HER receptor dimerization controlled with bivalent ligands

In this thesis, I show the ability of engineered bivalent ligands to control HER signaling by taking advantage of the dimerization-dependent mechanism of the four different receptors in the HER (EGFR) family. While monovalent wild-type ligands form HER homoe- and hetero-dimers according to the different members' relative expression levels, I hypothesize that bivalent ligands are able to drive dimerization of specific family members, independently of the cell's HER receptor composition. Using human telomerase reverse transcriptase (hTERT)-immortalized human mesenchymal stem cells (hTMSC), this study first confirms the bioactivity of the bivalent ligands: they exhibit an EC50 one order of magnitude lower than wild-type monomeric ligands and shown an avidity effect as shown from dose-response analysis of the pERK and pY-EGFR nodes. Then, I take advantage of known differences between EGFR hetero- and homodimer to investigate the bivalent ligands' ability to form selective dimers. I demonstrate that bivalent EGF is capable of inducing greater EGFR phosphorylation while preventing HER2 phosphorylation compared to wild-type EGF stimulation. Furthermore, the kinetics of EGFR activation by bivalent EGF differs from wild-type EGF with the appearance of a second activation peak at 20 minutes. Controlling the HER signaling network with these bivalent ligands has potential applications in tissue engineering, cancer therapy and in fundamental studies of the ErbB receptors activation mechanis

Protein engineering design principles for the development of biosensors

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015.Cataloged from PDF version of thesis.Includes bibliographical references.Investigating protein location and concentration is critical to understanding function. Reagentless biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can detect analytes of interest with high temporal and spatial resolution. However, because these biosensors require laborious empirical screening to develop, their adoption has been limited. Hence, we establish design principles that will facilitate development. In this thesis, we first develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. Following these guidelines, we use the human tenth type III fibronectin domain to engineer new binders against several ligands of the EGFR receptor. Using these binders and others, we design and characterize biosensors based on various target analytes, scaffolds and fluorophores. We observe that analytes can harbor specific binding pockets for the fluorophore, which sharply increase the fluorescence produced upon binding. Furthermore, we demonstrate that a fluorophore conjugated to locally rigid surfaces possesses lower background fluorescence. Based on these newly identified properties, we design biosensors that produce a 100-fold increase in fluorescence upon binding to analyte, about a 10-fold improvement over the previous best biosensor. In order to improve the methodology of reagentless biosensor design, we establish a method for site-specific labeling of proteins displayed on the surface of yeasts. This procedure allows for the screening of libraries of sensors for binding and fluorescence enhancement simultaneously. Finally, we explore an alternative sensor design, based on competitive inhibition of fluorescence quenching.by Seymour de Picciotto.Ph. D

Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment-sensitive fluorophore or Förster resonance energy transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We here develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. These concepts are broadly relevant to reagentless biosensor modalities.National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant 1 U54 CA112967)National Institutes of Health (U.S.) (R01 EB 010246

Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions

Quantifying protein location and concentration is critical for understanding function in situ. Scaffold conjugated to environment-sensitive fluorophore (SuCESsFul) biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can, in principle, detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins, and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best-reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors. Keywords: solvatochromism; Sso7d scaffold; sensors; protein engineering; directed evolutionNational Science Foundation (U.S.) (Grant MCB-115803)National Institutes of Health (U.S.) (Grant U54CA112967)National Cancer Institute (U.S.) (Grant U54CA112967)National Institutes of Health (U.S.) (Grant R01 EB 010246

Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools

Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology