Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

Abstract

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green f luorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonf luorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen f luoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⅐HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green f luorescence ( max ‫؍‬ 509 nm) under near-UV light. Both f luorescence excitation and f luorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.The bioluminescent jellyfish Aequorea victoria produces a greenish luminescence from the margin of its umbrella by using two proteins: a Ca 2ϩ -binding protein, aequorin (21.4 kDa) and a chromophore-bearing green fluorescent protein (GFP; 27 kDa) (1). On binding Ca 2ϩ , aequorin undergoes an intramolecular reaction, yielding a blue fluorescent protein in the singlet excited state, which transfers its energy by resonance to GFP. The acceptor of energy in GFP is a chromophore consisting of an imidazolone ring structure (2-4). A phenolate anion of the chromophore in the singlet excited state is the emitter of the green light (4). Chemical studies of GFP and expression of cDNA for GFP in prokaryotes and eukaryotes have shown that the chromophore is a product of a posttranslational modification of the primary structure, involving Aequorea GFP consists of 238 amino acid residues in a single polypeptide chain (7-9). The native molecule has been shown to regenerate its intrinsic fluorescence from the totally denatured state (10). GFP is a relatively large molecule to serve as a target for chemical synthesis because a 99-residue HIVprotease analogue is the largest protein synthesized thus far in a highly homogeneous form by the solid-phase method (11) and the 136-residue human pleiotrophin (12) is the largest protein that h...

Polarized Absorption Spectra of Green Fluorescent Protein Single Crystals: Transition Dipole Moment Directions

“…The unprecedented popularity of the green fluorescent protein from the jellyfish Aequorea Victoria stems from its endogenous cofactor, a p-hydroxybenzyledineimidazolinone group (Figure 1a; 1) that forms spontaneously as part of the protein folding process when the backbone of residues Ser 65 -Tyr 66 -Gly 67 cyclizes and dehydrates (2,3). After a further oxidation step, the resulting extended aromatic chromophore interacts strongly with neighboring residues within the 11-stranded -barrel can ( Figure 1b; 4,5) and exhibits intense fluorescence (Φ ∼ 0.79; 6).…”

Blue Fluorescent Amino Acids as In Vivo Building Blocks for Proteins

“…[36] Its fluorescence is possible only in the context of the properly folded protein. [37] In other words, the fluorescence of GFP is not an intrinsic property of the cyclyzed Ser-Tyr-Gly tripeptide since this sequence can be found in a number of other proteins. However, its cyclization is possible only in the context of the GFP structure.…”

Peptide synthesis: chemical or enzymatic

“…This technique is highly flexible with respect to the chemistry of coupling and the combination of the peptidic blocks. New strategies for synthesis in solution have been developed, going from the design of functional groups for the side chains and condensation of fragments for the synthesis of large molecules (Nishiuchi et al 1998) to the use of new coupling reagents (Hiebl et al 1999). …”