Synthetic control of green fluorescent protein

Abstract

Semisynthetic green fluorescent proteins (GFPs) can be prepared by producing truncated GFPs recombinantly and assembling them with synthetic beta-strands of GFP. The yield from expressing the truncated GFPs is low, and the chromophore is either partially formed or not formed. An alternative method is presented in which full-length proteins are produced recombinantly with a protease site inserted between the structural element to be removed and the rest of the protein. The native peptide can then be replaced by cutting the protease site with trypsin, denaturing in guanidine hydrochloride to disrupt the complex, separating the native peptide from the rest of the protein by size exclusion, and refolding the protein in the presence of a synthetic peptide. We show that this method allows for removal and replacement of the interior chromophore containing helix and that the GFP barrel is capable of inducing chromophore formation in a synthetic interior helix.

Figure 1

The 11 β-strands and α-helices of GFP represented as arrows and cylinders, respectively. The dashed line shows the loop insertion used to make GFP:loop:s11 (Fig 2A), and the dash-dotted line shows both the circular permutation and the loop insertion to make ih:loop:GFP (Fig. 2B). The N and C in the black circles show the new N and C termini after making the circular permutation to get ih:loop:GFP. The loop insertion sequence for both proteins is GTRGSGSIEGRHSGSGS, and the linker added between the native N and C-termini in ih:loop:GFP is GGTGGS.

Figure 2

Methods for replacing (A) β-strand 11 (or any of the 11 β-strands by circular permutation, c.f. Fig. 1) or (B) the interior helix (ih) that contains the 3 amino acids that become the GFP chromophore. In each case, a GFP with a loop insertion containing trypsin cleavage sites before the C or N terminal element that will be removed is formed recombinantly in high yield as a full-length folded protein with the chromophore formed. The steps shown are 1) digest with trypsin; 2) denature with guanidine hydrochloride and isolate the larger piece of GFP by size exclusion; and 3) dilute the larger piece of GFP out of denaturant into a solution containing a synthetic peptide.

Figure 3

Absorbance and fluorescence spectra of elements in method described in Fig. 2A for the removal and replacement of strand 11. GFP: loop:s11, GFP: loop:s11, and GFP: loop:s11•s11 and spectra are shown by dotted, dashed, and solid lines, respectively. The spectra of GFP: loop:s11•s11 and GFP with s11 covalently attached are nearly identical (data not shown). The absorbance spectrum of GFP: loop:s11 has a single band in the visible region unlike the parent protein with s11 covalently or noncovalently attached. All spectra are normalized by concentration so that the relative intensities of the absorbance spectra reflect differences in extinction coefficients and the emission spectra relative intensities reflect the difference in extinction coefficient and quantum yield upon excitation at 468nm.

Figure 4

Fluorescence excitation spectra of the elements shown in Fig. 2B. The native ih containing the chromophore incorporating serine 65 is removed, yielding a colorless protein ( ih:loop:GFP), followed by addition of a synthetic ih or a synthetic ih with the S65T mutation (ih S65T). When the original ih is removed and then reconstituted with a synthetic ih of the same sequence ( ih:loop: GFP•ih, solid red), the original spectrum is recovered ( ih:loop:GFP, dashed orange). In contrast, if instead it is reconstituted with ih S65T ( ih:loop:GFP•ih S65T, solid blue), the resulting excitation spectrum is identical to that of the protein in which the S65T mutation was introduced through mutagenesis (S65T ih:loop:GFP, dashed green). There is minimal fluorescence upon refolding ih:loop:GFP (solid black) in the absence of synthetic peptides.