last updated 7/3/06
The incredible complexity of biological systems derives to a large extent from the high degree of interactions amongst their constituent components. As such, the cell is often described as a complex circuit consisting of an interacting network of molecules. A key component of these networks are protein switches that serve to couple cellular functions. A switch recognizes an input signal (e.g. ligand concentration, pH, covalent modification) and, as a result, its output signal (e.g. enzyme activity, ligand affinity, oligomeric state) is modified. Examples of natural switches include allosteric enzymes which couple effector levels to enzymatic activity and ligand-dependent transcription factors that couple ligand concentration to gene expression. Creating artificial protein switches is an important goal of chemical biology. The ability to create novel switches or to modify existing switches by coupling hitherto uncoupled functions would enable the rewiring of the cellular circuitry to our own design. In addition, the ability to create protein switches has tremendous practical potential for developing novel molecular sensors, smart materials, and as a tool for elucidating molecular and cellular function.
We have developed a general approach for creating switches in which one selects from natural or engineered proteins with the desired input and output functions and, by combining the proteins in a systematic fashion, creates switches in which their functions were tightly coupled. Such an approach is inspired by the evolutionary mechanism of domain recombination – a major facilitator in the natural evolution of protein function. We reasoned that a diverse exploration of fusion geometries between two proteins would enable the creation of switches with superior properties. The structural space that we sought to explore can be conceptualized as rolling the two proteins across each others surface and fusing them through peptide bonds at the points where their surfaces meet. We developed a novel, homology-independent, combinatorial method for recombining genes that samples such a structural space. We have utilized this strategy to combine the enzyme TEM-1 β-lactamase and the ligand-binding protein maltose binding protein and create a family of allosteric β-lactamases that are modulated by maltose.
The major goals of our research program on molecular switches are (1) to create protein molecular switches through a combination of chemical and biological approaches that include molecular evolution and protein design, (2) to elucidate the mechanism of the switches created and (3) to develop switches for applications. For example, coupling a ligand-binding protein and a protein with good signal transduction properties would result in the creation of a molecular sensor for the ligand. Furthermore, switches that establish connections between cellular components with no previous relationship can result in novel cellular circuitry and phenotypes. We envision, for example, that such switches might establish connections between molecular signatures of disease (e.g. abnormal concentrations of proteins, metabolites, signaling or other molecules) and functions that serve to treat the disease (e.g. delivery of drugs, modulation of signaling pathways or modulation of gene expression) and therefore possess selective therapeutic properties.
*Kim, J. R. and M. Ostermeier. (2006) "Modulation of effector affinity by hinge region mutations also modulates switching activity in an engineered allosteric TEM1 β-lactamase." Arch. Biochem. Biophys. 446, 44-51. [PubMed] [PDF]
*Ostermeier, M. (2005) "Engineering allosteric protein switches by domain insertion." Protein Eng. Des. Sel. 18, 359-364. [PubMed] [PDF]
*Guntas, G., T. J. Mansell, J. R. Kim, and M. Ostermeier. (2005) "Directed evolution of protein switches and their application to the creation of ligand-binding proteins." Proc. Nat. Acad. Sci. USA 102, 11224-11229. [PubMed] [PDF]
*Guntas, G., S. F. Mitchell and M. Ostermeier. (2004) "A molecular switch created by in vitro recombination of non-homologous genes." Chem. Biol. 11, 1483-1487. [PubMed] [PDF] [commentary]
*Guntas, G. and M. Ostermeier. (2004) "Creation of an allosteric enzyme by domain insertion." J. Mol. Biol. 336, 263-273. [PubMed] [PDF]
Johns Hopkins University press release (11/29/04) on our protein molecular switch research.
An important goal of chemical biology is the ability to modulate the function of biological molecules and pathways. One attractive strategy for modulating protein function is through use of small molecule chemical inducers of dimerization (CID). This strategy utilizes a CID to induce proximity of two proteins (A and B) through facilitating the dimerization of domains attached to Proteins A and B. If bringing Proteins A and B together modulates a function then the CID can be used to control that function in a dose-dependent manner. CIDs have been used to initiate signaling pathways by dimerizing receptors on the cell surface, to translocate cytosolic proteins to the plasma membrane, to import and export proteins from the nucleus, to induce apoptosis, to regulate gene transcription and to induce protein splicing. However, this strategy can only be applied to those proteins whose function is modulated by changes in the oligomeric state or proximity of two or more proteins.
Inspired by enzymatic two-hybrid systems (called PCAs) for identifying and interrogating protein-protein interactions, we are exploring a strategy for engineering control of monomeric proteins. In this strategy a monomeric protein is split into two fragments that cannot assemble into an active protein unless protein domains attached to these fragments associate. By controlling the association of these 'conditional heterodimers' we hope to control the protein's function. We have developed a structure-independent, systematic method of creating conditional heterodimers with a large difference in function between the "on" and the "off" state. This was achieved through incremental truncation, a method for creating a library of every one-base truncation of a gene. We have have also found that it is possible to split an enzyme and create an enzyme that has wildtype abilities in vivo. In fact, the catalytic activity of one of these heterodimers exceeded that of the parental enzyme by 18-fold (see figure). Kinetic characterization of this conditional heterodimers demonstrated that the effects of bisection and mutations can be highly non-additive and, quite surprisingly, that bisection and reassembly alone can result in significant (140-fold) improvements in enzymatic activity.
*Paschon, D., Z. S. Patel and M. Ostermeier. (2005) "Enhanced catalytic efficiency of aminoglycoside phosphotransferase (3')-IIa achieved through protein fragmentation and reassembly." J. Mol. Biol. 353, 26-37. [PubMed] [PDF]
*Choe, W., S. Chandrasegaran and M. Ostermeier. (2005) "Protein fragment complementation in M.HhaI DNA methyltransferase." Biochem. Biophys. Res. Commun. 334, 1233-1240. [PubMed] [PDF]
*Paschon, D.E. and M. Ostermeier. (2004) "Construction of protein fragment complementation libraries using incremental truncation." Methods Enzymol. 388, 103-116. [PubMed]
The creation of proteins with desired properties is an important goal of biotechnology. Owing to the difficulties of a purely rational design approach, the use of combinatorial methods and molecular evolution to engineer proteins with desired properties has increased dramatically. Central to these combinatorial strategies are (a) methods to construct combinatorial DNA libraries and (b) methods that facilitate identification of those rare proteins with desired properties in these large libraries. Both of these areas are under active research in our group.
*Durai, S., A. D. Bosley, A. B. Abulencia, S. Chandrasegaran and M. Ostermeier. (2006) "A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions." Combinatorial Chemistry and High Throughput Screening 9, 301-311. [PubMed] [PDF]
*Bosley, A. D. and M. Ostermeier. (2005) "Mathematical expressions useful in the construction, description and evaluation of protein libraries." Biomol. Eng. 22 57-61. [PubMed] [PDF]
*Ostermeier, M. (2003) "Synthetic gene libraries: in search of the optimal diversity." Trends Biotechnol. 21, 244-247. [PubMed] [PDF]
*Ostermeier, M. (2003) "Theoretical distribution of truncation lengths in incremental truncation libraries." Biotechnol. Bioeng. 82, 564-577. [PubMed] [PDF]