Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
2001-05-16
2004-07-13
Gakh, Yelena G. (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Reexamination Certificate
active
06762056
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to the field of sensors for the detection and investigation of proteins, pathogens and other biologically important molecules, and more particularly to a bionanotechnology method for rapidly determining the surface topology of protein molecules.
Determining the surface topology of proteins is vitally important to broad fields of science including pharmaceutical research and genomics, as well as fundamental research into biological processes. Surface topology determines how proteins interact with each other, since proteins of complementary shape tend to bind together while proteins of non-complementary shapes do not. This lock-and-key binding of proteins provides the mechanisms by which enzymes, antibodies and nucleic molecules accomplish their vital biological functions, as well as the mechanisms that enable viruses and bacteria to infect cells. Knowing the shape and surface characteristics of a protein can help explain its function in a cell. Also, knowing the surface topology of a protein permits the development of drugs and sensors that will bind with that particular protein, enabling more effective medications and diagnostic tests for the treatment of human diseases.
Understanding the surface topology of biologically significant proteins is an important step towards the development of new medicines and medical treatments. Once the shape and chemical structure of a protein is known, pharmaceutical researchers can design a complementary molecule that will fit into the surface folds of the protein and bind to it just as an antibody binds to an antigen. Then pharmaceutically active molecules can be attached to the binding molecule to deliver a specific medication to a particular cell, bacterium or virus.
Understanding the surface topology of proteins is also essential to the development of sensitive diagnostic and protein assay methods. With the rapid advances of biotechnology and genetic research, which are elucidating the key roles and the presence of particular proteins that can be characteristic of certain disease states, great emphasis has been placed on developing very sensitive assays and sensor methods capable of detecting minute quantities of biologically significant molecules. The detection and quantification of specific proteins associated with a given disease, such as a given cancer, may enable much earlier detection and treatment. Furthermore, the ability to detect nucleic acid sequences encoding particular proteins enables many beneficial medical and commercial applications, including agricultural product screening and development. However, in many applications, methods used to detect particular proteins still require an understanding of the binding sites and surface topology of the protein.
The primary method used to develop an understanding of protein surface topologies is protein X-ray crystallography. X-ray crystallography makes use of the diffraction of X-rays by protein crystals to determine the precise three-dimensional arrangement of atoms within the protein molecule. Sophisticated analysis software and algorithms permit researchers to translate the diffraction patterns into three-dimensional structural information, and from that to identify the surface topology of the protein.
Before a protein can be studied with X-ray crystallography, the protein must be isolated, purified and crystallized. Because of the molecular complexity of proteins, obtaining suitable crystals for crystallography can be quite difficult, extremely time consuming and labor intensive. In response, a number of methods and equipment designs have been developed for crystallizing proteins (see, for example, U.S. Pat. No. 5,961,934, issued Oct. 5, 1999; U.S. Pat. No. 5,643,540, issued Jul. 1, 1997; U.S. Pat. No. 5,597,457, issued Jan. 28, 1997; U.S. Pat. No. 5,419,278, issued May 30, 1995; and U.S. Pat. No. 5,096,676, issued Mar. 17, 1992, having methods for forming protein crystals suitable for crystallography) and for streamlining the effort required to identify and reproduce appropriate conditions for crystallization of proteins (see, for example, U.S. Pat. No. 5,641,681, issued Jun. 24, 1997, showing a method for obtaining conditions for growth of high quality protein crystals). A number of physical and chemical factors can impact upon protein formation and crystal growth, so that, for example, it has been proposed that protein crystals be produced in a satellite in earth orbit having zero gravity.
Thus, there is a need for a less time consuming and expensive method for determining the surface topology of proteins, to benefit medical and biological research and facilitate the development of new medications.
Furthermore, there is a need for a means to quickly identify potential binding sites on specific proteins. In many pharmaceutical and diagnostic commercial applications, only potential binding sites on the surface of a protein, rather than a detailed model of all atomic coordinates, are of interest. Identifying binding sites by current methods requires sophisticated analyses of the three-dimensional structure of the protein involving complex numerical modeling. Thus, a less expensive and more direct method for determining potential protein-binding sites would be of economic value.
There is also a need for reliable diagnostic devices to diagnose individuals who are infected, for example, by a new or rapidly mutating pathogen, such as human immunodeficiency virus (HIV), influenza virus, or malaria, or other disease states that have evaded simple diagnostic tests, such as certain forms of cancer or pre-cancerous conditions.
SUMMARY
In one embodiment, the invention provides a method for discovering protein adsorption sites on a surface, comprising: providing a test surface having a surface topology comprised of a random distribution of randomly shaped features of a size from about 10
−10
meters (one Angstrom) to about 10
−8
meters (10 nanometers) in width, height, depth and spacing; exposing the test surface to a solution of a substantially purified protein, the solution remaining in contact with the surface sufficiently long to enable protein molecules to adsorb to adsorption sites; removing the solution with unadsorbed protein molecules from the test surface; and identifying the protein adsorption sites by detecting the presence of adsorbed protein molecules, to locate protein molecules adsorbed to the test surface.
A related method further includes removing the protein molecules adsorbed to the test surface, and measuring the surface topology of the identified adsorption site.
In accordance with a related embodiment, the method further comprises analyzing statistically the surface topology of a statistically significant number of identified adsorption sites to determine a most probable adsorption site topology. Measuring the surface topology further comprises using one or more of the group consisting of: a microcantilever, an atomic force microscope, a scanning tunneling microscope, a magnetic resonance force microscope, a thermomechanical atomic force microscope, a multi-tip atomic force microscope, and microparticles coupled to protein molecules adsorbed to the test surface. The method in various embodiments further comprises storing the most probable adsorption site topology in a computer database.
Another related method includes detecting the presence of adsorbed protein molecules using a microcantilever. Yet another related method includes detecting the presence of adsorbed protein molecules using, for example: an atomic force microscope; a scanning tunneling microscope; a magnetic resonance force microscope; a thermomechanical atomic force microscope; is a multi-tip atomic force microscope; or coupling a microparticle to protein molecules adsorbed to the test surface.
Another related method includes using a plurality of different surface coatings deposited on the test surface, each coating having a characteristic resiliency, and further comprising determining which of said surface coatings is de
Gakh Yelena G.
Guterman Sonia K.
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
Protiveris, Inc.
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