Arthur Glasfeld - US grants

The computer acquired through this project was a Silicon Graphics 4D/25G graphics workstation with 12 MB of memory and two 380 MB hard disk drives. The graphics and calculational capabilities of the computer are being used to support two biochemistry courses, a course in computational chemistry, and the senior thesis program. The workstation filled a gap that existed between what the chemistry undergraduates were taught and modern chemical research. The rapid growth of information regarding molecular structure in biochemistry has given rise to the need for a more effective method of representing large molecules in three dimensions than was available at the institution. The graphics workstation provided such a method. Using available software packages, students are introduced to the intricacies of macromolecular structure throughout the one year biochemistry course. An additional need arose from the lack of instruction that undergraduates received in computational chemistry. The acquisition of the workstation permitted the development of a course designed to teach the theory and practice of molecular calculations. To teach such a course effectively, the Chemistry Department required a more powerful computer than was available. The Silicon Graphics workstation provided not only the necessary processing power, but also the advantage of graphics capabilities to assist in the presentation and interpretation of results from sample calculations. The senior thesis program also made extensive use of the graphics workstation. Student projects that were affected included site- directed mutagenesis studies, enzyme inhibitor design and mathematical modelling of the relationship between structure and mutagenicity. The institution contributed in an amount slightly in excessof the NSF funds.

isomerase; enzyme mechanism; carbohydrates; carbohydrate metabolism; arabinose; biotransformation; fucose; genetic manipulation; genetic strain; biochemical evolution; sugar phosphates; hydrogen transport; enzyme substrate; radiotracer; Escherichia coli; mutagens; nuclear magnetic resonance spectroscopy; Salmonella typhimurium; site directed mutagenesis;

Organic chemistry and biochemistry students will construct, manipulate, and analyze computer generated visual models of chemical systems on a frequent and routine basis. These exercises will make use of computer-based molecular modeling tools and compatible chemistry modeling software. The workstations will enable faculty to lead modeling sessions for large groups of students, and will ensure easy access for students engaged in independent modeling exercises. This project is unique because it does not view computational chemistry as an end in itself. Rather, state-of-the-art computational techniques will be used to develop student "chemical intuition", and to provide a means for employing modeling, with an emphasis on structure visualization and analysis, as a learning activity.

The project involves purchase of a powder diffractometer, a camera station, and the computer hardware and software necessary for their operation. The instrumentation will be used to establish a modern x-ray diffraction facility which will enhance students' practical experience, strengthen and expand existing programs at the college, and improve the breadth of available laboratory experience for student research. It will be used in both Chemistry and Physics laboratory courses that range from introductory experimentation to advanced student research projects, and will impact virtually every science major at the College.

With this award from the Major Research Instrumentation (MRI) Program, the Department of Chemistry at Reed College will acquire a 400 MHz nuclear magnetic resonance (NMR) spectrometer. This equipment will enable researchers to carry out studies on a) enzyme mechanisms through magnetization tansfer; b) synthesis of transition-state mimics; c) natural product synthesis; and d) mechanistic and structural studies of sugar isomerases.

Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful tool available to chemists for the elucidation of the structure of molecules. It is used to identify unknown substances, to characterize specific arrangements of atoms within molecules, and to study the dynamics of interactions between molecules in solution. Access to state-of-the-art NMR spectrometers is essential to chemists who are carrying out frontier research.

With support from the Major Research Instrumentation (MRI) Program, the Department of Chemistry at Reed College will acquire an isothermal titration calorimeter. This equipment will enhance research in a number of areas including a) the determination of the affinity of metal ion-dependent regulatory proteins for cognate and non-cognate metal ions and the stoichiometry of combination; b) studies on enzyme mechanisms; and c) investigations into biologically active analogs of the polypeptide antibiotic bacitracin.

Calorimetry is an extremely useful tool in modern analytical chemistry. Calorimetric analysis is used to measure the thermal energy (heat) exchange that occurs during molecular interactions and reactions. Thus it can provide a very reliable and sensitive method for determining the thermodynamic properties of materials such as changes in heat capacity of liquid and solid samples. Reed College is an undergraduate institution, and the availability of these instruments will have a strong impact on undergraduate research.

Manganese is an essential nutrient for all organisms and is a limiting nutrient for many pathogenic bacteria, which respond to low manganese concentrations by producing proteins associated with virulence. Metalloregulatory proteins such as the streptococcal cell adhesion regulator (ScaR) from Streptococcus gordonii and manganese transport regulator (MntR) from Bacillus subtilis are responsible for mediating the cellular response to varying manganese concentrations by binding DNA at cognate operator sequences. ScaR and MntR are related by sequence similarity and function to the diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae, which is an iron-dependent regulatory protein. The work proposed here is directed at understanding how ScaR and MntR bind manganese specifically over iron, despite their structural similarities to DtxR and the chemical similarities between manganese and iron. Also, it is of interest to determine how the manganese activates MntR and ScaR for DNA binding. A variety of biophysical techniques will be used. X-ray crystallography will be the principal technique, and the primary aims of this proposal are to determine the crystal structures of MntR and Scar alone, bound to metal ions and in ternary complexes with manganese and DNA. In addition, quantitative studies will be performed to measure the affinity and specificity of MntR and ScaR, and site-directed mutants of these proteins, to their cognate metal ions and operator sequences. In this way, a fuller description of the activation and specificity of ScaR and MntR will be obtained.

[unreadable] DESCRIPTION (provided by applicant): In many bacteria, the production of transporter proteins involved in manganese uptake is under the control of manganese-activated transcriptional regulators. The streptococcal cell adhesion regulator (ScaR) from Streptococcus gordonii and manganese transport regulator (MntR) from Bacillus subtilis are specifically activated for DNA-binding by manganese. The mechanism of these proteins can be separated into two features: (1) the specific recognition of manganese, and (2) the transition from an inactive to active form upon manganese binding. The goal of this work is to define the underlying molecular mechanisms by which MntR and ScaR are able to selectively recognize and respond to manganese as an allosteric effector. X-ray crystallography, spectroscopic techniques, including fluorescence spectroscopy and EXAFS, and solution binding assays will be used to investigate the interaction of MntR and ScaR with metal ions. Site-directed mutagenesis will be used to create variants of these proteins, which will in turn be evaluated for changes in conformation and metal-binding properties. Manganese is an essential nutrient for all organisms and can be a limiting nutrient for many pathogenic bacteria, including S. gordonii, which respond to low manganese concentrations by producing proteins associated with virulence. Metalloregulatory proteins, such as MntR and ScaR, that control production of these virulence factors may be useful targets for antibiotic development. A deeper understanding of their structure and function will be important in efforts to disrupt manganese homeostasis in bacteria. Manganese is an essential nutrient for many disease-causing bacteria, and disruption of manganese uptake into the cell can have significant effects on virulence. This research is intended to provide a molecular-level understanding of how bacteria specifically recognize and respond to varying levels of manganese. That understanding may open the way to future antibiotic development. [unreadable] [unreadable] [unreadable]

This project provides scholarships to able and financially needy students majoring in chemistry, physics, biology, mathematics, and interdisciplinary fields. The project emphasizes supporting students through formal and informal interactions with faculty and among students, seminars, and an opportunity for dedicated funds for their thesis research project, covering supplies and/or travel, when they become seniors.

Intellectual Merit: The academic programs into which the students go are strong, and there are academic support activities. The project targets attrition between the first and second year of college, and it provides incentives for students to declare and remain in a STEM major. The project builds on a successful scholarship project that is just being completed.

Broader Impact: The project is increasing the number and diversity of students who complete a STEM major and go on to work in the field or to further education.