Paul Smith - US grants

The bis(2,6-dioxopiperazine), ICRF-193, is potent non-DNA damaging inhibitor of the decantenation activity of topoisomerase II. The mechanism of inhibition is being interpreted in terms of an ATP-modulated protein-clamp model. ICRF-193 inhibits cell division but allows cell cycle traverse and progression to polyploidy with a delay at the G2 checkpoint. We will use conventional FCM methods to examine cell cycle regulation in conjunction with the various cyclins, including A and B1 and fluorescence lifetime analysis will be performed on cellular-bound ICRF-123 to analyze its interaction with chromatin.

Abstract
IIS-9912077
Smith, Paul A.
University of South Carolina
$35,070 - 4 mos.

This is a standard workshop award. This proposal requests support for a workshop to be held in Columbia, South Carolina, on September 23-25, 1999, to unite researchers in the
area of film and video preservation and digitization with experts in film and video history. The workshop will explore the full scope of material needing preservation and access that are film-based but not traditional major motion pictures. Attendees will include scholars in the humanities, libraries, archives, film history, computer science, and representatives from organizations specializing in film preservation (such as George Eastman House). The workshop would bring together a rather scattered community and look at a new problem, the extension of digitization techniques and digital libraries to
motion picture film outside the context of major studio features.

This project has as its goal the integration of research and educational activities. The research activities involve the determination of thermodynamic binding parameters for a number of biologically important ion-pairing interactions involving ammonium and guanidinium groups. These determinations will be made in aqueous solution using a series of cationic cyclodextrin derivatives and appropriate anionic guests. Methods employed for these studies will include titration calorimetry, NMR spectroscopy and molecular modeling.

With this Award, the Organic and Macromolecular Chemistry Program supports the research and educational activities of Professor Paul J. Smith of the University of Maryland Baltimore County. Professor Smith will integrate research and education by involving students in the study of chemical problems that are at the interface between chemistry and biology. The research exploring the binding of biologically important ions will indeed have important ramifications in biochemistry and in molecular design.

Smith, Paul E.
MCB-0090483

Cosolvents can have dramatic effects on the properties of peptides and proteins in solution. They are often used to enhance the stability of proteins or to denature them in order to study protein folding. Exactly how cosolvents affect peptides and proteins is unknown. An understanding of these effects will greatly improve our current ideas concerning the nature of the denatured state of proteins. In turn, this will help to clarify the protein folding process by providing a well defined initial state from which one can rationalize the effects of temperature or denaturants. Experimental studies of the denatured states of proteins are limited due to the many conformations available to the protein, and an inability of techniques to determine the interactions between proteins and cosolvent molecules with atomic level resolution.

In principle, computer simulations can provide atomic level resolution of denatured proteins and their interactions with cosolvents. However, computer simulation experiments
are only as reliable as the force fields which describe the interactions between the different species. In this proposal, it is shown that current cosolvent force fields need to be improved
in order to quantitatively reproduce the experimental properties of cosolvent mixtures with water. A combination of molecular dynamics simulation and the Kirkwood-Buff theory of mixtures will be used to provide sufficient data for comparison with experiment, and to ensure the quality of the force field. Both effective pair potentials and polarizable models will be investigated for sodium chloride, guanidinium chloride, urea, and 2,2,2 trifluoroethanol; which represent common cosolvents displaying a range of effects on peptides and proteins. The improved force fields will then be used to fully characterize, with atomic detail, the properties of these solutions. This is the first step in determining their different effects on proteins, and is absolutely essential if one is to have any confidence in computer simulation results for these systems.

The aim is to emulate the reaction processes used in nature for lignin polymerization, the setting of mussel glue, and insect shell hardening. These polymerization and curing processes are enzyme-initiated and then they progress in a "domino" fashion. In this project, it is proposed to use tyrosinase to convert natural phenolic precursors into reactive o-quinones, which can then serve as agents for polysaccharide crosslinking and protein-polysaccharide coupling. To elucidate more mechanistic details, a combined biochemical and mechanical screening program has been devised.

It has been established that peptide and protein aggregation lies at the heart of many diseases. Hence, a detailed understanding of exactly when and how peptides or proteins tend to aggregate would be beneficial to a wide range of researchers studying a variety of health related issues. This proposal outlines a program for the systematic study of peptide aggregation using a unique combination of approaches including: currently available experimental thermodynamic data;the theory of preferential interactions as characterized by Kirkwood-Buff (KB) integrals;a simple model for peptide aggregation using preferential interactions between different functional groups in solution;and computer simulation. A theory and model is developed and demonstrated that can decompose and quantify the interactions between different amino acid side chains using existing experimental data on activity coefficients of small peptides, thus enabling predictions of the tendency for aggregation of any small peptide. Computer simulations are proposed to investigate the atomic level details of the aggregation process. A new peptide and protein force field (KBFF) that can reproduce the experimental KB integrals will be completed and used for the simulations. Aim 1. To quantify and characterize the interactions between functional groups observed in peptides. Analysis of existing experimental data will be performed in aqueous solution to determine preferential interaction (PI) parameters for different amino acid and small peptide systems. A simple model of the Pis will be developed and will then be used to isolate and quantify the Pis between different function groups on those peptides. Aim 2. To produce an accurate force field specifically designed for the study of preferential interactions in biomolecular systems. The KBFF approach will be extended to include all amino acid side chains. Aim 3. To understand the role of cosolvents in modifying intermolecular interactions. The addition of cosolvents to a solution of a solute and solvent changes the interactions between the solute molecules. This provides a tool for investigating the strength of intermolecular interactions common in biological systems and how they may be modified. We will focus on the effects of urea and NaCI during our initial studies.

? DESCRIPTION (provided by applicant): Studies of protein denaturation provide critical information concerning the forces that stabilize protein structure and other assemblies. This has a significant effect on our general understanding of the structure/function aspects of proteins that will directly impact our understanding of many diseases that are related to protein unfolding, misfolding, and aggregation. While the vast majority of studies have focused on the native state of proteins, the role of the denatured state is of equal importance. It is now generally accepted that the denatured state ensemble (DSE) is not random in nature but can possess residual native and non-native structure. While our knowledge of the properties of the native state has been advanced by a variety of experimental and simulation results, our understanding of the denatured state remains somewhat simplistic primarily due to the difficulties obtaining atomic level data from experiment, and our inability to determine the thermodynamic properties of the DSE from simulation. It is proposed that a combination of recent theoretical developments using Fluctuation Solution Theory, coupled with computer simulation approaches, can provide reliable data concerning the similarities and differences between denatured states generated by changes in pressure, temperature and composition at the residue level. There are three major aims to the proposed project. Aim 1: To Determine Residue Based Contributions to Protein Thermodynamics. Aim 2: To Determine the Thermodynamic Properties of Amylin and Mutant Amylin DSEs. Aim 3: To Determine the Effects of Environment on the Thermodynamic and Aggregation Properties of Small Peptide Sequences Derived from Amylin. The results of these studies will provide valuable information concerning the nature of the denatured state and the consequences for the role the DSE may play in a variety of diseases.