Martin Guthold - US grants

Martin Guthold joined the project December 1, 1997 to work with external biologists using the nanoManipulator in their investigations. He will also be continuing his investigations of DNA. We have initiated an investigation of DNA and protein-DNA complexes using the nanoManipulator and complementary biochemical studies. High-resolution images of DNA in air can now be obtained routinely. In initial pushing experiments of DNA adsorbed on mica in air, we were able to rupture DNA in a controlled matter. After proper calibration of the lateral force constant of the cantilever we will determine the rupture force of DNA. Experiments to determine the frictional force, the friction coefficient and the activation energy to move DNA on the surface are currently pursued. We have determined the binding energy of a 25 bp DNA fragment for adsorption onto mica for one set of conditions. The free energy of adsorption for this fragment was determined to be about 4.2 kT, the entropic energy loss was calculated to be about 1 kT, and the enthalpy of binding was, therefore, about 3.2 kT. Binding experiments will be done for different buffer and substrate conditions and for DNA fragments of different length. We are developing a theoretical framework to describe the adsorption of DNA to a substrate. Using a simple model, we have derived formulas for the length dependence of the free energy, enthalpy and entropy of adsorption. A complete model including the aforementioned parameters plus frictional parameters and the activation energy for motion on the surface will be developed in the future.

GRINCH II, Direct Map Interpretation. In the early 1980s Tom Williams, while a member of the GRIP project, built the GRINCH system for the ab-initio interpretation of electron density maps. Williams' system was based on work by Drs. Jonathan Greer, Carroll Johnson, and Stanley Swanson for representing electron density by marking the ridges of high local density. At least 50 copies of this system were disseminated to the crystallographic community. Graduate student Juraj Horacek of the current GRIP project team was re-examining this ridge line approach to ab-initio electron density interpretation using new, more powerful ridge line finding mathematics developed in our department by David Eberly for the analysis of medical images. Our new approach is distinguished from prior work in that we do not simply connect critical points but actually trace the ridges of high density through an interpolated function. This should help us avoid some kinds of common errors in map interpretation. We expect this work to lead to both new representations for electron density and to new methodologies for its interpretation as a molecular structure. Horacek decided to abandon or postpone his Ph.D. work in order to enter Cornell Business School. He worked on the project from May until September. He may work on it again this summer. We shall see. We do not now expect another student to pick up this work; if not, this subproject will be abandoned.

Supplement to develop full fledged distributed nanomanipulator to be operated remotely over Internet 2.

No work on this progress this year, and none is planned for the coming year. It will not hereafter be included in our reporting. [unreadable]

Fibrin fibers, the structural component of blood clots, have the mechanical task of stemming the flow of blood. Surprisingly, despite the importance of this task, the mechanical properties of fibrin fibers are largely unknown. The applicant is developing a novel technique in which the tip of an atomic force microscope is used to stretch these tiny fibers. With this technique, the applicant will determine the comprehensive mechanical properties of fibrin fibers.
Societal benefits. 1) The technique will enable investigations of many other nanoscopic fibers, thus enabling new discovery. 2) The proposed investigation of the properties of fibrin fibers will provide an entirely new understanding of blood clots and, thus, heart attacks, strokes, thrombosis and wound healing. This may potentially benefit millions of people who suffer and die from those diseases. 3) The project will provide to theoreticians and network modeler the needed data to construct realistic models of blood clots. 4) Preliminary data showed that fibrin fibers are extremely elastic and extensible, making them attractive candidates for new material science and bioengineering applications. 5) Teaching. The research is conceptually easy to understand and has clear medical implications. It has already been incorporated into three courses at Wake Forest University. Wake Forest University is a dedicated teaching institution, in which teaching is highly regarded. The applicant has received the Reid-Doyle teaching award. The material was used for a Science kids show. 6) Training and education. The project includes a minority Graduate student and undergraduate students. The applicant has a strong track record in including undergraduate students in research.

The primary research objective of this grant is to advance the fundamental understanding of the different physical and mechanical properties of cells as they advance through different stages of neoplastic transformation from normal to the metastatic state. Since recent reports indicate there is significant ambiguity about how these properties change for different cancer cells, the investigators plan to measure these properties for a single line of cells, and to determine whether the changes vary for different cellular components: i.e. whether the change in physical properties is due to a change in the cytoskeleton, the cell membrane, the cytoplasm, or a combination of these elements. Measurements using a wide array of techniques from physics and cellular biology will be applied to the different cellular structures. In addition, the investigators will create a biophysical computer model that brings together all measurements to account for the observed variations in physical properties at each state of neoplastic transformation.

This work will help to establish and disseminate new protocols and techniques for determining the differences in physical properties between cancerous and noncancerous cells. A team of physicists and biologists will train graduate and undergraduate students to work in this highly interdisciplinary field. Particular emphasis will be given to the education of minorities, through the connections to the MARC U* STAR programs of NC A&T University and Winston-Salem State University.