Welcome to Milan Mrksich's Group and the
Laboratory for BioInterface Science and Engineering
Overview. My group’s interests overlap chemistry, biology and engineering, with an emphasis on the design and synthesis of materials that are biologically active and in applications of the materials to relevant problems in the biological and medical sciences. Much of our work uses self-assembled monolayers of alkanethiolates on gold to prepare model surfaces that are structurally defined, yet that can have complex compositions and present the ligands in spatially-organized patterns. We pioneered the design of ‘dynamic substrates’ that present ligands whose activities can be switched on and off in response to electrical or optical signals, particularly for studies that address the responses of adherent cells to changes in the extracellular matrix. These mimics of the extracellular matrix have led the way to the discovery of novel ligands that mediate cell adhesion. We have also developed robust surface chemistries for preparing biochip arrays and that are compatible with new analytical methods for analyzing the arrays. For example, we have developed the SAMDI method, which uses mass spectrometry to analyze the arrays, and we have extended this method to the first label-free approach for high throughput screening, to the functional annotation of recently sequenced genes and towards an understanding of the networks that regulate protein acetylation. Finally, a recent program is creating defined systems for exploring biochemical reactions to understand the role that localization of enzymes and substrates play in controlling reaction networks.
Mimics of the Extracellular Matrix. Most cells are adherent and must attach to and spread on a protein matrix in order to survive, proliferate and control signaling processes. This extracellular matrix comprises several glycoproteins and glycosoaminoglycans that present a variety of ligands that interact with cell-surface receptors to mediate adhesion and intracellular signaling pathways. In a well-established example, extensive work has shown that the family of integrin receptors interacts with peptide ligands in the matrix and is central to maintaining cell adhesion and coordinating mechanical properties of the cell. The identification of new ligands from the matrix and the cell-surface receptors with which they interact, followed by understanding the signaling events that are triggered by these interactions, remains a difficult aim.
We have advanced the use of self-assembled monolayers to create mimics of the extracellular matrix. These surfaces allow excellent control over the composition and densities of peptide and protein ligands and are also designed to prevent non-specific adsorption of proteins, giving excellent control over the ligand-receptor interactions that operate between the cell and substrate. This approach has led to several findings of new ECM ligands for cell surface receptors. For example, we found that the platelet receptor, in addition to binding the canonical RGD ligand, binds peptides having the arginine residue replaced by hydrophobic residues. This finding points to a new strategy for the development of anti-thrombotic agents, since the AGD peptide can selectively inhibit the aggregation of platelets by fibrinogen without disrupting the adhesion of endothelial cells. In another example, we used peptide arrays to investigate the basis for a8b1-dependent adhesion to the protein nephronectin and found that the FEI tripeptide is a selective ligand for the a8 integrins. We have also used monolayers to characterize the influence of matrix ligands on cellular activities. In one example, we showed that monolayers presenting a high affinity ligand promote osteogenesis of a mesenchymal stem cell culture, but monolayers having a low density of ligand promote an adipogenesis program. This switch stems from the ability of high affinity ligands to sustain greater forces applied by the acto-mysoin cytoskeleton. In related work, we have shown how the shapes of cells can be engineered—using monolayers that are patterned with adhesive ligands—to promote contractility, and therefore osteogenesis, in adherent cells. Together, these examples show how the use of molecularly-defined substrates can be used to identify ligand-receptor interactions that mediate adhesion and to understand the roles these interactions play in regulating cell behavior.
BioChips. The development of oligonucleotide arrays, and the ability to globally profile gene expression in cells, has transformed the study of biological processes. The success of DNA arrays has motivated considerable work over the past decade to develop arrays comprising other classes of biomolecules, including peptides, proteins, oligosaccharides, and small molecules. Yet, these applications have proven significantly more challenging and still await a robust technology for profiling biochemical activities. One challenge stems from the difficulty in immobilizing molecules and ensuring that they are all active, present at a uniform density across the array, and not confounded by non-specific interactions of the sample with the biochip. We have developed self-assembled monolayers as a platform for preparing arrays that address these challenges and have shown that they permit quantitative assays of biochemical activity. A more significant limitation stems from the challenges in detecting the products of enzyme-mediated reactions of the immobilized molecules. The conventional approaches use fluorescent or radioisotopic labels—sometimes in conjunction with antibodies—to observe the products of a biochemical reaction. The labels can interfere with the enzyme activity and give false positive or negative results, can lead to substantial development times for new assays, and prevent the identification of unanticipated activities.
We have made a significant contribution to this challenge by developing a ‘label-free’ approach to characterizing biochip arrays. We found that the monolayers could be characterized directly with matrix-assisted laser desorption-ionization mass spectrometry. When the monolayers are irradiated with the laser the bond between the sulfer atom and the gold substrate is cleaved, releasing the full alkanethiolate (or the analogous disulfide) into the gas phase where its mass can then be detected. We have shown that this ‘SAMDI’ technique provides a straightforward measure of a broad range of enzyme activities—including kinase, protease, methyltransferase, ligase, and others—using a single format that relies on detection of the reaction products according to their change in mass. By avoiding the use of labels, SAMDI is compatible with enzyme substrates that have a greater relevance to endogenous substrates, is applicable to enzyme activities that are otherwise difficult to measure in label-dependent formats, and has the benefit that it can identify unanticipated activities. We have applied the SAMDI method to functionally annotate recently sequenced genomes. For example, with the Wang group, we identified eighty putative glycosyltransferases, which were expressed and tested against seven donors against an array presenting twenty five acceptors. This profiling experiment identified several new glycosyltransferases, including one that catalyzes a linkage for which enzymes haven’t been available. In a second example, we have used SAMDI to analyze the activity of lysine deactylases on arrays presenting four hundred peptide substrates. We have profiled most of the deactylases and have used the arrays to identify peptides that are selective substrates for several of the deacetylases. These substrates have enabled the profiling of deacetylases in cell lysates and nuclear extracts and represent a first example of measuring directly the endogeneous enzyme activities.