Raman spectra of the GFP-like fluorescent proteins

Ye Yuan,Ji Liu,Dianbing Wang,Jibin Zhang,Jian Chen,Xian-En Zhang

doi:10.1007/s41048-018-0072-0

Abstract

The objective of the study was to elucidate optical characteristics of the chromophore structures of fluorescent proteins. Raman spectra of commonly used GFP-like fluorescent proteins (FPs) with diverse emission wavelengths (green, yellow, cyan and red), including the enhanced homogenous FPs EGFP, EYFP, and ECFP (from jellyfish) as well as mNeptune (from sea anemone) were measured. High-quality Raman spectra were obtained and many marker bands for the chromophore of the FPs were identified via assignment of Raman spectra bands. We report the presence of a positive linear correlation between the Raman band shift of C5=C6 and the excitation energy of FPs, demonstrated by plotting absorption maxima (cm−1) against the position of the Raman band C5=C6 in EGFP, ECFP, EYFP, the anionic chromophore and the neutral chromophore. This study revealed new Raman features in the chromophores of the observed FPs, and may contribute to a deeper understanding of the optical properties of FPs.

Highlights

The Raman spectrum provides a ‘‘fingerprint’’ of the vibration and rotation of molecules
Raman spectroscopy with 785-nm excitation was used to acquire the Raman spectra of fluorescent proteins (FPs)
This excitation wavelength selectively enhances the intensity of vibrational bands originating in the chromophore, and thereby avoids certain issues associated with strictly on-resonance Raman experiments such as fluorescence, photoisomerization, or sample degradation (Bell et al 2000)

Summary

Introduction

The Raman spectrum provides a ‘‘fingerprint’’ of the vibration and rotation of molecules. The conformation and structure of biomacromolecules such as DNA, protein chains, membrane proteins and lipids, as well as other structural data related to such molecules can be obtained using Raman spectroscopy (Bunaciu et al 2015; Carey 1982; Tu 1982; Tuma 2005; Xu 2005). Analysis of the Raman spectra of GFP and its mutants revealed that. Femtosecondstimulated Raman spectroscopy showed that skeletal motions are related to proton transfers which makes GFP in the fluorescent form (Fang et al 2009). The Raman spectra of the red fluorescent protein, eqFP611, from the sea anemone, Entacmaea quadricolor, revealed photoinduced cis–trans isomerization of the chromophore (Davey et al 2006). Resonance and pre-resonance Raman spectra of the photochromic fluorescent protein, Dronpa, demonstrated enhanced Raman band selectively for the chromophore, yielding important information on the chromophore structure (Higashino et al 2016)

Objectives

Methods

Results