Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 128

Vibrational Spectroscopy of Fluorescent
Proteins: A Tool to Investigate the Structure
of the Chromophore and Its Environment

Valentina Tozzini and Stefano Luin

Abstract The design of fluorescent protein (FP) mutants with tailored properties
benefits from the comprehension of chromophore structure, interactions, energy
landscapes, and dynamics. Vibrational spectroscopy can often provide detailed
information on these characteristics for proteins in their natural aqueous environ-
ment, during their (photo)dynamics and without the need of crystallization. Here
we will review the experimental and theoretical techniques that have been used to
analyze the relations between vibrational spectra and different structural, photo-
physical, and chemical properties of FPs, in particular the ones able to selectively
address the chromophore and its close environment, like (pre)resonance Raman,
difference-IR absorption measurements, and their computational simulations. Start-
ing from the preliminary results aimed at identifying vibrational modes in the
neutral and anionic GFP chromophore, we will discuss selected experiments that
unraveled, often thanks to comparison with theoretical studies, the structure of
the chromophore in some FP mutants, the impact of cis - trans isomerization and
different protonation states in reversibly photoswitchable proteins, and the struc-
tural changes and proton-transfer pathway in the photoexcited state.

Keywords Density Functional Theory Fluorescent protein chromophores GFP
IR absorption Raman Time-resolved spectroscopy Vibrational spectroscopy

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
2 Experimental Techniques ................................................................... 136
2.1 IR Absorption ......................................................................... 136
2.2 Raman and SERS . . ................................................................... 138

V. Tozzini and S. Luin ( * )
NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12,
56127 Pisa, Italy
e-mail: [e-mail]