Vibrations‐determined properties of green fluorescent protein

Abstract

The physicochemical characteristics of the green fluorescent protein (GFP), including the thermodynamic properties (entropy, enthalpy, Gibbs' free energy, heat capacity), normal mode vibrations, and atomic fluctuations, were investigated. The Gaussian 03 computational chemistry program was employed for normal mode analysis using the AMBER force field. The thermodynamic parameters and atomic fluctuations were then calculated from the vibrational eigenvalues (frequencies) and eigenvectors. The regions of highest rigidity were shown to be the beta-sheet barrel with the central alpha-helix, which bears the chromophore. The most flexible parts of the GFP molecule were the outlying loops that cover the top and bottom of the beta-barrel. This way, the balance between rigidity and flexibility is maintained, which is the optimal relationship for protein stability in terms of Gibbs' free energy. This dual-schemed structure satisfies the requirements for GFP function. In this sense, the structure of GFP resembles a nanoscale drum: a stiff cylinder with flexible vibrating end(s).

Physicochemical properties of blue fluorescent protein determined via molecular dynamics simulation

“…The most flexible residues are located in the loops connecting the β‐strands (Figure 12A), which is to be expected, because the side chains of these residues point radially outward from the protein, preventing stiffening due to binding. The RMS fluctuations calculated by method of vibrational analysis38 show similar correlations to experimental data. Also, we can see the expected decrease in RMS fluctuations for almost all flexible parts of the protein at an applied pressure of 1000 MPa.…”

Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus

“…The emission of increasingly brighter fluorescence indeed seems to strongly correlate with the enhanced stiffness of the encapsulated chromophore, by preventing dissipation of the excited state energy through isomerization of neighboring residues during the excited state144647. Enhanced stiffness of the chromophore, which is combined with a slight tilt in the bright bfloGFPa1, could also decrease the non-coplanarity of the residues, which is known to increase QE in GFP114849.…”

“…clearly indicative of a high flexibility region), while being <10 Å 2 for bfloGFPa1. The low B-factor for bfloGFPa1 clearly indicates that the chromophore and its surrounding have limited structural flexibility (hence high stiffness), which is known widely in the literature to be associated with greater QE (e.g.,294652). …”