Jacquelyn Fetrow, George Holzwarth, Daniel Kim-Shapiro, Keith Bonin, Martin Guthold, Fred Salsbury, Jed Macosko, and Howard Shields direct programs in the rapidly changing area of biological physics.
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The primary interest in Jacquelyn Fetrow's laboratory is understanding the relationship between protein structure, motion and function. A better understanding of the inter-relationship between these features will be valuable in the design of better and more specific compounds for the pharmaceutical drug discovery process. Dr. Fetrow and student, Anne Jeffers The members of Dr. Fetrow's group conduct research in three distinct, but related, areas: 1) computational analysis of functional sites in proteins, with a goal of automated identification of mechanistic and specificity determinants at molecular functional sites; 2) development of methods to model biological networks from experimental time course data, with the goal of understanding how networks of molecular signals cause or influence the behavior of biological systems; and 3) analysis of molecular dynamics and motion in proteins, with the goal of understanding the molecular mechanisms by which long-range communication occurs in these molecules. The research is interdisciplinary, involving students from math, computer science, physics, chemistry and biology and collaborations with many experimental researchers.For more information, see Prof. Fetrow's research page.
The primary research project in George Holzwarth's lab is measuring the drag force and mechanical work required for fast transport of vesicles in living cells and to relate this cellular task to the known limitations of motor proteins, especially kinesin. In buffer, kinesin takes about 100 8-nm steps/s. Each step takes only 50 ms steps; the maximum of steady force is 6.5 pN. In a cell, the viscoelastic drag force is 10,000-100,000 times greater than in buffer. Does kinesin develop the same force and pull with the same quick steps in these two environments? We are measuring vesicle transport in differentiated PC12 cells, which are a good model system for neurons. Some of our experimental results on vesicle movement can be found here.
Video-enhanced differential interference microscopy(VE-DIC) is another area of research in Holzwarth's lab. We improved VE-DIC by inserting a computer-controllable variable retarder into the optical train of a DIC microscope and switching the DIC imbalance from +R to -R in alternate frames. By subtracting alternate images, one automatically subtracts background and improves signal(contrast).. The system allows observation of low-contrast dynamic features in living cells as well as cellular components such as microtubules. For details, see the Holzwarth group.
The use of light (especially polarized light)to study the structure and function of biological macromolecules has also been a major area of study in Daniel Kim-Shapiro's research. A theoretical and experimental approach to the understanding of how light interacts with matter (and, in particular, how matter changes the polarization state of light) is taken to gain structural information not readily obtained by other biophysical methods. Measurements of the absorption, scaStudent research in the Kim-Shapiro labttering and fluorescence of macromolecules are made. New information regarding macromolecular structure leads to new insight concerning their function. Additional information is gained by monitoring macromolecular structure dynamically while the structure is changing due to some perturbation using rapid mixing (stopped-flow) or laser photolysis techniques.
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The use of these biophysical techniques are being applied to the study of the kinetics and mechanism of sickle cell hemoglobin depolymerization. Sickle cell anemia is disease characterized by a mutant form of hemoglobin (the oxygen transporting molecule in the red blood cell) which polymerizes when the red blood cell is without oxygen but which can depolymerize when the red blood cell is reoxygenated at the lungs. When the hemoglobin polymerizes the red blood cell becomes rigid and leads to microvascular occlusion that is responsible for much morbidity and mortality. The kinetics of depolymerization is therefore not only useful in understanding the physics of polymerization and depolymerization(and how this affects cell rigidity), but it is also important in understanding the clinical severity of the disease. Another more recent application in this laboratory to sickle hemoglobin has been in an effort to understand how the drug hydroxyurea - now being prescribed to patients, is working and how nitric oxide may benefit patients. These projects involve rheological measurements and various forms of spectroscopy.
The goals of Keith Bonin's research on molecular motors are to characterize their motion and to measure their energy efficiency. This will help us understand motor proteins at a fundamental level and could be a factor in understanding the effect of some diseases on the operation of these motors at the molecular level. This work is a collaboration with Prof. George Holzwarth, a biophysicist in the department, and neurobiologists at the Wake Forest University School of Medicine. They have recently setup a research class microscope to implement a magnetic technique for replication of vesicle motion in cytoplasm. A novel magnetic-field gradient design is being incorporated into the experimental setup to apply significant forces to magnetic beads in cytoplasm. They will track the motion of vesicles and magnetic beads in cells to deduce the motor protein efficiency in these cells. They will also try to estimate the number of protein motors that act at one time by studying stall forces experienced by our magnetic beads in real cells. In addition, they hope to study size effects by investigating forces and energy efficiency as a function of bead diameter.
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The research in Dr. Guthold's lab is at the interface of Biophysics and Nanotechnology. In particular, Dr. Guthold is using a modified Atomic Force Microscope - the so-called nanoManipulator - to image and manipulate biological molecules. Currently, DNA molecules, fibrin fibers (major component of blood clots) and others biopolymers are investigated. The goal of this research is to determine physical properties (rupture force, elasticity, strength, etc…) of individual biomolecules and to put the results in a biological context. In another project, a novel instrument is being developed that will allow the user to select single aptamer molecules on a surface (chip), pick them up individually and analyze them. Aptamers (from apt: fitted, suited; Latin aptus: fastened) are oligonucleotides, typically about 15 to 100 nucleotides in length, that have a demonstrated capability to specifically bind molecular targets with high affinity. Thus, aptamers are similar to monoclonal antibodies with the distinct difference that they are composed of nucleotides rather than amino acids and they are much smaller than antibodies. Since they were first described in 1990, dozens of aptamers, binding to such diverse targets as thrombin, viral proteins, organic and other molecules, have been identified. In our methodology, individual aptamers with desirable binding properties will be identified and isolated from highly diverse oligonucleotide libraries. The isolated, single oligonucleotides will then be amplified, sequenced and characterized. This method utilizes a unique combination of single molecule techniques and biochemical methods, thereby advancing both fields. The proposed methodology is particularly compelling for use in drug discovery. For example, we anticipate the discovery of molecules that can bind specifically to cancer cells or to pathogens (e. g., viruses). In the future, other, non-biological samples from our condensed matter group will also be investigated.
Fred Salsbury's research focuses on computational biophysics and chemical physics. The members of Dr. Salsbury's group conduct research in computational molecular biophysics; broadly construed. Their research projects involve, in varying combinations, understanding the molecular physics behind biological function, improving the computational methods used in biological physics, and applying physics-based tools to systems of biomedical interest.
The underlying questions in molecular physics that drive the research in Dr. Salsbury's group are 1) how do proteins modulate molecular function through conformational change, 2) how does communication occurs within and between proteins and other macromolecues, and 3) how do molecular interactions, which are altered through conformational change, especially electrostatics, control function.
To address these questions, method development has to occur at times, especially to improve the range of problems addressable by computational physics. Dr. Salsbury is best-known for his work in developing Generalized Born solvation models, and implicit models of protonation, especially through the constant-PHMD method. Currently, Dr. Salsbury's group is more interested in developing tools to analyze and use simulations to better address the underlying questions of interest to his group, and to develop model protein systems for such questions
Dr. Salsbury's group is remarkable in that in addition to developing new methods, and studying model systems, a primary focus of their work is the study of macromolecules and macromolecular complexes of real practical and biological interest. Their recent focus has been on DNA repair proteins (MSH2/MSH6), redox proteins (PRXs) and metallo-beta-lactamases. Four specific areas are currently being explored, which focus on the underlying questions of interest to Dr. Salsbury's group : 1) using physics-based measures, especially electrostatics, to understand the molecular function of proteins with an eventual aim of improving bioinformatic tools by incorporating physical measures (PRXs); 2) studying conformational change and long-range communication in macromolecules (all projects) 3) understanding the molecular and physical basis for multifunctional proteins (MSH2/MSH6) and 3) developing theraupetics that act via conformational and functional selectivity, i.e., by selecting specific conformations that activate specific cellular functions (MSH2/MSH6). Most of this work is multidisciplinary and so Dr. Salsbury's group collaborates extensively with experimental biomedical researchers. This work is or has been funded by the NIH, NSF, TSI, and GAC
Jed Macosko and his research group focus on understanding the mechanics of protein machines. They use techniques, including atomic force microscopy (AFM), single molecule fluorescence microscopy and video-enhanced differential interference contrast light microscopy (VE-DIC), to study how protein motors use chemical fuel to power their work cycle. One of their strategies in this effort is to use force and temperature to slow down or speed up the movements of the motors. This gives us information about the potential energy surface corresponding to the rate limiting mechanical transition in the molecular motor. Their long-term goal is the identification of precise mechanical-chemical couplings in molecular machines and the characterization of the overall pathways of their physical motion.
Although retired, Howard Shields, continues with his research. Electron paramagnetic resonance (EPR) techniques are being used to study irradiation damage in keratin proteins, defects in ionic solids, and structural phase changes in polymeric chains containing metallic ions. EPR which detects unpaired magnetic moments is ideal for studying damage resulting from exposure of keratin proteins to ionizing X-rays or UV light. In the proteins, the initial action of ionizing irradiation is the random breaking of bonds to form positive and negative defects. However, these defects are not stable, and in many cases, several different molecular configurations or radicals are detected as the unpaired electrons migrate to sulfur-containing residues where the unpaired electrons are trapped by sulfur atoms. The kinetics of the migration of unpaired spin to the sulfur atoms is being studied in both native proteins and model compounds. The model compounds are amino acid crystals containing small amounts of sulfur traps. These studies are designed to determine the mechanism by which the damage or unpaired electron migrates through the solid from the initial site to the stable sulfur trap.
Jacquelyn Fetrow and Fred Salsbury are also members of the Center for Structural Biology, an interdisciplinary organization with interests in structural and computational biology, comprised of faculty from the departments of Biochemistry, Chemistry, and Physics.