of the protein. In addition, the binding of the ligand to one site of a protein, can also alter the structure of other regions of the polypeptide [See generally, Kempner,
FEBS
326:4-10 (1993)]. Indeed the relative orientation and motions of domains within many proteins are key to the control of multivalent recognition, or the assembly of protein-based cellular machines. Therefore, it is not surprising that there has been a long and continuous effort to determine the structures of nucleic acids and proteins, not only in their resting state, but also in their more dynamic state in their native environment.
In recent years it has become apparent that there is a large but finite number of protein structural domains that are shared throughout nature. These domains are used by the proteins to carry out their biological roles. One such pair of domains are the src homology domains SH
2
and SH
3
. Eukaryotic cellular signal-transduction pathways that are initiated by transmembrane receptors with associated tyrosine kinases rely on these two small protein domains for mediating many of the protein-protein interactions that are necessary for transmission of the signal [Cantley et al.,
Cell
64:281-302 (1991); Schlessinger et al.,
Neuron
9:383-391 (1992); Pawson et al.,
Curr. Biol
. 3:434-442 (1993)]. These domains were first discovered in cytoplasmic (non-receptor) protein tyrosine kinases such as the src oncogene product, thus leading to the term ‘src homology domains’ [Sadowski et al.,
Mol. Cell. Biol
. 6:4396-4408 (1986)].
The unique importance of these domains became clear with the discovery of the crk oncogene product, which consists of little more than an SH
2
and an SH
3
domain fused to the viral gag protein, but is capable of transforming cells [Mayer et al,.
Nature
332:272-275 (1988)]. SH
2
and SH
3
domains have been identified in molecules with distinct functions that act downstream from the receptors for, among others, epidermal growth actor (EGF), platelet-derived growth factor (PDGF), insulin and interferon, and the T-cell receptor [Koch et al.,
Science
: 252:668-674 (1991)].
An important aspect of the role of protein domains such as the SH
2
and SH
3
domains is their ability to recognize particular amino acid sequences in their target proteins: SH
2
domains bind tightly to phosphorylated tyrosine residues [Anderrson et al.,
Science
; 250:979-982 (1990); Matsuda et al.
Science
248:1537-1539 (1990); Moran et al.
Proc. Natl. Acad. Sci
. USA 87:8622-8626 (1990); Mayer et al
Proc. Natl. Acad. Sci
. USA: 88:627-631 (1991); Songyang et al.
Cell
72:767-778 (1993)] whereas SH
3
domains bind to proline rich segments forming a short helical turn in the complexes [Kuriyan and Cowburn,
Annu. Rev. Biophys. Biomol. Struct
., 26:259-288 (1997), the contents o
Cowburn David
Fushman David
Xu Rong
Brusca John S.
Kim Young
Klauber & Jackson
The Rockefeller University
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