Spectral Perturbations of the Aequorea Green‐fluorescent Protein

Abstract

Abstract— In the jellyfish Aequorea, the green‐fluorescent protein (GFP) functions as the in vivo bio‐luminescence emitter via energy transfer from the photoprotein aequorin. Accumulated evidence has indicated that the Aequorea GFP is a relatively inflexible protein. Present evidence, however, indicates that the chromophore environment is readily accessible to a variety of external perturbants. Native Aequorea GFP has an absorbance maximum at 395 nm and a shoulder at 470 nm. In low ionic strength buffer at neutral pH and room temperature the 395/470 nm absorbance ratio is about 2.0. We show that this ratio is highly variable depending upon temperature, ionic strength, protein concentration, and pH. A maximum ratio of 6.5 (at a protein concentration of 18.6 mg/m/) and minimum of 0.42 (at a pH of 12.2) have been measured. In the latter case, the resulting absorption and excitation spectra resemble those of Renilla GFP in spectral shape (but not wavelength maximum). In all cases as the perturbant is varied the resulting spectra pass through a sharp isosbestic point, suggesting a relatively simple two‐state mechanism. These spectral perturbations are fully reversible. On the basis of these results, we suggest that the chromophore binding site is conformationally flexible. pH‐Dependent changes in the near‐UV and visible circular dichroism spectra plus spectrophotometric titration of tyrosine residues lend additional support to this hypothesis.

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

“…2 and 3), reflects the temperature induced protein unfolding. The melting temperature value determined here of 60°C is smaller than T≈78°C, observed for the A-GFP (at pH=7) by Ward et al [27]. Above 60°C the A-GFP structure collapses and its emission rapidly decreases due to the accentuation of intrinsic non-radiative and dynamic quenching events.…”

Structural basis of fluorescence quenching in caspase activatable‐GFP

“…The measured melting temperature (T m ) for GFP of 76 C, closely agrees with published value of 78 C based on loss of fluorescence. 15,16 The curves for both GFP 1-10 and CA-GFP cannot be fit to a twostate melting model but show similar stability profiles to one another. These data suggest that CA-GFP adopts an ensemble of structures that have more similar thermodynamic properties to GFP [1][2][3][4][5][6][7][8][9][10] than to mature GFP.…”

“…Chromophore maturation in split GFP occurs with similar kinetics to CA-GFP. 4,15 We hypothesized that CA-GFP might adopt a stable, partially folded structure similar to that of GFP [1][2][3][4][5][6][7][8][9][10] [ Fig. 1(C)].…”

“…15 N-labeled CA-GFP and GFP were obtained by growth in M9 minimal media using 15 N ammonium chloride as its sole nitrogen source (Cambridge isotopes). 31 A 20 ml LB culture was inoculated from a dense overnight culture.…”

An Alternate Proton Acceptor for Excited-State Proton Transfer in Green Fluorescent Protein: Rewiring GFP

“…In wild-type GFP (wtGFP), excitation into either the neutral (λ max 395 nm, A) or anionic (λ max 475 nm, B) forms of the chromophore results in very efficient green (λ em 508 and 504 nm, respectively) fluorescence. [14][15][16] The 508 nm emission following excitation of A arises from an anionic species formed in the excited state (I*) by deprotonation of the chromophore and concomitant protonation of the E222 carboxylate group (Figure 1). The directly excited B* and indirectly excited I* states differ in that the former has an environment optimized for the anionic form while the latter retains the unrelaxed neutral environment.…”