# Protein/Chromphore Interactions via Protein Design: Interrogation and Application

> **NIH NIH R01** · MICHIGAN STATE UNIVERSITY · 2020 · $104,173

## Abstract

Project Summary
 We began our program by re-engineering members of the Fatty Acid Binding protein family to be mimics of
rhodopsin, through rational design principles. In the process of achieving these goals we became convinced that the
characteristics of these fantastically stable proteins rendered them ideal vehicles for a variety of other applications. With
their small size, large binding pocket that could accommodate a variety of unrelated structures, high expression yield,
resistance to structural misfolding due to mutations, and propensity for crystallization, we found these proteins as ideal
tools for sensor and imaging applications. The story of their development into unique fluorescent proteins continues
with this proposal as a result of two fundamentally important observations, translated to the two major aims of this grant.
First, having suitably engineered the binding pocket, and chosen the appropriate chromophore as a partner, we
demonstrate the creation of a protein/chromophore complex as an imine, which upon photo-irradiation experiences
Excited State Proton Transfer (ESPT) to generate the iminium. Critical to the design of the chromophore is that iminium
protonation generates a highly conjugated system capable of Intramolecular Charge Transfer (ICT). ICT fluorophores are
typically red-shifted and highly fluorescent. Thus, photo-irradiation of a blue absorbing complex leads to an excited red-
shifting species, which fluoresces with a Large Stokes Shift (LSS). Second, we have realized the ability to create a
parallel suite of photo-switchable fluorochromes, where the fluorescence output can be rapidly and photo-chemically
switched between `ON' and `OFF' states. Such fluorescent systems are the essential tools required for ultra-high
resolution microscopy, a technology that has the potential to revolutionize our understanding of biological phenomena if
the proper fluorochromes can be developed. They are also essential in biological imaging applications that require
spatio-temporal control.
 The approach to these goals involves the precise, structure-based design and optimization of both protein and
fluorophore to find the ideal system. We will optimize ESPT of a protein/chromophore complex as a photobase, a
photoacid, and also in what we suggest to be a dual-ESPT mode, requiring both photoacid and photobase activity
during the single photo-excitation event. This would convert a ground state neutral imine to a zwitterionic, highly
polarized, conjugated excited state that will emit far in the red from the wavelength of excitation. The design of photo-
switching fluorophores married to the appropriate protein environment that supports and promotes the structural change
in the chromophore, will optimize the characteristics necessary for obtaining a desired photo-switch, such as red-shifted
emission and high brightness. Furthermore, `ON' and `OFF' kinetics will be optimized, as rapid rates are advantageous in
many imaging applications.

## Key facts

- **NIH application ID:** 10197274
- **Project number:** 3R01GM101353-06S1
- **Recipient organization:** MICHIGAN STATE UNIVERSITY
- **Principal Investigator:** BABAK BORHAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $104,173
- **Award type:** 3
- **Project period:** 2013-09-20 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10197274

## Citation

> US National Institutes of Health, RePORTER application 10197274, Protein/Chromphore Interactions via Protein Design: Interrogation and Application (3R01GM101353-06S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10197274. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*