Designing Chemical- and Light-Activated Protein Switches for Regulating Peptide Functions

Abstract

Peptides serve important biological functions including neuromodulation, hormonal regulation, cell signaling, protein localization, and enzyme inhibition. The ability to modulate peptide functions with precision is invaluable in biological research. Genetic tools have offered precise control over biological systems with cell-type specificity, and chemogenetic and optogenetic techniques have expanded this control, providing high temporal and spatial resolution through small molecules and light as signaling inputs. While these methods have been extensively applied to regulate protein functions, their application in controlling peptide functions is less explored. This thesis describes the engineering of chemogenetic and optogenetic protein domains for regulating peptide functions. These domains offer versatile control over various peptides, modulating biological processes through orthogonal signal inputs from small molecules and light. A directed evolution platform for optimizing these domains is also introduced. For chemogenetic control over peptides, we developed the chemically activated protein domains (CAPs) for controlling the accessibility of both the N- and C-terminal portions of functional peptides. CAPs were developed through directed evolution of an FK506 binding protein (FKBP). By fusing a peptide to one or both CAPs, the peptide’s function is blocked until a small molecule displaces them from the FKBP ligand binding site. CAPs are generally applicable to a range of short peptides, including a protease cleavage site (TEVcs), a dimerization-inducing heptapeptide (SsrA), a nuclear localization signal peptide (NLS), and an opioid peptide (enkephalin), with a chemical dependence up to 156-fold. We show that the CAPs system can be utilized in cell cultures and multiple organs in living animals. The second light, oxygen, voltage sensing domain from Avena sativa phototropin 1 (AsLOV2) has been widely applied to modulate the activity of various peptides by light. However, due to geometry restrictions, AsLOV2 is not applicable for peptides whose functions requires fusion-free N-terminus. We re-engineered AsLOV2 using circular permutation strategy to generate cpLOV. This modification allows modulation of the C-terminal accessibility of functional peptides while leaving the N-terminus unfused. Using the same strategy as CAPs and showcased by TEVcs, functional peptides can be fused to both AsLOV2 and cpLOV tandemly to reduce the basal activity and tune the dynamic range. To further optimize these chemical- and light-switchable protein domains, we established an efficient yeast surface based directed evolution platform. This platform simultaneously exhibits activation and leakage signals on the same yeast cell, enabling further optimization of CAPs' caging efficiency. The improved CAPs were then applied to regulate three neuropeptides: enkephalin, pituitary adenylate cyclase-activating polypeptide (PACAP), and α-melanocyte-stimulating hormone (α-MSH), showcasing their broad applicability in modulating peptide functions. Potential future work includes the optimization of the developed switchable protein domains, expanding the scope of using CAPs to modulate other neuropeptides, and development of orthogonal switchable protein domains. This thesis contributes significantly to the field of peptide function modulation, offering novel chemogenetic and optogenetic tools and methodologies that have profound implications for biological research.