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Originally published In Press as doi:10.1074/jbc.M209174200 on September 30, 2002

J. Biol. Chem., Vol. 277, Issue 49, 47596-47602, December 6, 2002
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Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains*

Andrea T. Da PoianDagger , John E. Johnson§, and Jerson L. SilvaDagger

From the Dagger  Departamento de Bioquímica Medica and Centro Nacional de Ressonancia Magnetica Nuclear de Macromoleculas, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil and the § Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, September 6, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and time-resolved fluorescence approaches. We compared the environment and dynamics of tryptophan side chains of empty capsids and ribonucleoprotein particles of two icosahedral viruses from the comovirus group: cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV). We found a great difference between tryptophan fluorescence emission spectra of the ribonucleoprotein particles and the empty capsids of BPMV. For CPMV, time-resolved fluorescence revealed differences in the tryptophan environments of the capsid protein. The excited-state lifetimes of tryptophan residues were significantly modified by the presence of RNA in the capsid. More than half of the emission of the tryptophans in the ribonucleoprotein particles of CPMV originates from a single exponential decay that can be explained by a similar, nonpolar environment in the local structure of most of the tryptophans, even though they are physically located in different regions of the x-ray structure. CPMV particles without RNA lost this discrete component of emission. Anisotropy decay measurements demonstrated that tryptophans rotate faster in empty particles when compared with the ribonucleoprotein particles. The increased structural breathing facilitates the denaturation of the empty particles. Our studies bring new insights into the intricate interactions between protein and RNA where part of the missing structural information on the nucleic acid molecule is compensated for by the dynamics.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Virus assembly is a puzzle when viewed from the perspectives of thermodynamics and kinetics. Virus assembly touches questions related to protein folding, protein-protein interactions, and protein-nucleic acid interactions. As previously shown for several viruses, these questions are quite linked in many bacteria, plant, and animal viruses (1-5). Although much has been learned about the structure of many macromolecular assemblies, in most cases the direct correlation with function is not possible because of the lack of information about the dynamics of the system (4). In solution, icosahedral viruses can be viewed as molecular "quasicrystals," and they are good models for understanding the linkage among protein folding, protein-nucleic acid interactions, and macromolecular assembly.

Comoviridae is a family of icosahedral plant viruses characterized by a divided genome. This genome consists in two single-stranded, positive-sense RNA molecules, which are encapsidated into distinct particles. The capsids contain equimolar amounts of a large (L) and a small (S) proteins.1 The two RNA molecules are referred to as RNA 1 and RNA 2, with ~6.0 and 3.5 kb, respectively (6). Isolation of comovirus from infected plants results in a mixture of the two ribonucleoprotein particles, as well as empty shells. Thus, three different particles can be separated by gradient ultracentrifugation from preparations of these viruses: the top (no RNA), the middle (containing RNA 2), and the bottom (containing RNA 1) components. Although these components differ in RNA content, they have the same protein composition.

Large crystals of cowpea mosaic virus particles (CPMV), the type member of the comovirus family, were obtained for the first time by White and Johnson (7). A preliminary analysis at 3.5-Å resolution has shown that the CPMV structure is very similar to that of animal picornaviruses (8). One coat-protein heterodimer forms each of the 60 asymmetric units of a P = 3 capsid (1). The purified middle component of bean pod mottle virus (BPMV), another member of the comovirus family, was also crystallized (9). Its x-ray diffraction map at 3.0-Å resolution was used to construct a complete three-dimensional model of the capsid (10). In this study it was possible to detect well ordered RNA, which interacts with the 60 asymmetric units of the particle. An analysis of these data made possible the construction of a model for the RNA packing in BPMV middle component (11).

Protein-nucleic acid interactions are important for many biological functions (12, 13). There are only a few high resolution structures of protein-RNA complexes that have shed light into the understanding of the nature and mechanisms of protein-RNA recognition (14). The recent atomic resolution of the larger subunit of the ribosome was a landmark for the understanding of protein-RNA interactions (15, 16). Hydrostatic pressure has been utilized to study protein folding, multimolecular assembly, and protein-nucleic acid interactions (17-20). We have focused on the role of the linkage between protein-protein and protein-nucleic acid interactions for the recognition process by transcription factors (21-23) and by viral coat proteins (24-27). We have found that, in the case of CPMV, the presence of the viral RNA greatly stabilizes the capsid against pressure denaturation and promotes capsid reassembly after pressure release (25). The observation of reassembly only in the presence of the viral RNA was attributed to strong interactions between protein and the nucleic acid in this virus (25, 26).

Here, we characterize the linkage between dynamics and stability of the three components of two members of the Comoviridae family: BPMV and CPMV. The steady-state and time-resolved fluorescence emission properties of tryptophans are strong tools to unveil the dynamics and conformation of proteins (28, 29). We show that the presence of the nucleic acid in virus capsids modifies the tryptophan environments and its fluorescence properties in the capsid proteins. We also observe a reduction in the overall dynamics of the particle with RNA, which is directly related to the increase in capsid stability.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals-- All reagents were of analytical grade. Distilled water was filtered and deionized through a Millipore water purification system. The experiments were performed at 20 °C in the standard buffer: Tris 50 mM, NaCl 150 mM, pH 7.5.

Comovirus Samples-- Bean pod mottle virus (BPMV) was isolated and purified by extraction from mixtures of chloroform and 1-butanol, as described by Semancik and Bancroft (30). Bil mutant of the yellow strain of cowpea mosaic virus (CPMV) was purified as described elsewhere (31, 32). The three components were separated by equilibrium centrifugation in a self-forming 40% cesium chloride gradient at pH 7.0 (32).

Steady-state Fluorescence Measurements-- Fluorescence measurements were recorded on ISS200 or on ISSK2 spectrofluorometers (ISS Inc., Champaign, IL). The average energy of the fluorescence emission was measured by the center of spectral mass < nu p> ,


⟨<UP>&ngr;</UP>⟩<UP>=&Sgr;&ngr;<SUB>i</SUB> · </UP>F<SUB><UP>i</UP></SUB><UP>/&Sgr;</UP>F<SUB><UP>i</UP></SUB> (Eq. 1)
where Fi stands for the fluorescence emitted at wavenumber nu i and the summation is carried out over the range of appreciable values of F.

Time-resolved Fluorescence Measurements-- Lifetime measurements were performed by a multifrequency cross-correlation phase and modulation fluorometer that uses the harmonic content of a high repetition rate, mode-locked Nd-YAG laser. This laser is used to synchronously pump a dye laser, whose pulse train is frequency-doubled with an angle-tuned frequency doubler (33, 34). Lifetime measurements were also done on the ISSK2 phase and modulation fluorometer (ISS Inc., Champaign, IL). Techniques used for phase fluorometry lifetime measurements and data analysis have been fully described previously (35-38). The quality of fits was assessed by chi 2 values and by plots of weighed residuals. The excitation wavelength was 295 nm, and the emission was observed through a long wavelength pass filter with a cutoff at 320 nm.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fluorescence Properties of CPMV Components: Steady-state and Dynamic Measurements-- The coat protein heterodimer of CPMV has 14 tryptophanyl residues, 9 in the large subunit and 5 in the small subunit. Intrinsic fluorescence spectra of the three components of CPMV were compared in Fig. 1. No differences were observed when the intrinsic fluorescence spectra of bottom and middle components of CPMV were compared with CPMV top component spectrum. The fluorescence emission spectra of all the three components presented an average energy of emission of 29,200 cm-1. This result suggests that the presence of the RNA in the capsid does not affect the average exposure of tryptophans to the aqueous solution.


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  		Fig. 1.   Tryptophan fluorescence spectra of cowpea mosaic virus components. The top component (open circle ), middle component (triangle ), and bottom component (). The excitation wavelength was 280 nm.

Although the fluorescence emission spectra obtained for the three components of CPMV were very similar, the lifetimes of excited state of tryptophanyl residues differed significantly for each component. Fig. 2 shows the fits and the respective errors obtained for the phase and modulation data for the three components of CPMV. The best fits were obtained using a sum of one discrete exponential component and a Lorentzian distribution of lifetimes. The values of the fractions, the lifetimes, and the center and width of the distribution, as well as the chi 2 values for each fit, are summarized in Table I. This table also shows a large increase in chi 2 value when decay models based on two discrete exponential components or one Lorentzian distribution were utilized. The best decay models for top, middle, and bottom components are shown in Fig. 3. Whereas for the top component the exponential decay corresponds to a small fraction and a short lifetime, for middle and bottom components a discrete exponential decay with longer lifetime was predominant. The larger fraction of top component decay corresponds to a distribution of lifetimes, suggesting more heterogeneous environments for tryptophans in the absence of the RNA. The presence of the RNA in the capsid leads to a distribution with shorter lifetimes and an increase in the fraction corresponding to an exponential decay, suggesting a decrease in heterogeneity as a consequence of protein-nucleic acid interactions. This effect was slightly more pronounced for the middle component.


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  		Fig. 2.   Tryptophan lifetime measurements for the three component of CPMV. A, phase (closed symbols) and modulation (open symbols) in the frequency range of 2-300 MHz were measured for the CPMV top component (open circle , ), middle component (triangle , black-triangle), and bottom component (, black-square). The lines correspond to the best fits for each set of data, using one discrete exponential and one component Lorentzian distribution. B, C, and D, errors obtained for measurements in each frequency for the top, middle, and bottom components, respectively. Excitation was at 295 nm and emission was observed through a WG 320 filter. The protein concentration was 0.1 mg/ml.

                              
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Table I
Results of continuous distributions and discrete exponential analysis for lifetime data
The errors correspond to the average of three different experiments. The lifetimes as well as the center and the width of the distributions are given in nanoseconds.


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  		Fig. 3.   Lifetime distributions of the three components of CPMV. Lifetime analysis was performed using one discrete exponential and one component Lorentzian distribution. A, top component; B, middle component; and C, bottom component. The excitation wavelength was 295 nm, and the emission was observed through a WG 320 filter. The protein concentration was 0.1 mg/ml.

Rotation Measurements: Dynamic Depolarization of Tryptophan Fluorescence-- To compare tryptophan mobility in each component, anisotropy decay measurements were performed. The rotations of the tryptophanyl residues of the three components of CPMV were measured using a pulsed-laser multifrequency phase-fluorometer (34, 38). Fig. 4 shows the differential phase and modulation data for each component. Least-square analysis of the data was performed as described by Gratton et al. (38). The best fits to the data were obtained using a model that assigns two rotational motions (Table II). Although the major tryptophan movement in the top component was that related to the shorter rotation correlation time (phi 2), the longer rotational motion (phi 1) is predominant when the RNA was present in the capsid (middle and bottom components). These data suggest that the tryptophans rotate faster in the top component than in the ribonucleoprotein particles.


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  		Fig. 4.   Dynamic depolarization of tryptophans in CPMV components. A, differential polarized phase angles (closed symbols) and modulation ratios (open symbols) were measured for top component (open circle , ), middle component (triangle , black-triangle), and bottom component (, black-square). Curves represent the least-squares fits for the data. Excitation was at 295 nm, and emission was observed through a WG 320 filter. The protein concentration was 0.1 mg/ml.

                              
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Table II
Anisotropy decay measurements
The rotation correlation times (theta ) are given in nanoseconds. r0 = 0.291.

Relation between Increased Dynamics and Lower Pressure Stability of the "RNA-lacking" Particles-- The increase in protein dynamics results in an increase in configuration entropy. However, a more flexible protein implies a rise of the average exposure of the protein surface to the solvent, which may result in a lower stability. High pressure provides a remarkable way to evaluate the relation between dynamics and stability (17, 18), especially because of the direct relation between protein flexibility (due to volume fluctuations) and its isothermal compressibility (39). Pressure is well known to perturb the native state of proteins by causing the infiltration of molecules of water (17, 40, 41). Fig. 5 shows how the top component particle is much less stable against pressure than the ribonucleoprotein components. Thus, RNA seems to stabilize the particle by restraining the protein flexibility and by decreasing the solvent access.


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  		Fig. 5.   Pressure stability of the three components of CPMV. Pressure-induced denaturation curves of top component (), middle component (black-triangle), and bottom component (black-square) in the presence of 1.5 M urea. The samples were excited at 280 nm, and the emission was measured from 300 to 420 nm. The protein concentration was 50 µg/ml.

Comparison of the Fluorescence Properties of CPMV and BPMV Components-- The x-ray structure of bean pod mottle virus (BPMV), another member of the comovirus family, has also been determined (10). For the middle component of this virus, it was possible to detect well ordered RNA, which interacts with the coat proteins in a pocket formed in each of the 60 asymmetric units of the particle.

The coat proteins of BPMV and CPMV show extensive homology (42). The homology between the proteins drops to a low value only in two places: around the cleavage site between the L and S proteins and at the carboxyl terminus of the S protein. The coat protein heterodimer of BPMV has 16 tryptophanyl residues, 7 in the large subunit and 9 in the small subunit (43). 12 of these residues are in the same position as those found for the sequence of the CPMV proteins. Intrinsic fluorescence spectra of the three components of BPMV were compared in Fig. 6. The spectra obtained for the bottom and middle components were quite similar, showing a value of the center of mass of ~29,700 cm-1. On the other hand, the top component spectrum was greatly shifted to higher wavelengths, showing a center of mass value of 28,948 cm-1. This result suggests that, although the tryptophans are more exposed to the aqueous medium in the top component, they occupy a more nonpolar environment when the RNA is present in the capsid.


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  		Fig. 6.   Tryptophan fluorescence spectra of BPMV components. The top component (open circle ), middle component (triangle ), and bottom component (). The excitation wavelength was 280 nm.

Fig. 7 compares intrinsic fluorescence spectra of the BPMV and CPMV bottom components and the BPMV top component. The CPMV spectrum shows an intermediate value of the center of mass, suggesting that the tryptophanyl residues in CPMV are more exposed to the aqueous solution than they are in BPMV ribonucleoprotein particles, although they are less exposed to the medium than in the top component of BPMV.


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  		Fig. 7.   Comparison of tryptophan fluorescence spectra of BPMV and CPMV. Tryptophan fluorescence spectra of the BPMV bottom () and top (open circle ) components and the CPMV bottom component (black-square). The excitation wavelength was 280 nm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Each of the 60 asymmetric units of comovirus capsid is formed by a heterodimer of L and S coat proteins. The L protein has been divided into two domains (designated B and C) apparent from x-ray crystallography studies of CPMV and BPMV (8, 10). The S protein consists of a single domain (the A domain) (Fig. 8). The three beta -barrel domains (A, B, and C) are a variation on the canonical eight-stranded antiparallel beta -barrel fold observed in protein subunits of icosahedral RNA viruses that infect plants, mammals, and insects (1). Both coat proteins of BPMV and CPMV show extensive sequence homology as well as structural similarity (42). The heterodimer of CPMV coat proteins has 14 tryptophanyl residues. Most of these residues (12) are in the same position as in BPMV heterodimer (Fig. 8). The other two tryptophans of CPMV are all in the B domain, whereas the other four tryptophans of BPMV are all in the A domain. The fluorescence spectra of all components of CPMV are shifted to higher wavelengths when compared with the spectra of BPMV ribonucleoprotein particles. This indicates that the tryptophans in CPMV are more exposed to the aqueous solution.


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  		Fig. 8.   Structures of CPMV (A) and BPMV (B) coat protein heterodimers stressing the clusters of aromatic residues. Red, tryptophan side chains; blue, tyrosine side chains; yellow, phenylalanine side chains.

Time-resolved fluorescence studies revealed significant structural differences between ribonucleoprotein particles and empty capsids of CPMV. The data suggested that the top component presented more heterogeneous environments for its tryptophans than the middle and bottom components, as shown by the predominant decay as a distribution of lifetimes in empty capsids. The presence of the RNA in the middle and bottom capsids increased the fraction of exponential decay. This is probably related with an increase in homogeneity in tryptophan environments due to interaction with the RNA, either by direct tryptophan-RNA interaction or by changes in protein conformation promoted by the presence of the nucleic acid.

Intrinsic fluorescence spectra of BPMV reflect the average molecular environment of the 16 tryptophan residues present in each heterodimer. We found that, despite the great heterogeneity related to this large number of tryptophans in this virus, the presence of the nucleic acid in the interior of the capsid increased drastically the average energy of tryptophan fluorescence emission. This result could be explained by protein-RNA interactions strongly modifying tryptophan environments. X-ray crystallography of the middle component of BPMV revealed that 33% of the RNA is in close contact with the proteins in the capsid (10). However, no tryptophan was found close to the RNA-binding pocket. Thus, packaging of the RNA indirectly alters tryptophan environments in this virus, probably by restricting segmental motions. Raman spectroscopy studies indicated that some minor changes occur in BPMV subunits as a consequence of RNA packaging in the virions (44). In this work, the authors estimated that only 5% of the secondary structure of the coat proteins was altered by RNA encapsulation. In addition, they found that only a few of the tryptophan residues of the ribonucleoprotein components (two) exist in a more hydrophobic environment as compared with the particle without RNA, consistent with our data. The fluorescence data suggest that strong modifications should occur in the average environments in the presence of RNA.

Fig. 8 shows the backbone structure and the aromatic residues of CPMV and BPMV. There is a clear proximity between tryptophan side chains and other aromatic residues ("aromatic clusters"), which makes the electronic properties of the tryptophans highly sensitive to segmental and rotational motions. The presence of aromatic clusters may present a good explanation for why half of the emission is fitted to a single discrete decay for the ribonucleoprotein particles of CPMV. RNA would restrict the conformational phase space of the tryptophan side chains. Aromatic interactions are crucial to protein folding and protein recognition (45-49). Burley and Petsko (45-47) have estimated that favorable energies of about -1 to -2.5 kcal/mol exist for each aromatic-aromatic interaction. The relatively low values of these energies would make them susceptible to be broken by the thermal energy (room temperature). The clusters of aromatics found in the structure of CPMV subunits may contribute strongly to the stability of the ribonucleoprotein complex as compared with the empty particle, and it is probably related to the highly entropic stability of the particles (26). The aromatic confinement, with several possibilities of interactions, would also lead to an increase in the residue protein entropy (50, 51), which may be a significant factor for the higher entropy and stability of the native particles. The importance of tryptophan side chains to entropically stabilize the native structure of a virus capsid was described for P22 procapsid shells (52). Recently, the stabilization of poliovirus by capsid-binding antiviral drugs was also shown to be due to entropic effects (53).

A great stabilization of the CPMV capsid is conferred by the presence of the RNA: empty capsids dissociate much easier than the middle and bottom components (Fig. 5). This coupling between protein association and binding to the nucleic acid has already been shown for the interaction between operator DNA and Arc repressor (21), Lex A repressor (23), and E2 DNA-binding domain of human papillomavirus (22). The middle component of CPMV is slightly more stable to pressure-induced disassembly than the bottom component, although presenting a smaller RNA. Time-resolved fluorescence data obtained here revealed that middle component of CPMV presented more homogeneous environments for its tryptophans. This result, together with the pressure stability data, clearly indicates stronger interactions between proteins and RNA in this component. Computation studies on the energetics of icosahedral viruses perceive the importance of stabilization induced by RNA (54).

We also found that the coat proteins remain bound to the nucleic acid after pressure disassembly of the capsid in comoviruses (26, 55), picornaviruses (56), and nodaviruses (27), suggesting again the strong interactions between protein and nucleic acid in virus assembly. These interactions can be disrupted by decreasing temperature to negative values (-15 °C) under pressure, revealing their entropic nature (26). The two RNAs of the comovirus have little sequence homology (57, 58) apart from the 5' and the 3' termini, suggesting that the interactions between proteins and nucleic acid are probably sequence-independent. In the case of BPMV, no significant differences were found between subunit conformations in the middle and bottom components by Raman spectroscopy studies (44). In fact, it was possible to cocrystallize all the three components in the same crystal, which diffracts to at least 3.0-A resolution.

The pressure-dissociated coat proteins of the top component assume a partially denatured conformation, with characteristics of a molten globule conformation (5). A faster rotation in the nonpolar core of the empty capsids suggests a more flexible conformation in the absence of the protein-RNA interactions, which supports our previous data on the increasing facility to denature this form to a molten-globule structure. Indeed, the Arc repressor dimer, which is also denatured by hydrostatic pressure to a molten globule conformation (34), is gradually stabilized by increasing glycerol concentration in the medium, suggesting that a loss of structural flexibility promoted by decreasing water concentration makes it more difficult for the protein to denature to a molten globule conformation (40). The interactions between proteins and nucleic acid in viral capsids probably lead to a similar loss in structural flexibility, resulting in a great stabilization, which is crucial to protection against extracellular environments.

    ACKNOWLEDGEMENTS

We gratefully acknowledge Emerson Gonçalves for competent technical assistance. We are indebted to the Laboratory of Fluorescence Dynamics of the University of Illinois for use of the lifetime facility.

    FOOTNOTES

* This work was supported by grants from Programa de Núcleos de Excelência, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Apoio a Pesquisa do Estado do Rio de Janeiro, by an international grant from the Howard Hughes Medical Institute (to J. L. S.), and by a grant from the National Institutes of Health (to J. E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 5521-2562-6756; Fax: 5521-2270-8647; E-mail: jerson@bioqmed.ufrj.br.

Published, JBC Papers in Press, September 30, 2002, DOI 10.1074/jbc.M209174200

    ABBREVIATIONS

The abbreviations used are: L and S proteins, large and small proteins; CPMV, cowpea mosaic virus; BPMV, bean pod mottle virus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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