Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 129

2.3 Nonlinear Techniques: CARS and Time-Resolved
Pump-and-Probe-Based Techniques ................................................. 140
3 Theory: The Density Functional Theory Framework ..................................... 143
3.1 Calculation of the Vibrational Frequencies .......................................... 145
3.2 Calculation of the Vibrational Spectra ............................................... 146
4 Results on Fluorescent Proteins and Their Chromophores ................................ 150
4.1 Identification of Vibrational Modes of Model Chromophores . . . . . . . . . . . . . . . . . . . . . . 150
4.2 Dependence of Vibrational Modes on Chromophore Structure and Its Interactions
Inside Various Fluorescent Proteins ................................................. 154
4.3 Raman Study of the Chromophore States in Photoswitchable Mutants . . . . . . . . . . . . 157
4.4 Time Resolved Vibrational Spectroscopy for Analyzing ESPT . . . . . . . . . . . . . . . . . . . . 161
5 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

1 Introduction

Vibrational information is specific to the chemical bonds and symmetry of a mole-
cule; therefore, every molecule has its distinctive vibrational spectrum, a fingerprint
by which the molecule can be identified. For this reason, vibrational spectroscopy
techniques can in principle be used both for studying the presence and localization
of molecular species and for following structural changes within chemical, phy-
sical, or biological processes. Indeed, the interest of vibrational spectroscopy of
the green fluorescent protein (GFP) and its mutants was driven by the interest on the
structural changes accompanying its mechanism of fluorescence and its complex
photophysics [ 1 - 4 ], and on the role of the protein matrix in modulating the spectral
properties of the chromophore. This knowledge was and still is necessary to help
the design of GFP mutants with novel and engineered spectroscopic properties.
The structure of a molecule cannot be directly determined by its vibrational
spectrum, as is the case, e.g., for X-ray crystallography. However, the comparison
of experimental results on GFP mutants with theoretical calculations and with the
vibrational spectra of isolated chromophores homologues (like the HBDI shown
in Fig. 1a ) give to vibrational spectroscopy the sensitivity to detect important
structural details such as the protonation state and the effect of light absorption
on the structure of bound chromophores, e.g., the eventually induced isomerization.
Moreover, vibrational spectroscopies are nondestructive techniques that often do
not require any lengthy sample preparation (like crystallization) and allow moni-
toring the results of ongoing (photo)reactions of molecules in their natural environ-
ment, i.e., aqueous solutions for proteins.
In this chapter, we will review the results of vibrational spectroscopy in GFP
mutants, with an emphasis on the results on their chromophore in different envi-
ronments. In Sect. 2 , after a short general introduction on vibrational properties of
molecules, we will present the experimental techniques that have been most
used to analyze vibrational spectra of fluorescent proteins. The theoretical methods
for calculating the vibrational spectra of fluorescent protein chromophores will be
reviewed in Sect. 3 , with particular attention on methods based on the time-
dependent density functional theory (TDDFT). Section 4 is dedicated to the