UMSI 2001 Annual Report: Leonard J. Banaszak, Associate Fellow

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College of Biological Sciences and Medical School Index

Leonard J. Banaszak, Associate Fellow

Structure and Function Studies of Biological Macromolecules

The link between the biological function and the conformation of protein molecules is still not well established. To make the connection and to understand the chemical properties of proteins, this research team studied the conformation of selected protein molecules by macromolecular x-ray crystallography. Such direct conformational determinations by x-ray diffraction methods are computationally intensive and require the use of supercomputer resources. However, the results proved startling in that thousands of connected atoms may be positioned according to their orientation in living organisms. Since each protein molecule has a specific task, studies continued to develop information on the structure: task relationship. Because a human being has somewhere between 30,000 or 40,000 different proteins, structural biology is divided into different areas related to the tasks of the various proteins.

This team of scientists was made up of graduate and undergraduate students, post-doctoral fellows and staff scientists. A variety of studies were undertaken, each focusing on a structure: task problem in biology. In terms of their overall focus, three areas stand out:

1.  Using the structural biology tools, lipid transport and metabolism were studied. Toward this goal, the researchers' efforts focused on understanding the atomic basis for fat transport and breakdown. Not only were molecular structures determined, but also attempts are now being made to redesign proteins that bind lipids, as well as to explain the result of certain genetic disorders.

2.  Many proteins are also classified as enzymes or living chemical catalysts. Once the molecular structure is determined, further studies may reveal the atomic mechanisms by which the chemical reactions are catalyzed and the molecular changes that are necessary to control the catalytic reaction.

3.  In cells found in higher organisms, both in plants and animals, some proteins are synthesized and then must be "directed and translocated" to an appropriate organelle. An organelle is a subparticle within a cell generally responsible for some overall physiological function. For example, chloroplasts in plants contain the proteins responsible for photosynthesis. In animals and plants, mitochondria are organelles responsible for energy production. Each contains a different array of proteins. Using x-ray crystallography, studies were made to determine how different proteins are sorted to their appropriate organelle within the cell.

  In group (1), studies focused on adipocyte lipid binding protein (ALBP), cellular retinoic acid binding protein (CRABPI), lipovitellin (LV), microsomal triglyceride transfer protein complexed with protein disulfide isomerase (MTP:PDI). To illustrate the magnitude of the determination and visualization problem, lipovitellin is a molecule that contains 1500 amino acids of which there are 20 different kinds. The "average" amino acid contains seven atoms not including hydrogens. Hence, to determine the molecular structure, the positions of about 10,500 atoms must be located.

  Still in group (1) the researchers partially accounted for species variation and the accommodation of different ligands in the family of proteins, called the fatty acid binding proteins (ALBP, CRABPI, etc.), In the crystal structure of rat liver fatty acid binding protein (LFABP) and an alignment of amino acid sequences of all known species were used to demonstrate two groups of sub-classes. Based on estimates at neutral pH and the electrostatic field calculated using the crystal coordinates, some evidence of changes that occur in going from holo- to apo-forms was obtained. LFABP, unlike other family members, has two fatty acid binding sites. The two cavity sites were reviewed and arguments for interactions between the sites were developed. Finally, hypothetical models were built of complexes of LFABP and heme, and LFABP and oleoyl CoA. In both cases, the stoichiometry was one to one and the models exhibited why this is likely.

  By changing specific atoms in the overall structure, attempts have been made to understand how two protein homologues may have different binding affinity. For example, one family member may bind fatty acids (ALBP) while another prefers retinoic acid (CRABPI). By careful studies of the molecular structures, attempts have been made to change the properties of one protein into another.

		In the group (2) area, projects on the enzymes malate dehydrogenase (MDH), short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD), phosphoglycerate dehydrogenase (PGDH), isocitrate dehydrogenase (IDH), and isocitrate dehydrogenase kinase/phosphatase (IDHKP) were undertaken. With the goal of understanding the molecular basis for catalytic activity and catabolite control, a number of crystal structures have been determined and refined, taking advantage of the Supercomputing Institute's resource-the Biomedical Supercomputer Laboratory (BSCL). Each study was carried out by a team member and several aspects of the work were totally dependent on supercomputing time. SCHAD offers a good example of how a structural study proceeds. After the initial three-dimensional structure was determined, further studies focused on determining the binding sites for the compounds that are catalytically changed by the protein. A series of x-ray studies showed that a small rearrangement of the conformation took place during a catalytic cycle. It also described the molecular basis for human disorders that result from single site mutations in the enzyme. Enzymes may be regulated in different ways. In the case of the enzyme, IDH, it is inhibited by phosphorylation. The organism senses the need to turn off the catalyst (IDH) and another protein modifies IDH by adding 4 additional atoms. Hence a catalyst containing thousands of atoms is temporarily inhibited by a relatively simple chemical modification. Computational resources of the Supercomputing Institute/BSCL were used extensively in the comparison of the IDH from different organisms. Another enzyme, PGDH, is controlled by binding the end product of the pathway. Studies describing three-dimensional changes that accompany the binding process were nearing completion. Eventually these results will explain a major issue in the control of biological catalysts. In the group (3) category, structural studies focused on the comparison of molecules that may be translocated with those that are not. Again, these protein molecules contain thousands of atoms and the resources of the Supercomputing Institute are in constant demand.
Crystal structure of Bs-isocitrate dehydrogenase: an illustration of the enzyme isocitrate dehydrogenase from B. subtilis with bound citrate and propylene glycol. The dimeric molecule contains over 7500 atoms excluding hydrogen. The ribbon representation simplifies viewing. The catalytic activity of IDH from E. coli is controlled by phosphorylation. Bs-IDH, which is structurally similar to the E. coli homologue, can be made inactive in vitro by E. coli IDH kinase/phosphatase.

Research Group

Joseph Barycki, Research Associate

Jessica Bell, Graduate Student Researcher

Kathryn Bixby, Research Associate

Bryan Cox, Graduate Student Researcher

Paula M. Dalessio, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania

Ed Hoeffner, Staff

Amy Reese-Wagoner, Graduate Student Researcher

Ira J. Ropson, Department of Biochemistry and Molecular Biology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania

Satinder Singh, Graduate Student Researcher

James R. Thompson, Adjunct Faculty

Todd Weaver, Department of Chemistry, University of Wisconsin-La Crosse, La Crosse, Wisconsin

Brian C. Yowler, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania

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