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Leonard J. Banaszak, Associate Fellow

Structure and Function Studies of Biological Macromolecules

Proton binding curve for HEW lysozyme. The graph illustrates the correlation between the experimental and evolutionary derived titration curves for lysozyme. The solid continuous curve is generated using the result of the theoretical pKs and the Henderson-Hasselbalch relationship. The pKs derived from these computations determine the electrostatic field in and around the molecule. Although this is a model study, its wider application to other disease related proteins may have positive effects in the fields of drug design and other biotech areas.

Research Group

Joe Barycki, Research Associate
Jessica Bell, Graduate Student Researcher
Kathryn Bixby, Research Associate
Kent Brown, Graduate Student Researcher
Bryan Cox, Graduate Student Researcher
Jessica Flynn, Undergraduate Student Researcher
Ed Hoeffner, Staff
David T. Mitchell, Research Associate
Jeramia Ory, Graduate Student Researcher
Amy Reese, Graduate Student Researcher
Satinder Singh, Graduate Student Researcher
Jim Thompson, Adjunct Faculty Collaborator
Todd Weaver, Department of Chemistry, University of Wisconsin, La Crosse, Wisconsin

These studies involve biochemistry, molecular biology, and X-ray crystallography for structure determination and functional characterization of proteins. Supercomputing resources are used for X-ray crystallographic determination and refinement of several protein structures. The structural studies include adipocyte lipid binding protein (ALBP), cellular retinoic acid binding protein (CRABPI), liver fatty acid binding protein (LFABP), lipovitellin, and microsomal triglyceride transfer protein (MTP). Projects on enzymes [malate dehydrogenase, isocitrate dehydrogenase, short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD), and phosphoglycerate dehydrogenase] are also being pursued.

The research has already lead to a number of X-ray structures of intracellular lipid binding proteins. The goal of this study is to carry out in depth structure/function studies via protein engineering so that the ligand specificity within this protein family is definable by a systematic set of chemical rules. Possible reasons for the low lipid specificity of LFABP have already been suggested. Current work is attempting to obtain crystals of the apo-protein and other ligands bound to the protein.

The understanding of ligand specificity in ALBP and CRABPI is already greatly increased with the completion of several crystal structures-both in apo-form and in complex to different lipids. Structural data and site-directed mutagenesis is used to convert fatty acid specificity of ALBP to a high-affinity retinoic acid site, imitating CRAPBI's unique retinoid specificity. To date, three data sets of one mutant has been collected and the models are being refined.

The crystal structure of MTP, a heterodimeric polypeptide involved in transport of neutral lipids and phospholipids between membranes, is being solved. It is required for the assembly of plasma, very low density lipoproteins in the liver, and chylomicrons in the intestine. The smaller subunit of MTP has been identified as protein disulfide isomerase. The precise in vivo roles of the two subunits are not yet clear. A domain of MTP has sequence similarity to that of a highly helical segment of the lipovitellin lipoprotein.

A complete X-ray structural analysis of lipovitellin, a lipoprotein containing 30-35 molecules of bound phospholipid per monomer, has been undertaken. Lipovitellin is a large protein whose function is the storage and transport of lipids. The lipid is bound in a central cavity surrounded by a tear-drop shaped series of beta-sheets along the sides of the lipid moiety. There appears to be electron density present in the current maps due to lipids and chemical composition analysis of the lipid portion. Since complete sidechain assignments for the lipid-lining beta-sheets are available, current work lies in modeling lipid molecules visible in the electron density map obtained from crystals at 100K.

Phosphoglycerate dehydrogenase (PGDH) catalyzes the first committed step in the serine biosynthetic pathway. Serine, an amino acid, regulates the rate at which this enzyme works by binding to one of the three domains that are part of a single subunit. Using recombinant techniques, the serine binding domain has been purified separately from the remaining two domains. The two domain protein contains the unregulated active site. By studying the molecular structures of these now separate pieces, the hypothesis is that it will become possible to piece together the molecular mechanics of PGDH regulation.

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Isocitrate dehydrogenase (IDH) kinase/phosphatase is a bifunctional regulatory enzyme of the glyoxylate bypass in E. coli that inactivates isocitrate dehydrogenase via reversible phosphorylation at serine 113 when the organismÕs sole source of energy is limited to acetate or some other 2-carbon-containing compound. Kinetic and photoaffinity labeling experiments have suggested that the kinase, phosphatase, and perhaps even the ATPase activities are all located at a single catalytic site. In a parallel project, examination is being done on the substrate specificity of the enzyme for B. subtilis isocitrate dehydrogenase. Kinetic experiments have shown the amino acid sequence around the serine phosphoryation site is identical to that of the E. coli homologue, but the B. subtilis version is an extremely poor substrate of E. coli kinase/phosphatase. Thus, the crystal structure of B. subtilis IDH, which is near completion, might shed light on the arrangement of active site amino acids in the kinase/phosphatase.

This information is available in alternative formats upon request by individuals with disabilities. Please send email to alt-format@msi.umn.edu or call 612-624-0528.

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