Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

Abstract

Green fluorescent protein (GFP) is widely used as a biomarker in living systems; however, GFP and its variants are prone to forming low-affinity dimers under physiological conditions. This undesirable tendency is exacerbated when fluorescent proteins (FP) are confined to membranes, fused to naturally-oligomeric proteins, or expressed at high levels in cells. Oligomerization of FPs introduces artifacts into the measurement of subunit stoichiometry, as well as interactions between proteins fused to FPs. Introduction of a single mutation, A206K, has been shown to disrupt hydrophobic interactions in the region responsible for GFP dimerization, thereby contributing to its monomerization. Nevertheless, a detailed understanding of how this single amino acid-dependent inhibition of dimerization in GFP occurs at the atomic level is still lacking. Single-molecule experiments combined with computational microscopy (atomistic molecular dynamics) revealed that the amino group of A206 contributes to GFP dimer formation via a multivalent electrostatic interaction. We further showed that myristoyl modification is an efficient mechanism to promote membrane attachment of GFP. Molecular dynamics-based site-directed mutagenesis has been used to identify the key functional residues in FPs. The data presented here have been utilized as a monomeric control in downstream single-molecule studies, facilitating more accurate stoichiometry quantification of functional protein complexes in living cells.

Keywords: N-myristoylation; molecular dynamics; single molecule; stoichiometry.

Figure 1

Single-molecule imaging of GFP and GFP-A206K. (A) Typical single-molecule fluorescence imaging of GFP on the coverslip surface. (B) One- and two-step photobleaching of GFP. Traces are arbitrarily shifted along the y-axis for display clarity. (C,D) Fluorescence intensity and photobleaching step distributions of GFP spots in 0.17 μg/mL and 0.57 μg/mL solutions, respectively. (E) Typical single-molecule fluorescence imaging of GFP-A206K on the coverslip surface. (F) One- and two-step photobleaching of GFP-A206K. Traces are arbitrarily shifted along the y-axis for display clarity. (G,H) Fluorescence-intensity and photobleaching step distributions of GFP-A206K spots in 0.17 μg/mL and 0.57 μg/mL solutions, respectively.

Figure 2

Analysis of the potential dimerization interfaces of wild-type GFP and GFP-A206K. (A) Representative structures of the most populated interfaces formed between wild-type GFP molecules in a water solution. (B) Top view of the GFP dimer by 90° rotation. (C) Representative structures of the most populated interfaces formed between GFP-A206K molecules in a water solution. (D) Top view of the GFP-A206K complex by 90° rotation.

Figure 3

The folding population landscapes of GFP-206A and GFP-A206K projected onto the first two principle components. (A) Free energy landscape between the first and second principal components for GFP-206A. Regions of low free energy are indicated in cold colors (green to blue) and regions of high free energy in hot colors (orange to red). (B) Representative structures of the five highly-populated regions for GFP-206A. (C) Free energy landscape between the first and second principal components for GFP-A206K. (D) Representative structures of the five highly-populated regions for GFP-A206K.

Figure 4

Atomic MD simulations reveal how the myristoyl group anchors GFP to a POPC membrane. (A) Snapshots showing the process of myristoyl group insertion into the lipid bilayer. The myristoyl moiety is highlighted in green and displayed in Corey-Pauling-Koltun (CPK) mode. (B) Binding free energy contributions of per-residue (olive green) and each POPC lipid (purple) to the formation of the protein-lipid bilayer complex. Error bars were not included since the standard errors were always considered in GROMACS algorithms. (C) Contour plot of the relative free energy landscape (FEL) determined from principal component analysis (PCA) using the first and second principal components for the myristoylated GFP-POPC system. The energy scale is given in kJ/mol, with red indicating the high free energy regions and blue the low free energy regions, corresponding to unfavorable and favorable conformations, respectively. Representative structures of the three highly-populated regions, I (50 ns), II (70 ns), and III (90 ns), with the energy minimum are also shown in (A). (D) Polar binding energy contribution of each residue in the myri-GFP-POPC system. (E) Nonpolar binding energy contribution of each residue in the myri-GFP-POPC system. The residues with the most favorable (<−10 kJ/mol) contributions and unfavorable (>10 kJ/mol) are labeled.

Figure 5

The 3D isocontours generated using an independent gradient model (IGM) represent the non-covalent interactions (NCIs) formed between GFP molecules. Green areas correspond to λ2 ≈ 0 (weak). λ2 is one of the three eigenvalues of the electron-density Hessian matrix, with λ1 ≤ λ2 ≤ λ3. All isosurfaces are colored according to a blue-green-red scheme over a range of −0.1 < sign(λ2)ρ < 0.1 a.u. (A) The interface region and isosurface map between two GFP monomers. (B) The interface region and isosurface map between two GFP-A206K monomers. (C–J) The key non-covalent interaction and isosurface map of residues near A206 at the GFP-dimer interface. (K) The non-covalent interaction and isosurface map between K41 and N146 of GFP-A206K.

Figure 6

Per-residue energy contribution plots. Binding free energy contribution of each residue to the interactions between GFP monomers (A) and GFP-A206K monomers (D). Polar binding energy contribution of each residue to the interactions between GFP monomers (B) and GFP-A206K monomers (E). Nonpolar binding energy contribution of each residue to the interactions between GFP monomers (C) and GFP-A206K monomers (F). The residues with the most favorable (<−10 kJ/mol) and unfavorable (> 10 kJ/mol) contributions are labeled.