Preparation and Fluorescence Anisotropy Study of a Ribonuclease-Lucifer Yellow Conjugate Malone, Christine1; Sumida, John2; Pusey, Marc3. 1. USRA, 4950 Corporate Drive, Suite 100, Huntsville, AL 35806 256-544-2709, christine.malone@msfc.nasa.gov 2. USRA, 4950 Corporate Drive, Suite 100, Huntsville, AL 35806 256-544-0669, john.sumida@msfc.nasa.gov 3. NASA/Marshall Space Flight Center, Biophysics SD48, Huntsville, AL 35812 256-544-7823, marc.pusey@msfc.nasa.gov Abstract We have prepared a chemical derivative of ribonuclease A (RNase) with lucifer yellow (LY). The rotational dynamics of the LY-RNase conjugate were characterized by steady state and time resolved fluorescence techniques. Steady state anisotropy measurements were performed at varying viscosities at 10oC and 20oC, and the rotational correlation time of both RNase and the covalently linked LY probe were determined by time resolved frequency domain measurement. Our data suggest that the fluorophore is rigidly bound at 10oC. Introduction Fluorescent spectroscopy is proving to be a powerful tool to study the protein crystal nucleation and growth process. While most proteins are intrinsically fluorescent, working at crystallization concentrations necessitate the use of covalently prepared derivatives added as tracers. This approach requires that these derivatives do not markedly affect the crystal packing. We have previously prepared a number of fluorescent conjugates of chicken egg white lysozyme, and now have initiated the synthesis of fluorescent derivatives of RNase. Previous work, which we have been able to duplicate, has shown that the probe iodoacetamide-5-(2-aminoethyl)aminonapthalene-1-sulfonic acid, (IEDANS), can be bound to one of the active site histidine residues, where it is apparently bound with no independent motion.(1,2) We have now succeeded in using the reactivity of the active site histidines to incorporate the fluorescent probe lucifer yellow, (LY) into RNase A. Such a derivative can be used for fluorescence anisotropy and energy transfer studies of protein crystal nucleation. We have used this probe to characterize the rotational dynamics of RNase A. Purpose It has been suggested that protein crystallization proceeds through the ordered self assembly of monomeric protein units.(3,4,5) In order to characterize this process, fluorescence studies of various protein systems have been initiated. At present the

research strategy is two-fold. Firstly, steady state and time resolved methods are being used to observe and quantitate resonance energy transfer between spectrally matched protein-fluorophore conjugates. This work yields solution based structural information about self-assembled precrystallographic assemblies. Secondly, both steady state and time resolved fluorescence techniques are being used to observe and quantitate the rotational dynamics of the protein and assemblies in solution. These measurements yield information regarding the dimensions of the self-assembled species in solution. In this paper, the covalent binding of an exogenous fluorophore, lucifer yellow, to RNase at the HIS 12 residue is presented. Additionally using the fluorescence of the fluorophore, the rotational dynamics of the protein-fluorophore system is characterized in order to determine: (1) the degree of rigidity of the bound LY; (2), the rotational correlation time of RNase; (3), the rotational diffusion coefficient of RNase; (4), the molecular volume of the protein-fluorophore system. Materials and Methods RNase A, (bovine pancreas Type 1-A, Sigma) was purified by ion-exchange chromatography.(6) Conjugates of LY (iodacetamide, dipotassium salt, Molecular Probes), were prepared based on the method of Jullien and Garel.(7) The reaction mixture contained 7.5×10-4 M RNase and 0.001M LY in 200mM sodium cacodylate, pH=5.5. The reaction proceeded for 48 hours at room temperature, with continuous stirring, and was protected from light. Gel filtration was used to remove unbound fluorophore. LY derivative was purified from nonderivatized protein by cation exchange, (CM-Macroprep, BioRad), chromatography, (Figure 1.jpg). Protein was eluted with a salt gradient from 0 to 0.3M NaCl in 10mM sodium cacodylate buffer, pH=6.5. Major peak fractions with the highest degree of labeling were collected. Labeled protein yield was determined to be approximately 82%. For the steady state anisotropy measurements, solutions of glycerol and buffer, (0.05M Tris buffer with a pH = 7.5), were prepared in order to vary the viscosity. Temperature was also varied by using circulated water from a water bath set to –20oC in conjunction with a stream of cold air also precooled by the water bath. Using both circulated water and cooled air, an effective temperature of 10oC +/- 2oC was achieved inside the sample compartment. The temperature inside the sample compartment was monitored using a Digisense Thermistor with a thermocouple. Viscosities at 10oC were corrected from their room temperature values using both a four parameter correlation, (8), and a two parameter correlation for glycerol-water solutions of fixed composition, (9). Since the latter correction provided a more continuous range of corrected viscosity values for the data collected, the two parameter correlation was used in the final analysis. The fluorescence anisotropy was determined using the ISS K2 frequency domain fluorimeter. The anisotropy was measured as a function of wavelength by collecting plane polarized excitation spectra. This was done by setting the relative positions of the

excitation polarizer and emission polarizers at 90o and 90o; 90o and 0o; 0o and 0o; and 0o and 90o, respectively. These positions were designated HH, HV, VV, and VH; H refers to the horizontal position and V to the vertical position of the polarizer. Excitation spectra were collected for each orientation and the anisotropy was determined using equation 1, where I(λ)VV and I(λ)VH, are the intensity excitation spectra collected with the excitation and emission polarziers set to 0o and 0o, and to 0o and 90o respectively. G is a correction factor which corrects for polarization dependent sensitivity of the detector and emission optics. For these measurements this factor should be close to unity since there was no monochromator in the emission channel, only a 515nm long pass filter. The G factor was determined by measuring the horizontally polarized excitation when the emission polarizers were set to the horizontal and vertical positions, equation 2 Results Shown in Figure 2.jpg are the excitation spectra collected at all four polarizer orientations along with the resulting anisotropy spectrum for the LY free probe measured in 100% glycerol. In the long wavelength regions the anisotropy reaches a maximum of 0.33 which is equivalent to the limiting anisotropy for this compound. Using equation 3, where β is the angle between the excitation and emission transition dipole moments, the angle β was determined to be 16.78o for the unbound fluorophore. The anisotropy spectrum for LY bound to RNase at HIS 12, RNase-LY, is shown in the Figure 3.jpg. As in the previous figure the excitation spectra for each polarizer orientation are shown along with the resulting wavelength dependent anisotropy. In this case, the anisotropy approaches a maximum of 0.4 in the long wavelength region, indicating that for the bound probe, the excitation and emission dipoles are colinear. This may reflect Equation 3 2 12 −β=

cos3rr o Equation 2 HHHV)I( )I(Gλλ

= Equation 1 ( ) ( )

( ) ( )VHVV VHVV 2GII GI-I rλ+λ

λλ=

either the slower rotation of the bound RNase-LY system, or a change in the absorption and emission properties of the fluorophore upon binding, or a combination of both. Figure 3.jpg indicates that upon covalent attachment to RNase, the LY polarization spectrum at 325nm undergoes a 5nm redshift, and decomposes into two distinct polarization spectra representing two distinct transition moments. Furthermore, the decrease in the peak amplitude at 325nm is a reflection of the quenched emission observed for the bound LY versus the unbound form of the fluorophore. The spectra in the 360nm to 500nm range are unchanged. The spectral shifts observed in the 325nm region are most likely due to the interaction of the fluorophore with the protein which absorbs in this region. The increase in the anisotropy from 0.33, for the unbound fluorophore, to 0.40, for the bound fluorophore, is indicative of the differential effect that binding has on the two transition moments observed. Since the anisotropy is at a maximum value for the bound fluorophore, the absorption and emission dipoles must be colinear. Consequently, excitation and emission presumably occur from the same electronic state.(10) In contrast for the free fluorophore absorption and emission are characterized by the fractional contributions of two different electronic states. The spectra in the 360nm to the 500nm region are unchanged because RNase does not display any absorption in this region. The rotational dynamics of the RNase-LY system were characterized by both steady state and time resolved methods. For a series of glycerol water mixtures, the anisotropy of the bound fluorophore was measured by observing the emission through a 515nm long pass filter at 10oC and 20oC. The results of these steady state measurements are plotted in Figure 4.jpg. Using the perrin relationship, equation 4, the anisotropy displays a linear relationship to the rotational correlation time, θ, (see Figure 5.jpg); τ is the lifetime, and r0 is the limiting anisotropy. Since the correlation time is related to the hydrodynamic volume, equation 4 can be rewritten as equation 5. Equation 4 00 rr1r1 ⋅θτ+= Equation 5 h0 Vη TRτr1r1 ⋅

⋅⋅+=

where the correlation time, θ, = η⋅Vh/R⋅T; η is the viscosity. This data then yields a value for the hydrodynamic volume at 10oC equal to 1.8×104 Å3/molecule, and at 20oC, the hydrodynamic volume is determined to be 1.2×104 Å3/molecule. Figure 4.jpg not only provides information regarding the hydrodynamic volume of the protein, but also provides information regarding the rotational dynamics of the fluorophore bound to the protein as well as the rotational dynamics of the protein as a whole. The graph shows data collected at two temperatures, 10oC and 20oC. At high viscosity, the 10oC data sets converges to a value of 1/r = 2.5 which corresponds to r0 = 0.4; the 20oC data set converges to a value of 1/r = 2.7, (see inset in Figure 5.jpg). As the viscosity is decreased, the anisotropies measured at 10oC extend linearly away from the y-intercept, however in contrast, the 20oC data shows an increase before displaying linearity, such that the y-intercept is offset to a value of 2.9 which corresponds to an apparent r0 = 0.34, reminiscent of the value obtained for the free fluorophore in glycerol. This offset in the 20oC data reflects relative mobility of the bound fluorophore that is not observed for the same protein-fluorophore combination at 10oC. This data indicates that at 10oC the bound fluorophore is held rigidly in place. To further quantitate these rotational motions, frequency domain measurements were performed to extract the rotational correlation times of RNase and the segmental motion of LY. The results are illustrated in Figure 6.jpg. The steady state measurements suggest that the room temperature anisotropy in viscous solution is dominated by the rotational motions characteristic of the free unbound fluorophore, r0-apparent = 0.34. Recall that for the unbound fluorophore r0-free probe = 0.33. The time-resolved measurements performed in the frequency domain support this initial observation. The shift to larger phase angles observed at higher modulation frequencies is indicative of rapid rotational motions of the bound LY. The anisotropy decay was fit using equation 6. Two exponentials were found to be suχ2=0.320. From this, two correlation shorter 100ps time constant is assignefluorophore, LY. The longer 6.7ns timRNase, and yields a rotational diffusio5, the rotational correlation time furth

Equation 6 fficient to fit the decay with a chi-squared value, times were resolved, θ1=6.7ns and θ2=0.1ns. The d to the segmental motion of the bound e constant is assigned to the rotational motion of n coefficient of 2.49×107 s-1. Using equation 3 and er yields a measure of the protein volume of ∑= θ

−j jt0 er)t(r

2.7×104 Å3/molecule. Since the time resolved measurement is not as sensitive to trivial factors such as scattered light and concentration effects, which affect the linearity of the steady state measurements, the volume derived from the time resolved measurements is thought to be a more accurate measure of the hydrodynamic volume at room temperature. Conclusion The purpose of this work was to characterize the rotational dynamics of RNase through the fluorescence anisotropic decay of the attached exogenous fluorophore, LY. The results presented above indicate that the RNase-LY system exhibits segmental motion of the fluorophore at 20oC which is distinguishable from the 6.7ns correlation time measured for the protein. Furthermore, the steady state anisotropy measurements indicate that these segmental motions are no longer apparent at 10oC. This data also suggests that the hydrodynamic volume decreases by a factor of 1.5 at the higher temperature. Whether this is a result of an actual change in the hydration of the protein, or merely a consequence of the segmental mobility of the fluorophore at this temperature will require further work. The results from this study along with the structural information gleaned from resonance energy transfer, will be used to further understand the self assembly mechanisms involved in the crystallization process.

List of Figures Figure 1: Cation exchange chromatography of LY and RNase-A conjugation reaction products. Elution with linear gradient of 0 to 0.3M NaCl in 10mM sodium cacodylate buffer, pH=6.5. Figure 2: Polarization excitation spectra and anisotropy spectrum, (yellow), of the unbound fluorophore, LY in glycerol. Figure 3: Polarization excitation spectra and anisotropy spectrum, (yellow), of the bound LY-RNase conjugate. Figure 4: Comparison of polarization excitation spectra unbound LY, (blue), and the bound LY-RNase conjugate, (yellow). Figure 5: Perrin plot for RNase in aqueous solutions of glycerol at 10oC and 20oC. Figure 6: Frequency domain fluorescence data showing the variation in phase angle and modulated emission as a function of the frequency of the modulated excitation light. The sample is the RNase-LY in 0.05M Tris at pH=7.5 and 7%NaCl.

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