CARB Research HighlightsProteins are the most complex molecules known. Although made from only 20 simple building blocks called amino acids, proteins usually contain thousands of atoms precisely arranged in a three-dimensional structure that is unique for each type. The specific sequence of amino acids that make up each protein is coded by a gene in the DNA of living cells. Proteins, the real "Business" molecules in the cell, perform many functions. They act as enzymes to catalyze the many thousands of highly specific biochemical reactions in the cell. Proteins also provide the structure that gives their shape and form and allows them to move, and they serve both as communication signals between cells and as regulators of biological activity.
Genetic Engineering has made possible the production of large quantities of proteins by cloning the appropriate gene sequence into suitable cells. Some of the most important biotechnology products now emerging are genetically engineered proteins that are used in a variety of medical and commercial applications. Many are hormone-like molecules with significant potential as therapeutic agents. While genetic engineering will continue to produce a variety of natural proteins for medical, industrial, agricultural and other commercial uses, the rapidly advancing field of protein engineering will significantly enhance our ability to produce novel proteins with improved properties.
Protein Engineering, the specific modification of the atomic structure of a
protein, can be used to alter and enhance the properties of the molecule.
Protein engineering has evolved from recent developments in the manipulation of
DNA (DNA synthesis and mutagenesis) that allow us to make precise changes to the
sequence of genes, thereby altering the structure and functions of the resulting
protein. The success of protein engineering will depend on the ability of
researchers to determine the detailed atomic structures of proteins, and to
understand the relationship between the structures and the properties of
proteins before and after engineering. To advance the field, CARB has
established programs in five critical research areas:
molecular biology,
macromolecular crystallography,
nuclear magnetic resonance spectroscopy,
physical biochemistry, and
computational chemistry and
modeling. The combined efforts of scientists in these areas, working toward
the common goal of understanding protein structure and function, will help
ensure that protein engineering becomes a commercial reality.
To re-engineer a protein or to understand its function requires a grasp of the protein's structure in atomic detail. The most powerful technique to gather that information is X-ray crystallography. In the past few years, significant advances have been made in X-ray instrumentation and CARB has a state-of-the-art X-ray diffraction laboratory. Deducing protein structure from X-ray data demands extensive computation. CARB is equipped with state-of-the-art computer and graphics hardware and software for use in solving protein crystal structures. The X-ray laboratory is completely computer controlled. The NIST central computing facilities and many in-house high performance graphics workstations are available for data analysis and structure refinement.
Faculty members doing research in this area are: Gary L. Gilliland, Osnat Herzberg and Roy Mariuzza
The magnetic dipoles of spinning atomic nuclei contain important structural information, such as the nature of the local molecular environment and the distances between atoms. NMR spectroscopy is a powerful tool for gathering this information and has long been used in chemistry to decipher the structure of small organic molecules. NMR technology can now be used to get structural information at near-atomic resolution for peptides and small proteins. NMR also provides information of the structure of proteins in solution and complements crystallography where the protein must be in a crystalline state. Indeed, for smaller proteins like therapeutically important growth and regulatory factors and for polypeptide hormones where crystallization may prove difficult, NMR methods offer a more rapid means of obtaining three dimensional structure information. The NMR laboratory at CARB is used for protein structure studies and for the development of new methods that will make NMR applicable to larger and more complex proteins and macromolecular complexes.
Faculty members doing research in this area are: Jim Ames, John Orban and John P. Marino
While crystallography and NMR spectroscopy are powerful structure probes, these techniques do not directly answer the questions of protein function and stability. For this CARB's physical biochemistry group focusses on what happens to the functional and physical characteristics of engineered proteins. This research area covers the study of enzyme activity and the use of various physical measurements such as fluorescence and absorption spectroscopy and analytical ultra-centrifugation to study the properties of proteins in solution. The stability of proteins and the thermodynamics of protein-ligand interactions is further analyzed using various microcalorimetric methods, including differential scanning calorimetry and titration site-directed mutagenesis, these physical methods provide valuable insight into the molecular details of protein structure and stability.
Faculty members doing research in this area are: Philip Bryan, Edward Eisenstein, Kevin D. Ridge, Frederick P. Schwarz and
The three-dimensional structure of a protein must be analyzed and related to the protein's function through the physical and chemical principles that govern molecular interactions and reactions. The analysis may be as simple as understanding the electrostatic and geometric factors that influence protein-substrate binding. Or it may be as complex as predicting an enzyme reaction mechanism that includes making and breaking of chemical bonds. The ultimate goal of protein modeling is to predict the complete three-dimensional structure of a protein from the amino acid sequence and use that structure to predict the protein's biological and physical properties. While success may be many years in the future, significant progress has been made in relating known protein structures to function and in predicting protein structures using homologous modeling and systematic conformational search methods. Computerized modeling and theory are used at CARB to guide protein engineering by predicting the effects of amino acid substitutions on structure, function, and stability. esearch projects include: the development of methods for determining structure from sequence using state-of-the-art search methods and Monte Carlo and molecular dynamical simulation; the development and evaluation of methods for modeling molecular recognition and protein-ligand binding in electrolyte solutions; and the use of quantum chemistry to predict and analyze enzyme mechanisms. Computer resources include many high-performance workstations and compute servers as well as access to the NIST central computing facilities.
Faculty members doing research in this area are: Michael K. Gilson, Morris Krauss and John Moult.
Basic to all research at CARB is the ability to clone and express genes to produce large quantities of engineered protein and nucleic acids for detailed biophysical studies. Molecular biology research at CARB focuses on the efficient use of prokaryotic cells to yield engineered proteins as well as the study of protein function in prokaryotic DNA regulation and cellular chemistry and metabolism. CARB has established molecular biology laboratories with state-of-the-art facilities for DNA chemistry and genetic engineering.
Faculty members doing research in this area are: Philip Bryan, Edward Eisenstein, Zvi Kelman, Roy Mariuzza and Kevin D. Ridge.
One of CARB's missions is to narrow the gap between discoveries in the research laboratory and the practical needs of industry. Close ties with industry will influence the direction of research at CARB and enhance the transfer of technology to strengthen Maryland's and the nation's industries in protein structure, function, and design. Industrial interactions with CARB may take many forms. Collaborative projects may be established with Visiting Industrial Fellows, support of postdoctorals, or direct support of CARB programs. CARB's exceptional facilities are available for collaborations. The formal nature of industrial relationships will be negotiated on a case-by-case basis.
Return to CARB Home Page
Please send comments to: webmaster-carb@nist.gov