Molecular Forces Determining the Strength of Receptor-mediated Cell Adhesion
D. Leckband,* T. Calvert
National Science Foundation, BES-9503045

Cell adhesion is mediated by contacts between chemical moieties on cell surfaces. In particular, the enhanced expression of certain glycolipids on cancer cells may determine their metastatic potential. The significance of specific glycolipid interactions, however, is linked directly to the strengths of the molecular forces governing the resulting adhesion. We are using direct force measurements, fluorescence microscopy, and light scattering to quantify the magnitudes and ranges of glycolipid-mediated adhesive forces and to determine the impact of those forces on the strengths of glycolipid-mediated membrane attachments. We are testing directly the role of membrane surface components in cell adhesion and the potential utility of therapeutics designed to block their interactions.

Biosensor Design and Performance - The Role of Transducer Surface Composition
D. Leckband,* R. Vijayendran, N. Lavrik
U.S. Office of Naval Research, N00014-96-1-339

Many biosensor designs are based on the selective binding of soluble analyte to immobilized receptors. The surface microenvironment can, however, significantly affect sensor performance. We are currently quantifying changes in protein-binding strengths in response to interfacial perturbations. Kinetic modeling of site-selective adsorption data have demonstrated that subtle interfacial perturbations impact sensor performance. Our objective is to determine the molecular basis of altered protein function by the surface microenvironment and to optimize sensor performance through the tailored manipulation of the transducer surface properties.

Molecular Basis of Protein Interactions
D. Leckband,* T. Calvert, S. Sivasankar, C. Yeung, A. Kloss
National Institutes of Health, 1R29GM51338

Protein surface topology plays a major role in modulating the rates of protein-binding events. We are using direct force measurements to probe the impact of local protein structural motifs on the forces that control the rates of protein collisions. In particular, we are investigating the impact of surface charge distributions and protein orientation on protein electrostatic surface properties and the resulting protein interactions. Use of both wild type and engineered proteins permits precise control of the surface region probed. Measurements are compared with theoretical calculations. The functional implications of these findings are being investigated by Brownian dynamics simulations and kinetic measurements.

Poly(ethylene oxide) Interactions with Proteins
D. Leckband,* N. Efremova, S. Sheth, M. Grunze (Heidelberg)
NATO Collaborative Research Grant

The unique biological activity of poly(ethlyeneglycol) is believed to be a function of intramolecular structure of the polymer backbone and of its unique interactions with water. This research program combines direct surface force measurements with several surface spectroscopies to elucidate the relationship between grafted polymer chains and short oligomers on surfaces, the structural content of the chains, and the interfacial properties that determine how these materials interact with the biological environment.

Chemical Communication between Cells and Engineered Bioscaffolds
D. Leckband,* B. Wheeler (Elect. & Comput. Engr.); T. Eurell and D. Gross (Vet. Biosciences)
U of I Campus Research Initiative

Patterned bioscaffolding materials are being developed for the directed growth of hippocampal neurons and endothelial cells. This cross-campus, interdisciplinary effort is aimed at controlling cell communication with engineered materials in order to direct cell growth and sustain function in artificial environments.

Surface Display of Antibodies in Yeast for Affinity Maturation
K. D. Wittrup,* E. T. Boder, J. V. Antwerp
Whitaker Foundation

A novel system has been developed to engineer proteins to have desirable binding properties. Genetic fusions to a cell wall protein allow the protein of interest to be tethered to the cell surface and probed for binding to fluorescently labeled targets. Introduction of diversity into a population by mutagenesis is followed by isolation of desirable mutations by flow cytometry and sorting.

Engineering Disulfide Formation Kinetics to Enhance Heterologous Secretion in Saccharomyces cerevisiae
K. D. Wittrup,* R. Raines, E. V. Shusta, W. S. Kwon
National Science Foundation, BES 95-31407

The purpose of this work is to measure and manipulate the redox regulation of yeast's secretory pathway in order to improve production yields of pharmaceutical proteins. Genetic, biochemical, and fermentor operation strategies will be implemented to alter the cellular processing of disulfides, covalent crosslinks which stabilize the folded structure of a protein.

Protein-folding Kinetics in the Endoplasmic Reticulum
K. D. Wittrup,* J. M. Kowalski, S. J. Bannister
National Institutes of Health, GM50673

The goal of this project is to examine the kinetics of protein processing in the secretory pathway in order to identify the mechanistic steps which limit the yield and rate of protein secretion. The ultimate goal is to use this information to rationally design improved production systems for phamaceutical proteins.