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Open Access Publications from the University of California

Site-Specific Surface Immobilization of Proteins and Development of Synthetic Fluorescent Sensors for Studying the Cell Biology of Metals

  • Author(s): Lee, Jessica Pui Yan
  • Advisor(s): Francis, Matthew B
  • Kumar, Sanjay
  • et al.
Abstract

As the major building block of life, proteins have an extraordinary range of structures and functions, which can be harnessed through their controlled immobilization. The covalent attachment of proteins onto surfaces has broad applications in biotechnology and materials science, but current strategies rely on nonspecific conjugation reactions. These reactions often affect protein properties and function. To address this problem, we have developed two site-specific immobilization strategies that target only the N-termini of proteins: one for the functionalization of hydrogels and one for the photopatterning of proteins on glass substrates. We have successfully applied the novel hydrogel material for studying substrate stiffness-dependent cell behavior. We envision that this site-specific immobilization strategy will be applicable to other hydrogel substrates and widely adopted in the field of mechanobiology.

Metal homeostasis is vital for the survival and health of all living organisms. Monitoring metal ion accumulation, efflux, trafficking and organelle distribution is essential for the understanding of roles of sodium and copper ions in cell biology. Synthetic fluorescent sensors have been used to investigate such molecular processes in high spatial and temporal resolution. We have developed novel sodium sensors, which show significant improvements over the commerically-available sensors in their sensitivities and affinities. They are able to reliably report sodium ion levels in live cells. We have also expanded the molecular imaging tool box with a series of photostable copper(I) sensors, which are able to report cellular copper(I) levels in real time.

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