Structural Biology
The activities of cells are highly regulated on several levels. Insofar as cancers result from a failure of regulation of cell division and growth, it is important to identify the mechanisms responsible for growth control. In large measure these involve interactions between macromolecules.
Our goal is to examine the properties of interacting molecules in the normal state and in the cancer state. This information is essential for rational design and optimization of new drugs for novel targets identified in the Molecular Targets Program.
Structural biology and biophysics are of use in studying the structure, dynamics, stability and molecular properties of biological molecules, and the consequences of their interactions with their targets. Major questions include:
- What are the conformations, dynamics and molecular properties of the individual components?
- How do these change on forming specific compared with non-specific complexes?
- What are the molecular and functional consequences of these changes?
- How strong and how fast are the interactions?
- How do these relate to the physiological function of the molecules?
In order to answer these questions, it is necessary to integrate structural (conformation) data, with information about the dynamics and energetics of all of the components. The work of the Structural Biology Program, due to the broad range of expertise involved, has consequently required the establishment in the Brown Cancer Center of shared core facilities which provide a broad range of technologies and methodologies, including nuclear magnetic resonance (NMR), optical spectroscopy, X-ray diffraction, thermodynamics, kinetics, computational strategies, metabolomics, synthetic chemistry and biochemistry. We also have established a facility for large-scale expression and purification of recombinant proteins necessary for studying the molecular mechanisms that will enable the development of in vitro screening.
Structure determination
Structure determination can be carried out at the Brown Cancer Center (BCC) by three complementary techniques: NMR, X-ray diffraction and computation.
NMR can be applied to the solution state or to the solid state. Expertise and instrumentation for both of these modalities are available at BCC. For example, high resolution NMR at 14.1 T and 18.8 T is used to determine the structures of small proteins in solution at high resolution, as well as measure their internal dynamics by relaxation techniques. This modality also is used for determining metabolites in complex mixtures such as crude extracts from cancer cells.
However, not all proteins are soluble in water, including important health-related ones such as those that are part of connective tissue (collagens, elastin) which are relevant to metastasis and in numerous skin diseases and found in plaques in the neurodegenerative diseases. These can be studied by solid-state NMR techniques, which provides essential molecular information that cannot otherwise be obtained. NMR Core
X-ray crystallography provides the highest resolution structural information, and is now possible for very large complexes. In conjunction with solution and computational approaches it is exceptionally powerful. Macromolecular X-ray crystallography has been introduced to BCC in a collaborative effort with the UofL Chemistry Department, A new facility for macromolecular diffraction has been established, including a rotating anode generator and area detector system. The facility is supervised by crystallographers Drs. Mashuta (Chemistry) and Hong Ye (Brown Cancer Center).
Computational biology is an increasingly important discipline in several respects. It is an essential component of structure determination based on experimental data, such as from NMR or x-ray diffraction. It also is used in structure modeling based on homologies or de novo prediction. Computational techniques can supply information that is simply not accessible to direct experimentation. Different expertise is available for calculating electrostatic properties of macromolecules and their charge interactions such as Poisson-Boltzmann calculations and structure determination and analysis approaches at the BCC Molecular Modeling Core Facility (Director: Dr. John O. Trent). Computational Core. These facilities provide expertise, hardware and software solutions to structure determination, and for calculating the properties of macromolecules that are not readily accessible to direct measurement. This adds value to the purely experimental data. In addition an increasingly important aspect of the research is in drug discovery using a variety of virtual screening approaches, which is producing lead compounds against targets identified at BCC.
Biophysics
In addition to structure determination of isolated components and complexes, it is essential to determine the energetics and kinetics of the interactions, and changes in physical properties of the components on forming the complexes. To this end, a biophysics core laboratory has been established at BCC where researchers can measure functional properties of their purified systems. This is achieved using a variety of biophysical methods including calorimetry, optical spectroscopy such as fluorescence and circular dichroism, rapid reaction kinetics and hydrodynamic techniques (Director: J. Brad Chaires). Biophysics Core
Protein Expression
All of these endeavors require substantial (mg) quantities of purified materials. To assist researchers in this area, a protein expression/purification core laboratory was established, which houses equipment for fermentation and protein purification as well as initial characterization. Protein Expression Core
Metabolomics
Investigating the differences between normal and transformed cells requires techniques to determine the concentrations and nature of the cellular contents. In parallel with gene chip and proteomics technologies, the Program is carrying out functional studies of the metabolome in normal and cancerous cells. Metabolomics uses methods of structural biology, especially NMR and mass spectrometry, to determine the nature and concentrations of metabolites, and the fluxes through particular metabolic pathways. This therefore provides a functional view of a cell type under certain circumstances. In addition to providing basic information about cell metabolism, it is valuable for assessing the influence of putative drugs on cell function to ascertain whether the desired effects are being achieved. This is done in parallel with other biological assays of cell function. Recently a Center for Regulatory Environmental Analytical Metabolomics (CREAM) was established at UofL (Director, T. W-M Fan) that is housed in the new Belknap Research Building. This Center for Regulatory and Environmental Analytical Metabolomics houses a wide range of state of state of the art mass spectrometry equipment, and focuses on stable isotope tracing methods for metabolomics.
At the right you will find links to information about Structural Biology research projects that are currently in progress. Selecting one of these project titles will lead to a general abstract of the project. From there, you can follow the "Technical Notes" links to more specific and detailed information.
Structural Biology
The activities of cells are highly regulated on several levels. Insofar as cancers result from a failure of regulation of cell division and growth, it is important to identify the mechanisms responsible for growth control. In large measure these involve interactions between macromolecules.
Our goal is to examine the properties of interacting molecules in the normal state and in the cancer state. This information is essential for rational design and optimization of new drugs for novel targets identified in the Molecular Targets Program.
Structural biology and biophysics are of use in studying the structure, dynamics, stability and molecular properties of biological molecules, and the consequences of their interactions with their targets. Major questions include:
- What are the conformations, dynamics and molecular properties of the individual components?
- How do these change on forming specific compared with non-specific complexes?
- What are the molecular and functional consequences of these changes?
- How strong and how fast are the interactions?
- How do these relate to the physiological function of the molecules?
In order to answer these questions, it is necessary to integrate structural (conformation) data, with information about the dynamics and energetics of all of the components. The work of the Structural Biology Program, due to the broad range of expertise involved, has consequently required the establishment in the Brown Cancer Center of shared core facilities which provide a broad range of technologies and methodologies, including nuclear magnetic resonance (NMR), optical spectroscopy, X-ray diffraction, thermodynamics, kinetics, computational strategies, metabolomics, synthetic chemistry and biochemistry. We also have established a facility for large-scale expression and purification of recombinant proteins necessary for studying the molecular mechanisms that will enable the development of in vitro screening.
Structure determination
Structure determination can be carried out at the Brown Cancer Center (BCC) by three complementary techniques: NMR, X-ray diffraction and computation.
NMR can be applied to the solution state or to the solid state. Expertise and instrumentation for both of these modalities are available at BCC. For example, high resolution NMR at 14.1 T and 18.8 T is used to determine the structures of small proteins in solution at high resolution, as well as measure their internal dynamics by relaxation techniques. This modality also is used for determining metabolites in complex mixtures such as crude extracts from cancer cells.
However, not all proteins are soluble in water, including important health-related ones such as those that are part of connective tissue (collagens, elastin) which are relevant to metastasis and in numerous skin diseases and found in plaques in the neurodegenerative diseases. These can be studied by solid-state NMR techniques, which provides essential molecular information that cannot otherwise be obtained. NMR Core
X-ray crystallography provides the highest resolution structural information, and is now possible for very large complexes. In conjunction with solution and computational approaches it is exceptionally powerful. Macromolecular X-ray crystallography has been introduced to BCC in a collaborative effort with the UofL Chemistry Department, A new facility for macromolecular diffraction has been established, including a rotating anode generator and area detector system. The facility is supervised by crystallographers Drs. Mashuta (Chemistry) and Hong Ye (Brown Cancer Center).
Computational biology is an increasingly important discipline in several respects. It is an essential component of structure determination based on experimental data, such as from NMR or x-ray diffraction. It also is used in structure modeling based on homologies or de novo prediction. Computational techniques can supply information that is simply not accessible to direct experimentation. Different expertise is available for calculating electrostatic properties of macromolecules and their charge interactions such as Poisson-Boltzmann calculations and structure determination and analysis approaches at the BCC Molecular Modeling Core Facility (Director: Dr. John O. Trent). Computational Core. These facilities provide expertise, hardware and software solutions to structure determination, and for calculating the properties of macromolecules that are not readily accessible to direct measurement. This adds value to the purely experimental data. In addition an increasingly important aspect of the research is in drug discovery using a variety of virtual screening approaches, which is producing lead compounds against targets identified at BCC.
Biophysics
In addition to structure determination of isolated components and complexes, it is essential to determine the energetics and kinetics of the interactions, and changes in physical properties of the components on forming the complexes. To this end, a biophysics core laboratory has been established at BCC where researchers can measure functional properties of their purified systems. This is achieved using a variety of biophysical methods including calorimetry, optical spectroscopy such as fluorescence and circular dichroism, rapid reaction kinetics and hydrodynamic techniques (Director: J. Brad Chaires). Biophysics Core
Protein Expression
All of these endeavors require substantial (mg) quantities of purified materials. To assist researchers in this area, a protein expression/purification core laboratory was established, which houses equipment for fermentation and protein purification as well as initial characterization. Protein Expression Core
Metabolomics
Investigating the differences between normal and transformed cells requires techniques to determine the concentrations and nature of the cellular contents. In parallel with gene chip and proteomics technologies, the Program is carrying out functional studies of the metabolome in normal and cancerous cells. Metabolomics uses methods of structural biology, especially NMR and mass spectrometry, to determine the nature and concentrations of metabolites, and the fluxes through particular metabolic pathways. This therefore provides a functional view of a cell type under certain circumstances. In addition to providing basic information about cell metabolism, it is valuable for assessing the influence of putative drugs on cell function to ascertain whether the desired effects are being achieved. This is done in parallel with other biological assays of cell function. Recently a Center for Regulatory Environmental Analytical Metabolomics (CREAM) was established at UofL (Director, T. W-M Fan) that is housed in the new Belknap Research Building. This Center for Regulatory and Environmental Analytical Metabolomics houses a wide range of state of state of the art mass spectrometry equipment, and focuses on stable isotope tracing methods for metabolomics.
At the right you will find links to information about Structural Biology research projects that are currently in progress. Selecting one of these project titles will lead to a general abstract of the project. From there, you can follow the "Technical Notes" links to more specific and detailed information.