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Record Count: 3
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header (Title, Principal Investigator, Institution, City, ST, Award Code, or
Pubs).
The focus is on cysteine-rich proteins that form metal thiolate
polymetallic clusters. A paradigm of this class is metallothionein (MT).
Polymetallic clusters with distinct properties are induced by Zn (II) and
Cu(I) ions. One goal is to determine the magnitude of structural
reorganization in MT depending on the type of cluster formed. A second
objective is to determine whether similar cluster structure alter the
tertiary fold and function of these proteins. Three classes of molecules
will be studied. First, a novel metallothionein implicated in
Alzheimer's disease will be investigated. The MT, designated as GIF for
Growth Inhibitory Factor, is active in inhibiting dendrite formation in
neurons induced by Alzheimer's brain extracts. The tangled outgrowths
of neurons that is characteristic of Alzheimer's disease may relate to
the low concentration of GIF in Alzheimer's brain tissue. We propose
experiments to determine which metallo-conformer of GIF is active in
reversing the Alzheimer's extract induced proliferation of neurons. A
series of experiments are proposed to map the segment of GIF responsible
for activity. We plan to characterize the metal clusters in GIF to
determine whether sequence differences in MT and GIF affect properties
of the polymetallic clusters. The second class of proteins includes two
fungal transcription factors. ACE and AMT1. Cu(I) binding to ACE! and
AMT1 activates the factors for transcriptional activation of MT genes in
Saccharomyces cerevisiae and Candida glabrata, respectively. We propose
to characterize the Cu(I) thiolate polymetallic clusters in these two
protein conformations. DNA binding sites of CuACE1 and CuAMT1 will be
characterized with the goal of elucidating the structure of the
transcriptionally active CuAMT1/DNA complex. The third class is the
cysteine-rich sequence motif, designated LIM. The metal centers in two
LIM-domain proteins, designated Cysteine-Rich Protein (CRP) and Cysteine-
Rich Intestinal Protein (CRIP) will be studied to determine whether LIM
proteins exhibit metal-induced conformational dynamics. A central
postulate is that the structure and function of these classes of proteins
are affected by the coordination chemistry of the metal centers. We
eventually want to determine the role of specific metal ion binding in
function. Molecules in these three classes exhibit a wide range of
physiological functions from regulation of DNA transcription (ACE1 and
AMT1), metal ion buffering (MT), inhibition of neuron outgrowth (GIF),
protein-protein interaction (CRP) and perhaps metal transport (CRIP).
DESCRIPTION (provided by applicant): The U.S. faces an enormous task in cleaning up hazardous wastes. Bioremediation via wild-type (Wt) and genetically engineered microorganisms (GEMs) has the potential of completely degrading waste material with little or no toxic byproducts. Bacterial adhesion and movement towards contaminants (termed "Chemotaxis") are two important factors that may affect the role of bacteria in the biodegradation of pollutants. However, the fundamental mechanisms governing these factors for wild type (Wt) and genetically engineered microorganisms (GEMs) are still poorly understood and have not been well defined because of the inability to measure basic physico-chemical properties of bacterial chemotaxic behaviors in the presence of chemoattractants. In this project, we propose to develop a whole cell biosensing system to measure chemotaxic behaviors of bacteria in real-time based on a novel design of dual mode electric impedance measurements. The specific aims are: 1) integrated whole cell biochip design and microfabrication that is capable of measuring electric impedance (ECIS) and acoustic impedance (AIA) responses; 2) real-time and simultaneous measurement of adhesion and chemotaxic behaviors of Wt and genetically engineered Pseudomonas putida KT 2440 on the integrated microfabricated working electrode; measurements of morphological (by ECIS), viscoelastic (by AIA), and velocity (by time-lapse video microscopy) properties of Wt and the GEM. 3) comparison of the proposed whole cell biosensing system with currently available chemotaxis detection techniques in terms of sensitivity and versatility; 4) quantification of the chemotaxic behaviors of Wt and GEM (alteration of various chemotaxis genes) in the presence of various chemotaxic substances, and comparison of parameter responses by analysis of the proposed biosensing system; 5) enhancement of the Biological Engineering program at Utah State by addressing the goals of the NIH AREA grant program.
We propose to develop artificial membranes containing nanoscale pores whose mechanical, electrical, and chemical properties are controlled at a nanoscale with organic molecules. The pores will be fabricated in 30-50 nm silicon nitride membranes, and as an integral component of Pt/glass nanopore electrodes. The goal of the proposed work is to covalently modify the inner walls of 5-50 nm wide nanopores with organic molecules whose size and shape are sensitive to external stimuli, such as pH, solvent polarity, pX (where X is an ion or a small molecule), external electric field, light, etc., thus producing responsive nanopores. Specific aims of the project are: (1) to develop the preparation of the nanopores in silicon nitride and in glass to the specified size; (2) to design the molecules for the responsive nanopores, and to optimize
the chemistry needed for their attachment to the nanopore walls; (3) to study the stimulus-response behavior of the attached molecules and their interactions within the confines of the nanopores; and (4) to study the transport dynamics within the resulting responsive nanopores. The pores have long-range applications in stochastic biosensor devices, in separations investigations of biomolecules, and in controlled drug release devices.
The proposed work is a highly innovative, design-driven effort that will lead to the creation and use of nanoscale devices with a host of biological and medical applications. This effort brings together such disciplines as organic synthesis, surface chemistry, analytical chemistry, material science, and electrical engineering.
Crisp Terms/Key Words: acidity /alkalinity, biological transport, biomaterial evaluation, biomaterial development /preparation, biomaterial interface interaction, electric field, lighting, ion, molecular polarity, nitrite, scanning electron microscopy, glass, silicon, stimulus /response, transmission electron microscopy, artificial membrane, atomic force microscopy, nanotechnology, small molecule
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