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Published online before print August 4, 2005, 10.1110/ps.051394605
Protein Science (2005), 14:2333-2343. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Three amino acids that are critical to formation and stability of the P22 tailspike trimer

Matthew J. Gage, Jennifer L. Zak and Anne Skaja Robinson

Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA

Reprint requests to: Anne Skaja Robinson, 259 Colburn Laboratory, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA; e-mail: robinson{at}che.udel.edu; fax: (302) 831-6262.

(RECEIVED February 3, 2005; FINAL REVISION May 31, 2005; ACCEPTED May 31, 2005)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation. We now show that the R549Q and R563K variants significantly delay the protrimer-to-trimer transition both in vivo and in vitro. Previously, variants that destabilize intermediates have shown wild-type chemical stability. Interestingly, both the R549Q and R563K variants destabilize the tailspike trimer in guanidine denaturation studies, indicating that they represent a new class of tailspike folding variants. R549Q has a midpoint of unfolding at 3.2M guanidine, compared to 5.6M for the wild-type tailspike protein, while R563K has a midpoint of unfolding of 1.8 M. R549Q and R563K also denature over a broader pH range than the wild-type tailspike protein and both proteins have increased sensitivity to pH during refolding, suggesting that both residues are involved in ionic interactions. Our model is that R563 and D572 interact to stabilize the adjacent turn, aiding the assembly of the dimer and protrimer species. We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation.

Keywords: P22 tailspike; protein folding; stability; mutation; ionic interaction

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051394605.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Protein folding is an intricate and complicated process, especially for large, multimeric proteins. Some multimeric assemblies fold and assemble in a concerted fashion, such as the adenovirus tail fiber (Van Raaij et al. 1999). With other assemblies, such as most icosahedral viruses, the monomers fold independently and then assemble to form the final quaternary structure. The overall stability of a multimeric assembly is often related to the contacts between subunits in the assembly. The increased stability that can be gained by subunit–subunit interaction has been proposed as one reason why thermophilic proteins are more stable then their mesophilic counterparts (Auerbach et al. 1998). Correct association between subunits is a critical requirement for proper function in many multimeric proteins.

The tailspike protein from the bacteriophage P22 is a useful model system for studying folding and oligomeric assembly. The P22 tailspike protein is a thermally stable trimer, with each monomer consisting of 666 amino acids. The fully folded protein is both SDS-and protease-resistant (Goldenberg and King 1981), which has aided in elucidation of the folding pathway of the protein. The structure of the protein can be broken into three main domains; the N-terminal domain, the {beta}-helix domain, and the C terminus (Fig. 1Go). The N-terminal domain functions in attachment of the protein to the head of the phage (Schwarz and Berget 1989; Chen and King 1991). The {beta}-helix domain consists of a 13-runged right-handed {beta}-helix made of three {beta}-sheets (Steinbacher et al. 1994). The contacts between the three {beta}-helices are bridged by ~80 ordered waters and there are no significant direct subunit– subunit interactions (Steinbacher et al. 1994). The C terminus consists of two prismlike structures, with each side of the prism created by the {beta}-sheet from each of the monomers (Steinbacher et al. 1994). In addition, the chains connecting the {beta}-helix domains and the C terminus are intertwined, providing the protein with its SDS resistance and thermal stability (Steinbacher et al. 1994; Kreisberg et al. 2002).



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  		Figure 1. Structure of P22 tailspike. (A) Ribbon diagram of the structure of P22 tailspike protein. The three subunits are colored red, blue, and yellow. The four main structural features are indicated on the structure. (B) C-{alpha} trace of the 109–666 subunit of the P22 tailspike protein. Two subunits are colored in light blue and one subunit is colored in black. The three residues that are the focus of this article are shown in ball and stick representation in the inset.

 
The folding pathway of the tailspike trimer is one of the few that has been characterized both in vivo and in vitro (Fig. 2Go) (Goldenberg and King 1982; Goldenberg et al. 1983; King and Yu 1986; Haase-Pettingell and King 1988; Seckler et al. 1989; Danner and Seckler 1993; Danner et al. 1993; Beibinger et al. 1995; Robinson and King 1997; Gage and Robinson 2003). In vitro, refolding is initiated by dilution and unfolded protein forms a folding-competent monomer species. Two folding-competent monomers associate to form a dimer species that then adds a third monomer to form a protrimer species. The protrimer species is SDS- and protease-sensitive, unlike the native trimer (Goldenberg and King 1982). It also migrates with a larger apparent molecular weight than the native tailspike protein by both gel filtration chromatography and nondenaturing PAGE (Benton et al. 2002). In addition, the protrimer species has been shown to contain a nonnative disulfide bond that dissociates during the protrimer maturation step (Robinson and King 1997). The protrimer then undergoes structural rearrangements to form the final native trimer species, though the exact nature of the rearrangements is poorly understood.



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  		Figure 2. Folding and aggregation pathway of the P22 tailspike protein. Unfolded monomer folds and forms either aggregate-prone monomer (bottom) or folding competent monomer (top). Two monomers are sequentially added together to form either a folding competent dimer or dimer aggregate. In the case of a folding competent dimer, the associations are formed through the C terminus, as shown. Addition of a third monomer through C-terminal interactions leads to formation of the protrimer species, which then forms the fully folded trimer. Aggregate formation is not limited to monomer addition.

 
There are over 60 different mutations in the tailspike gene that have been identified. The largest class of mutants is the temperature sensitive folding (tsf ) mutants. Tsf mutants are capable of forming nativelike trimer at permissive temperatures (20°C, in vivo), but form inclusion bodies at restrictive temperatures (37°C) and are clustered in the {beta}-helix domain, especially in loop regions (Fane and King 1987; Haase-Pettingell and King 1997). While these mutations affect the folding pathway of the protein, they do not significantly alter the thermostability of the full-length protein (Sturtevant et al. 1989). Mutations to each of the three C-terminal cysteines also affect folding of the tailspike protein but do not affect the stability of purified trimer (Danek and Robinson 2003). In addition, a number of "lethal" mutations in the C terminus lead to formation of misfolded chains and inclusion bodies in vivo.

We now report the identification of two amino acids in the C terminus of the protein that play a significant role in the folding and stability of the P22 tailspike trimer. Substitution of amino acids R549 and R563 altered in vivo trimer formation kinetics, resulting in long-lived protrimer intermediates, demonstrating that these mutations affect protrimer maturation. Purified R549Q and R563K trimer showed similar behavior in vitro. Both mutant proteins have much longer-lived protrimer species than exist for the wild-type tailspike protein. Furthermore, both mutant trimers demonstrated reduced chemical stability and increased sensitivity to pH during denaturation relative to the native tailspike trimer. The R563K trimer also demonstrated decreased thermal stability relative to the wild-type tailspike protein. In addition, both mutants showed increased sensitivity to pH during refolding, indicating the presence of a potential ionic interaction. We propose that R563 interacts with D572, positioning this residue in proper position to form an ionic interaction with R549 from an adjacent polypeptide chain. This intricate threefold interaction plays a key role in maturation of the protrimer and in the subsequent stability of the wild-type trimer.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Three C terminus mutations form both trimer and a stalled protrimer species in vivo
Recent results have demonstrated that correct formation of the C terminus is critical to formation of the native P22 tailspike trimer (Kreisberg et al. 2002; Gage and Robinson 2003). One position that has been shown to be critical in this process is arginine 563, which fails to form the tailspike trimer when mutated to glutamine (Kreisberg et al. 2002). Substitution of the arginine at amino acid 563 with glutamine results in a stable monomeric species that does not form detectable protrimer or trimer, indicating that this residue plays an important role in assembly of the tailspike protrimer. Close examination of the crystal structure shows that R563 lies within potential ion-pairing distance to D572, on the opposite side of a {beta}-turn (Fig. 1Go). In addition, a second potential interaction was identified between D572 and R549 from the adjacent subunit. Based on the work of King and colleagues (Kreisberg et al. 2002), we hypothesized that interaction of these three amino acids might be critical to formation and maturation of the protrimer species.

A series of mutations at positions 549, 563, and 572 were generated to study the role of these residues in assembly of the tailspike trimer (Table 1Go). Each of the mutant genes were expressed for 4 h at 20°C, the bacteria were lysed, and the soluble and insoluble fractions were collected. When the expressed protein was analyzed by SDS-PAGE, the R563K, R549Q, and R549L variants formed a small amount of trimer as well as a large amount of monomeric tailspike subunits while the R563Q, D572N, and D572E variants formed only a large amount of soluble monomeric protein (Fig. 3AGo; Table 1Go). SDS disrupts any subunit–subunit interactions that occur between folding intermediates, so any dimeric and trimeric intermediates appear as monomeric species. To determine if any of the mutant proteins were able to form protrimer, the same samples were analyzed by nondenaturing PAGE. The R563K, R549Q, and R549L proteins all formed protrimer, the slower mobility band on the nondenaturing gel, while wild-type protein forms primarily native trimer (Fig. 3BGo). The identity of the protrimer species was confirmed by Western blot analysis with anti-native tailspike antibody (data not shown). Surprisingly, the D572N protein also appears to form protrimer as well, though it is incapable of forming trimer (Fig. 3BGo). The difference between this mutation and the others will be addressed later.


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  		Table 1. Effects of amino acid substitutions at positions 549, R563, and D572
 


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  		Figure 3. R549Q, R549L, and R563K proteins are capable of forming trimer and protrimer. Protein expression was induced at 20°C using 1 mM IPTG and cells were grown for 4 h. Samples were collected, lysed, and separated on 10% SDS-PAGE (A) and 7% nondenaturing PAGE (B) as described in Materials and Methods. Monomer and trimer markers are in lanes 1 and 2. Lanes 3, 5, 7, 9, 11, 13, 15, and 17 are of the supernatant (S) from the lysis. Lanes 2, 4, 6, 8, 10, 12, 14, and 16 were of the pellet (P) from the lysis. The individual variants are indicated below the lanes in the gel. Lanes 3 and 4 show expression of the pET11a vector without the P22 tailspike gene.

 
Expression of the R563K, R549Q, and R549L genes produced lower yields of trimer relative to the wild-type protein, suggesting that trimer formation was altered by these amino acid substitutions. Furthermore, the appearance of a protrimer species on the native gel suggests that these three amino acid substitutions primarily affect the protrimer-to-trimer transition.

R563K and R549Q have spectroscopic properties similar to those of wild-type tailspike
Mutant R549Q and R563K trimers were purified to investigate the effects of the mutations in vitro. Fluorescence and circular dichroism (CD) spectra of the mutant trimers were measured to determine whether the amino acid substitutions resulted in any observable structural changes. When the CD spectrum of the two mutant trimers was measured from 260 to 190, the R563K trimer showed a single minimum at 220 nm, consistent with the wild-type tailspike (Fig. 4AGo). The R549Q trimer showed the same characteristic {beta}-sheet trough as the wild-type tailspike protein, though the minimum of the spectra was slightly shifted (222 nm) (Fig. 4AGo). The slight shift in the minimum of the R549Q spectrum might represent a very small perturbation of the trimer structure, though the general shape of the spectra is consistent with the wild-type tailspike protein.



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  		Figure 4. R549Q and R563K trimers have spectroscopic properties similar to those of the wild-type tailspike protein. Circular dichorism (A) and fluorescence emission (B) spectra of purified R549Q and R563K trimer. Spectra were recorded using 100 µg/mL protein diluted in 1x (Fluorescence) or 0.01x (CD) Tris Refolding Buffer. Filled squares represent wild-type tailspike, open circles represent R549Q, and filled circles represent R563K (CD). Each data point is the average of three trials. Fluorescence data is shown as smooth curves of data points for clarity.

 
The fluorescence emission spectrum of each protein was measured by excitation at 280 nm and measuring the emission spectrum from 300–400 nm. For all three proteins, the peak intensity was 342 nmand the center of mass was 346 nm (Fig. 4BGo). In addition, the emission spectra exhibited a shape similar to the spectra of the wild-type protein, indicating that the environment of the tryptophan residues is not perturbed by either of the amino acid substitutions. These results indicate that the overall structure of both of the mutant proteins is consistent with the wild-type tailspike protein, though it is not possible to rule out small local perturbations of structure.

R563K and R549Q exhibit stalled protrimer species and altered trimer maturation rates in vitro
Based on the in vivo expression results, we expected that both the R563K and R549Q trimers would have long-lived intermediates during in vitro refolding experiments. To test this, denatured R563K, R549Q, and wild-type tailspike protein were refolded at 20, 50, and 100 µg/mL for 24 h at pH 7.6 and 10°, 20°, and 30°C. Under these conditions, all the denatured wild-type tailspike protein will have formed either trimer or aggregate at 20° and 30°C and the reaction nearly reaches completion at 10°C (Fig. 5Go).



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  		Figure 5. Refolding of R549Q and R563K proteins demonstrate long-lived folding intermediates. Native PAGE analysis of in vitro refolding of wild-type tailspike (W), R549Q (Q), and R563K (K). Denatured protein was refolded at 10°C (lanes 3,12), 20°C (lanes 4–6), and 30°C (lanes 7–9) overnight at pH 7.6 and either 50 µg/mL (A) or 100 µg/mL (B) and run on 10% native PAGE gels.

 
Consistent with the effects seen in vivo, folding intermediates for both the R549Q and R563K proteins were much longer lived relative to the wild-type tailspike protein. Both proteins demonstrated a significant accumulation of protrimer after 24 h of refolding at both 10° and 20°C and form primarily aggregate at 30°C. The R563K protein also accumulated monomer and dimer intermediates as well, indicating that this amino acid substitution affects in vitro assembly as well as maturation of the protrimer species. In contrast, the R549Q protein stalled predominately as protrimer, suggesting that the principal role of R549 is during protrimer maturation.

To determine the severity of the effects of the R563K and R549Q substitutions on trimer formation, trimer formation was measured over a 7-d period at 20°C and a concentration of 50 µg/mL. While wild-type tailspike trimer formation reached a plateau within the first day (t1/2=2 h) with a yield of 42 µg/mL, both of the mutants showed much slower rates of trimer formation (Fig. 6Go). At longer time points (data not shown), the yields of both variants plateau at lower than wild-type yields. The R549Q protein had a t1/2 of 26 h and a yield of 28 µg/mL. In contrast, the R563K protein had a t1/2 of 46 h and a yield of 7 µg/mL, suggesting that the amino acid substitution at position 563 resulted in a much more severe folding defect than the substitution at position 549.



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  		Figure 6. R549Q and R563K proteins have slower refolding rates and reduced yields in refolding experiments. Denatured tailspike was refolded at 50 µg/mL and 20°C for 7 d. Samples were collected, quenched on ice, and analyzed by nondenaturing PAGE. Filled squares represent wild-type tailspike, open circles represent R549Q, and filled circles represent R563K. Each data point is the average of three trials. Error bars have been omitted for clarity, but were generally about 20% of the value.

 
Amino acid substitutions do not affect initial folding events
One possibility for the prolonged lifetimes is that the R549Q and R563K substitutions affect the initial folding events. Intrinsic fluorescence was used to measure the initial rate of folding for both mutant proteins. It has been shown previously that changes in intrinsic fluorescence can be correlated with the formation of the initial monomeric folding intermediate (Fuchs et al. 1991; Miller et al. 1998). Intrinsic fluorescence was monitored at 340 nm for 10 µg/mL refolding reactions of wild-type tailspike, R549Q, and R563K at 20°C. Table 2Go shows that the rates of monomer folding determined for all three proteins were very similar, indicating that the effects of the amino acid substitutions lie on later stages in the folding pathway.


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  		Table 2. Rates of monomer folding at 20°C
 
R549Q and R563K are more sensitive to pH of refolding than wild-type tailspike
Wild-type tailspike protein is able to fold over a fairly broad pH range (6.5 to 10.5, Figs. 5Go, 7Go). At lower pH, wild-type tailspike protein formed only aggregates, while at higher pH, folding stalled and long-lived monomer and dimer species were seen. The accumulation of folding intermediates was observed at pH values around the pKa of arginine (~11), suggesting the role of an ionic interaction for arginine in assembly and maturation of the trimer.



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  		Figure 7. R549Q and R563K proteins are more sensitive to refolding buffer pH then wild-type tailspike protein. Denatured tailspike protein was refolded at 50 µg/mL and 20°C overnight in phosphate refolding buffer. Refolding buffer pH is shown beneath each lane. Trimer, monomer, and aggregate species are indicated by arrows.

 
If R549 and R563 are involved in an ionic interaction related to assembly and maturation of the trimer, then changes in these amino acids would be expected to result in increased pH sensitivity during refolding. Purified R563K and R549Q protein was refolded for 24 h at 20°C over a range of pH values, and then analyzed by gel electrophoresis. At pH values of 8–9, both variants formed trimer, though at lower levels than that of wild type. In addition, the protrimer species was present, even after 24 h of refolding (Fig. 7Go). For both variants assembly was completely inhibited at higher pH values. The R563K variant showed a greater sensitivity to pH, folding only between pH 8 and 9.0.

Interestingly, theR563K variant refolding reactions contained monomeric and dimeric intermediates after 24 h at a much lower pH than the wild-type tailspike or the R549Q variant. Monomeric and dimeric intermediates appeared faintly at pH 8.0 and are pronounced at pH 8.5. This is consistent with a model where R563 forms an intrachain salt bridge with D572, and, when disrupted, is much less capable of associating readily. While in the R563K variant the charge has been maintained, the pKa of free lysine is lower (~9.5) and so would be expected to become deprotonated at lower pH. This suggests that the interaction between R563 and D572 is important for assembly as well as protrimer maturation.

R549Q and R563K substitutions destabilize the tailspike trimer
Most tailspike variants that are capable of forming trimer are nearly as stable as the wild-type tailspike protein (Sturtevant et al. 1989; Danek and Robinson 2003). Unlike the tsf mutants, where low temperatures can stabilize less favorable conformations, the R549Q and R563K substitutions disrupt an ionic interaction that appears to be critical to folding at all temperatures. It is possible that this ionic interaction may also be critical for stability of the trimer. To test this hypothesis, the stability of purified R549Q and R563K trimer was measured using Guanidine-HCl (GuHCl) denaturation. The midpoint of the stability curve for the wild-type tailspike protein was 5.6 M GuHCl (Fig. 8AGo). In contrast, the midpoint for R549Q trimer was 3.2 M GuHCl and for R563K trimer it was 1.8 MGuHCl, representing a dramatic decrease in chemical stability. The {Delta}G for this transition is not reported because the transition is not reversible, similar to wild-type tailspike. The difference in stability between the R549Q and R563K trimer is consistent with the differences observed in both trimer formation and pH sensitivity.



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  		Figure 8. (A) R563K and R549Q trimers are less stable than the wild-type tailspike trimer, though R563K is a more destabilizing substitution. Purified tailspike trimer was incubated for 1 h in varying concentrations of guanidine hydrochloride (G-HCl). Center of mass of the intrinsic fluorescence spectra was determined and fit by linear extrapolation as described in the Materials and Methods to determine the fraction of the protein unfolded at each concentration. Wild-type tailspike (solid square) has a midpoint of 5.6 M G-HCl, R549Q trimer (solid triangle) has a midpoint of 3.2 M G-HCl, and R563K trimer (open circle) has a midpoint of 1.8 M G-HCl. (B) Purified wild-type tailspike, R549Q, and R563K trimer were incubated at 200 µg/mL in 8 M urea at varying pH values for 1 h and then separated on an SDS-PAGE gel and Coomassie stained. Representative gels for wild-type tailspike and R563K are shown. (C) Trimer concentration was determined by densitometry and normalized to the native protein level (std). Trimer concentrations were plotted versus pH. The experiment was performed in triplicate and averaged. The error bars represent the standard deviation from the average of three trials. (Filled bars) Wild-type tailspike; (striped bars) R563K; (open bars) R549Q. Notice that R563K trimer is at least partially denatured at all pHs, while the wild-type tailspike and R549Q trimers are only denatured significantly at high and low pH. (D) Wild-type tailspike, R549Q, and R563K trimers were incubated at 65°C in 2% SDS for 2 h. Samples were collected and separated by SDS-PAGE and Coomassie stained. Time points of sample collection are shown below each lane.

 
Effective denaturation of wild-type tailspike trimer required 8 M urea at pH <4.0 or pH >10 (Fig. 8BGo). The pH range where effective denaturation occurs is near the pKas of aspartic acid and arginine, indicating that ionic interactions including those between R549, R563, and D572 may play a role in stabilizing the native protein against denaturation. Amino acid changes disrupting this interaction might therefore alter the pH range over which tailspike denaturation can efficiently occur. The ability of 8 M urea to denature purified R549Q and R563K trimer was measured at different pHs to determine the effects of these mutations on pH sensitivity during denaturation. Purified trimer at 200 µg/mL was incubated in 8 M urea at pH values between 3 and 11 for 1 h at 25°C. Samples were then analyzed using SDS-PAGE gel electrophoresis to determine the concentration of native trimer.

As shown in Figure 8 B and CGo, wild-type tailspike was completely denatured at pH 3 in 8 M urea and partially denatured at pH 4, 10, and 11, but there was no denaturation of the trimer between pH 5 and 9. R549Q denatured at a slightly broader pH range (pH 4, 5, and 10), but was not denatured between pH 6 and 9. In contrast, the R563K trimer was partially denatured over the whole pH range in the presence of urea, showing a much greater sensitivity to pH. This indicates that R563 plays a more significant role than R549 in stabilizing the protein against denaturation.

A further test of the effects of these two amino acid changes on the stability of the tailspike trimer is thermal denaturation. The first 110 amino acids of the tailspike chains lose structure prior to denaturation of the remainder of the trimer resulting in the appearance of a semistable unfolding intermediate (Iu) that has an increased electrophoretic mobility relative to the native trimer due to increased SDS binding by the unfolded portions of the trimer, thus increasing the charge-to-mass ratio (Chen and King 1991). Purified trimer was heated at 65°C with 2% SDS and aliquots were removed over time and quenched on ice. The N terminus of both the wild-type tailspike trimer and the R549Q trimer denatured over the course of 60 min, resulting in a molecular weight shift due to SDS binding to the unfolded N terminus (Fig. 8DGo). By the end of 2 h, the protein began to form monomeric species, seen by the large shift in mobility resulting from SDS binding of the unfolded chain. However, a significant amount of protein remained in a trimeric state. In contrast, R563K began to denature into a monomeric state immediately on shifting to higher temperatures (Fig. 8DGo), indicating that the 110–666 amino acid region of the R563K variant is not as stable as the wild-type trimer.

It is interesting to note that while the R549Q trimer showed decreased chemical stability compared to the wild-type tailspike, it did not show a similar decrease in thermal stability in either of the thermal experiments. This indicates that the interaction between R549 and D572 may not play a role in the thermal stability of the protein, only in the chemical stability.


   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Many extracellular proteins are extremely stable due to the harsh environment in which they exist. The proteins can be stabilized through ionic interactions such as in thermophilic proteins (Auerbach et al. 1998), through subunit–subunit interactions (Hiraga and Yutani 1997), or through disulfide bonds such as in BPTI (Bulaj and Goldenberg 1999). The P22 tailspike protein is an extremely stable protein that is stabilized by both ionic interactions and subunit–subunit interactions.

Although the individual subunits of the tailspike protein interact in only the last third of the native structure, previous studies had demonstrated that the C terminus plays a critical role in stabilization of the trimer (Kreisberg et al. 2002). This article has demonstrated that the interaction between R563, D572, and R549 from an adjacent chain play a critical role in maturation of the tailspike trimer and in the stability of the trimer. Substitution of R549 with either Leu or Gln or substitution of R563 with Lys resulted in a significantly reduced rate of protrimer maturation both in vivo and in vitro. In addition, purified R549Q and R563K trimer had reduced urea stability and increased sensitivity to pH.

Interaction between R549, R563, and D572
Analysis of the crystal structure around D572 shows that D572 lies within the interaction distance of both R563 and the R549 residue from an adjacent chain. It is our hypothesis that R563 positions the D572 side chain so that it can interact with R549 during protrimer maturation (Fig. 9AGo). Alteration of R563 to lysine shortens the side chain, repositioning the positive charge (Fig. 9BGo). This leads to a repositioning of D572, resulting in an altered loop conformation, disrupting the interaction of D572 and the adjacent R549. In contrast, substitution of R549 with glutamine disrupts its interaction with D572, but does not affect formation of the loop containing R563 and D572 (Fig. 9CGo). This accounts for the difference in stability exhibited by each of the mutations. Substitution of R549 with glutamine causes a certain amount of destabilization of the tailspike trimer due to the lost interaction with D572. In contrast, the R563K variant was destabilized from the same loss of interaction as well as additional destabilization due to the altered loop conformation.



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  		Figure 9. Interactions between R549, R563, and D572. (A) Schematic of the role of R549, R563, and D572 in folding and assembly of the P22 tailspike protein. The trimer maturation step shows two possible outcomes. The upper pathway is for the wild-type protein. The lower pathway is for the R549Qmutant, where mutation of R549 prevents proper association between the two subunits. (B) Schematic of effects of the R563K mutation on folding of the P22 tailspike protein. Mutation of R563 to K results in a repositioning of D572, which prevents proper maturation of the tailspike protein. (C) C{alpha} trace of the tailspike protein in the wild-type structure focused on the region containing R549, R563, and D572. Subunit 1, containing R563 andD572, is shown in black. Subunit 2, containing R549, is shown in blue. R549, R563, and D572 are shown in ball and stick representation. The bonding distances between the oxygen of D572 and the R549 and R563 nitrogen atoms are shown in red with the bond length indicated.

 
The results presented in this article support this hypothesis. First, trimer stability and rate of trimer formation are more significantly altered in the R563K variant than in the R549Q variant, suggesting that the R563K substitution affects additional steps in folding that are not affected by the R549Q substitution. Second, the R563K variant accumulated folding intermediates at lower pHs than seen for either wild-type tailspike or the R549Q variant, consistent with the altered pKa of the 563 side chain. This suggests that proper formation of this loop is critical for assembly of the protrimer. Third, the R563K, R549Q, and R549L variants showed the same phenotype in vivo. This suggests that all three substitutions are affecting the maturation process at the same stage. While R563K was capable of forming a stable nativelike loop, it likely affects the maturation of the protrimer by mispositioning the negative charge of the D572. Similarly, both of the R549 variants were unable to interact with D572, affecting the maturation reaction as well. Clearly, there are other interactions involved in the maturation process, as it was still possible for the protrimer to mature to trimer even though one of these interactions had been removed. However, the rate of maturation was strongly affected by the loss of this interaction.

Interestingly, the D572N variant was able to form protrimer while the D572E variant was not. Asparagine and aspartic acid are very similar, with the substitution of a single oxygen on the aspartic acid with a nitrogen. This nitrogen could allow for formation of a weak hydrogen bond between positions 563 and 572, but may prevent formation of a similar interaction with R549 leading to trimer maturation. However, substitution of aspartic acid with glutamic acid repositions the charge and may prevent correct formation of the loop. Similarly, substitution of R563 with lysine maintains charge, though the position of the charge is likely further from the aspartic acid. Substitution of R563 with glutamine would also be a much weaker interaction and essentially prevent the loop from forming properly.

This is a significant result as it shows the dramatic effect that substitution of a single amino acid can have on protein assembly. Tailspike has 666 amino acids in each subunit and by altering one simple interaction, the stability and rate of assembly are significantly impaired.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Materials
All chemicals used were obtained from major commercial suppliers. 14C-labeled L-amino acid mixture was purchased from Perkin Elmer. Restriction enzymes were obtained from New England Biolabs. Primers used for cloning and mutagenesis were ordered from Integrated DNA Technologies.

Site-directed mutagenesis
Mutagenesis was performed using the QuikChange Site-Directed Mutagenesis kit (Stratagene). Mutagenesis was performed according to instructions using a pET11a vector containing the full-length P22 tailspike gene as a template. The identity of each mutation was verified by complete sequencing of the gene.

Protein expression
Chemically competent Escherichia coli BL21(DE3) cells (Novagen) were transformed with a pET11a plasmid containing the appropriate gene and selected on Luria Broth (LB) (Sambrook et al. 1989)–Ampicillin (100 µg/mL) plates. Individual colonies were grown in LB media to an OD600 ~0.5 at 30°C for wild-type tailspike or 20°C for mutant tailspike protein and induced using 1 mM IPTG for 4–24 h. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris at pH 7.6, 5 mM MgCl2, 0.1% Triton X-100, 0.1 mg/mL lysozyme, 0.1 mg/mL DNase). After two freeze/thaw cycles (–80°C/ 20°C), the cell debris was removed by centrifugation at 12,000g. The supernatant and resulting pellet were separated for SDS and nondenaturing PAGE analysis. The pellet was resuspended in a volume of lysis buffer equivalent to the supernatant prior to analysis.

Protein purification
Large-scale protein preparation of full-length or mutant P22 tailspike was conducted as described previously (Danek and Robinson 2003; Lefebvre and Robinson 2003). Fractions from the hydroxyapatite column containing tailspike were collected and concentrated to 10–15 mg/mL using Vivaspin 20 mL concentrators (Vivascience). Purity was determined by SDS-PAGE for each of the proteins (>95% for all three proteins). Final concentration was determined by measuring the OD278 (1 OD278=1.017 mg/mL tailspike).

Tailspike refolding
Wild-type tailspike or mutant tailspike protein was denatured in 8 M Tris-Acid urea (pH 3.0) for 1 h at a concentration of 1 mg/mL. Denaturation temperature was identical to initial refolding temperature. Refolding was initiated by dilution of the denatured protein into refolding buffer (50 mM Tris at pH 7.6, 1 mM EDTA) to the final experimental concentration. Samples were removed at various time points and quenched on ice in 3x nondenaturing sample buffer (15 mM Tris at pH 7.6, 120 mM glycine, 30% glycerol, bromophenol blue). Previous studies (Speed et al. 1995) showed that no changes in the intermediates occur during this incubation. Samples from time points longer than 8 h were frozen until completion of the experiment. Time points up to 7 d were collected and analyzed by SDS-PAGE, and all values reached a plateau. Data in Figure 6Go show only the first 100 h to visualize the early differences in the kinetics of trimer formation. The half-time to completion is reported rather than absolute rates because first-order fits (which best fit the rate-limiting protrimer-to-trimer transition and have been used for previous tailspike mutant studies; Danek and Robinson 2003, for example) did not fit the variant data well.

pH refolding
Wild-type and mutant tailspike protein was denatured and refolded as described above using phosphate refolding buffer (50 mM phosphate, 1 mM EDTA) at different pHs. Refolding was performed at 20°C for ~24 h.

Chemical stability
Five microliters of tailspike protein were added to varying concentrations of guanidine-hydrochloride buffer and incubated for 1 h at room temperature. The fluorescence emission spectrum was measured from 300 nm to 400 nm (excitation wavelength=280 nm) using a Hitachi F-4500 Fluorescence Spectrophotometer. The center of mass of each spectrum was calculated using the formula:


where I is equal to intensity and {nu} is equal to the wavenumber. The percent unfolded curves were generated by fitting the center of mass versus GdnHCl curves to a two-state unfolding model using linear extrapolation (Pace and Shaw 2000).

SDS stability
Purified tailspike and mutant protein was diluted to 200 µg/mL in a 2% SDS solution preheated to 65°C. Samples were collected at various time points and mixed with ice-cold 3x SDS sample buffer. Protein was separated by SDS-PAGE, Coomassie stained, and analyzed by densitometry.

Urea denaturation
Wild-type and mutant tailspike protein was diluted to 200 µg/ mL in 8 M urea at various pH values and incubated for 1 h at 20°C. The samples were separated by SDS-PAGE, Coomassie stained, and analyzed by densitometry.


   Acknowledgments
 
We thank Jamie Fiske for assistance in purifying the R549Q mutant, Noelle Comolli for assistance with gels and quantitation, and Cameron Haase-Pettingell for assistance performing the thermal denaturation experiment and helpful discussion. This work has been supported in part by Howard Hughes Medical Institute Undergraduate Research Support (J.L.K.) and NIH P20 RR15588.


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