X-ray Diffraction and Time-resolved Fluorescence Analyses of Aequorea Green Fluorescent Protein Crystals *

The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P612$ or P6,22 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallelepipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, @ = 10S0, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Timeresolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein’s structure-function relationship.

The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P612$ or P6,22 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallelepipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, @ = 10S0, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Timeresolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.
The green fluorescent proteins (GFPs)' are a unique class of chromoproteins found in many bioluminescent hydrozoan and anthozoan coelenterates (1). These proteins have been characterized best from the sea pansy Renilla reniformis (2) and the hydromedusan jellyfish Aequorea victoria (3-5), where they serve as the in vivo bioluminescent emitters. In Renilla, energy is transferred from the single excited state of an oxyluciferin monoanion to the GFP by a radiationless process.
In contrast, in Aequorea the evidence seems to favor radiative transfer to GFP from the photoprotein aequorin.
The well characterized GFPs from both species have been purified to homogeneity and found to be acidic, globular proteins of molecular mass 27,000-30,000 daltons. These mol-* This work was supported in part by Navy Contract ONR- ecules are amazingly resistant to denaturing conditions and have been shown to be stable in 8 M urea (6). Even in vitro fluorescence is unaffected by prior treatment in 6 M guanidine HCl, 8 M urea, or 1% sodium dodecyl sulfate (3). In addition, GFP is very resistant to a variety of proteases (7).
The fluorescent and bioluminescent characteristics of GFP result from a covalently bound chromophore. The Aequorea GFP chromophore has been proposed by Shimomura (8) to be a cyclic tripeptide apparently derived from the primary structure of the protein. Because the chromophore in Renilla GFP is thought to be identical, the large difference in absorption spectrum maxima of Renilla and Aequorea GFPs (103 nm) is believed to result from differences in noncovalent interactions between the chromophore and other regions of the protein (3). Fluorescence polarization and oxygen quenching measurements suggest that the chromophore is held rigidly within a conformationally inflexible domain (9). However, changes in pH, ionic strength, and protein concentration do perturb the spectral properties of the protein (10).
We have prepared two crystal forms of Aequorea GFP suitable for structure analysis by x-ray diffraction. A structural model of GFP based on a single crystal x-ray diffraction analysis will be crucial to the resolution of a number of questions concerning the nature of the energy transfer mechanism, the notable stability of the molecule, and details of the structure and environment of the unique chromophore.
The most striking feature of the protein is its bright green fluorescence, which has been extensively studied (1,4,5,10,11). Both crystal forms fluoresce almost identically to the protein in solution (see below), suggesting that the conformation of the protein in the crystal does not vary significantly from that in solution. Moreover, the fluorescence may serve as a useful probe for the structure and dynamics of the crystalline protein (12)(13)(14).  15) were used to isolate the protein in the form of ammonium sulfate pellets. This preprocessed material was later purified using gel filtration and ion exchange chromatography as described elsewhere (4,5). All preparations yielded absorbance ratios Aaes/Am greater than 1.0. To separate isoproteins, selected samples were further purified by anion exchange chromatography, using an analytical Pharmacia LKB Biotechnology Inc. Mono-& fast protein liquid chromatography column, eluted with a 0-0.25 M NaCl salt gradient in 50 mM BisTris, pH 5.8.

Specimens of
Large single crystals from several purified GFP samples were prepared using "hanging drop" vapor diffusion techniques (16). Hexagonal and monoclonic crystals were prepared by equilibrating lo-.] native crystal form was collected on a Nicolet Imaging Proportional asymmetric unit, the monoc,inic crystal form is the preferred Counter system (Xentronics). The data were corrected and processed as described elsewhere? For all diffraction analyses, crystals were form for a structure determination' In addition, the large mounted in quartz capillaries that had previously been silanated by lattice constant in the hexagonal crystal I d W s it unwieldy to immersing in a 3% dichlorodimethylsilane/toluene solution, followed work with, although all reflections can be resolved easily with by sequential rinsing with toluene, ethanol, and water. the well collimated radiation of a synchrotron source. There-Precession photographs of both crystal types were taken at beam-metry with a = 93-3, = 66*5, = 45.5 A and fi = Crysta1 chromatic x-radiation with a wavelength of 1.63 A. A data set of the Based on resolution and the number of molecules in the Fluorescence emission and corrected excitation spectra of the protein in solution and in crystals were obtained on a Spex Fluorolog I1 fluorimeter. The fluorescence lifetime was determined on an ISS variable frequency phase fluorimeter as described previously (19,20). For all measurements single crystals were mounted in glass capillaries that exhibited minimal fluorescence even when excited at ultraviolet wavelengths. Further details of the experiments are given in the figure legends (Figs. 3 and 4).

RESULTS AND DISCUSSION
Crystals grew in 4-7 days. Crystal growth rate and crystal size were significantly increased when protein samples were further purified using ion exchange fast protein liquid chromatography. Hexagonal plates increased in thickness, and monoclonic parallelepipeds increased in all dimensions. No great increase was seen in Asss/Am ratio, indicating that the improved quality of crystals was due only to better separation of GFP isoproteins. Average crystal dimensions were 0.1 x 0.1 X 0.8 mm for the monoclinic parallelepipeds and 0.4 X 0.4 x 0.1 mm for the hexagonal plates (Fig. 1).
Principal net precession photographs of both crystal types appear in Fig. 2 fore, a native data set was collected f!om monoclinic crystals. Of the 14,682 reflections within 2.2 A, 10,267 were collected. Of these, 8,582 were observed to be greater than 2a above background level. A search for isomorphous heavy atom derivatives is in progress. Fig. 3 depicts the fluorescence emission spectra of Aequorea GFP in solution and from a single hexagonal crystal; the monoclinic form exhibited a similar spectrum. The minor apparent increase in intensity on the red edge of the crystal spectrum can be attributed to some reabsorption of the blue side emission by the highly concentrated protein in the crystal; the longest wavelength excitation band is superimposed to make this apparent. The similarity of the crystal and solution spectra suggests that, under these conditions, the conformation of the protein in solution differs little from that in the crystal.
The emissive lifetime of protein fluorescence can also provide important information about the fluorophore and its dynamics (13,14). The lifetime of the fluorescence from the monoclinic crystal form is extremely similar to that found in solution; the best fit to the phase data in Fig. 4 gives a single lifetime of 3.298 k 0.090 ns with a x* of 7.0 and a fractional intensity greater than 96%. By comparison, the protein in solution exhibits a lifetime of 3.150 Preliminary polarized excitation spectra taken from single crystals in different, fixed orientations (and corrected for the F. G . Prendergast The phase angle shift (A@) between the sinusoidally modulated excitation and emission is a simple function of the circular modulation frequency ( w ) and the lifetime ( T ) : tan A@ = UT (12). The crystal (approximately 0.5 mm in its longest dimension) in stabilizing solution in a glass capillary was excited with the modulated 442 nm beam from a HeCd laser (Lumonics 4214NB, e10 71 filter. The reference was the exciting light scattered off the milliwatts), and its fluorescence was observed through a Corning 3capillary, measured through a Corion 450 nm (20-nm bandpass) interference filter without moving the capillary. Modulation data were overtly artifactual and not further analyzed (18). Fitting the above data to two components (e.g. a mixture of emitters) yielded a protein structure and dynamics that often provides data complementary to x-ray crystallographic methods. Although Weber found that lysozyme exhibited a fluorescence lifetime in the crystal similar to that in solution,5 we know of no other fluorescence lifetime data collected from protein crystals. The fact that the emission spectra and fluorescence lifetime of Aequorea GFP in the crystal are nearly identical to those in solution suggests that the protein conformation under these circumstances is the same. Because of its strong absorption band, high quantum yield and photostability, Aequorea GFP represents a favorable case for studying protein structure dynamics using both fluorescence and x-ray methods. Finally, x-ray crystallography will provide the structure of the fluorescent moiety and especially its relation to the rest of the protein, which in turn will elucidate the structural basis of its remarkable fluorescent properties. A single crystal x-ray diffraction structure analysis is in progress.