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. 2004 Dec;13(12):3085–3091. doi: 10.1110/ps.04805204

Native and nonnative conformational preferences in the urea-unfolded state of barstar

Neel S Bhavesh 1, Juhi Juneja 2, Jayant B Udgaonkar 2, Ramakrishna V Hosur 1
PMCID: PMC2287308  PMID: 15537753

Abstract

The refolding of barstar from its urea-unfolded state has been studied extensively using various spectroscopic probes and real-time NMR, which provide global and residue-specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25°C. Complete backbone resonance assignments of the urea-unfolded protein were obtained using the recently developed three-dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13Cα and 13CO secondary shifts show positive deviations in the urea-unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to β-strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the β-strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.

Keywords: unfolded state, HNN, HN(C)N, secondary chemical shifts

A complete understanding of protein folding requires the characterization of all species populated along the folding coordinate, including the unfolded state, partially folded intermediate states, and the fully folded native state. The importance of the unfolded state ensemble in guiding the folding process is now being realized. Unfolded states of proteins, such as in high concentration of chemical denaturant, have been modeled traditionally as statistical random coils in which local interactions dominate conformational behavior (Smith et al. 1996). Molecular dynamic simulations of unfolded polypeptides in the past few years have, however, indicated that steric clashes can prevent a protein chain from adopting any arbitrary conformation, thus constraining the unfolded state ensemble (Pappu et al. 2000; Van Gunsteren et al. 2001), and probably favoring conformations having native-like dihedral angles (Shortle 2002). Recently, it has been suggested that the average interresidue distances in collapsed unfolded structures agree well with the statistics of the ideal random-flight chain (Zagrovic and Pande 2003).

There is increasing evidence now from high-resolution NMR experiments that unfolded states of proteins are not completely random (Juneja and Udgaonkar 2003). The parameters and time scales of these NMR experiments can provide considerable information about the ensemble-averaged structural properties of unfolded proteins (Shortle 1996; Dyson and Wright 2001 Dyson and Wright 2002). One type of structure detected by NMR in unfolded proteins is a hydrophobic cluster, usually formed by local side-chain interactions. The first reported instance of a hydrophobic cluster populated in high urea concentration was in the case of the N-terminal domain of the 434-repressor (Neri et al. 1992). More recently, hydrophobic clusters in urea-unfolded states have been reported for rat intestinal fatty acid binding protein (Hodsdon and Frieden 2001) and apomyoglobin (Lietzow et al. 2002; Schwarzinger et al. 2002). Similarly, hydrophobic clusters were also observed in HIV-1 protease tethered dimer unfolded by guanidine hydrochloride (Bhavesh et al. 2003). For these proteins, the initial collapse of the poly-peptide chain that occurs when refolding is initiated by dilution of denaturant is likely to be a consequence of transient interactions between hydrophobic clusters that persist in the unfolded state. Another type of structure detected by NMR in unfolded proteins is fluctuating secondary structure, for example in urea-unfolded barnase (Arcus et al. 1995; Wong et al. 2000). It has been suggested that the folding of barnase is initiated around the native-like local structures that persist in the unfolded state until enough stabilizing long-range interactions form a nucleation site onto which further secondary structure formation and final native packing of tertiary structure occur (Wong et al. 2000).

For some proteins, multiple types of structures have been detected in the unfolded state. For example, for the drkN SH3 domain, both the unfolded state under folding conditions (Uexch) and the unfolded state in guanidine hydrochloride are reasonably compact (Farrow et al. 1995, 1997; Choy et al. 2002). Detailed NMR studies of Uexch (Mok et al. 1999; Crowhurst et al. 2002; Crowhurst and Forman-Kay 2003) indicate the presence of multiple structures ranging from conformers with nonnative structure possessing long-range contacts to those with more compact structures maintaining native-like secondary structure. Consequently, it has been suggested that drkN SH3 might fold via multiple folding pathways, conformers with nonnative hydrophobic clusters folding via a hydrophobic collapse mechanism and those with native-like structures folding via a hierarchical condensation mechanism (Crowhurst and Forman-Kay 2003). On the other hand, when the unfolded state appears to be largely unfolded, as determined to be so for the chymotrypsin inhibitor 2 (Kazmirski et al. 2001), the protein is thought to fold in a more cooperative manner with the establishment of long-range contacts linked to collapse and secondary-structure formation (Kazmirski et al. 2001). Clearly, identification of any residual structure and hydrophobic clusters in the unfolded state of a protein plays a crucial role toward obtaining insights into its folding mechanism (Fersht and Daggett 2002; Daggett and Fersht 2003; Gianni et al. 2003).

NMR studies appear to indicate the persistence of native-like structure in the unfolded states of hen lysozyme (Klein-Seetharaman et al. 2002) and the Δ131Δ fragment of staphylococcal nuclease (Shortle and Ackerman 2001; Ackerman and Shortle 2002). The native-like core in the unfolded state of hen lysozyme is stabilized by the involvement of a tryptophan residue in nonnative long-range tertiary interactions (Schwalbe et al. 1997; Klein-Seetharaman et al. 2002). In the case of the Δ131Δ fragment of staphylococcal nuclease, which has been characterized extensively as a model for the unfolded state of the protein under nondenaturing conditions (Alexandrescu et al. 1994), the values observed for the dipolar couplings have been interpreted to indicate a native-like topology that might arise from local steric interactions between side chains and the protein backbone (Srinivasan and Rose 1999; Pappu et al. 2000; Ohnishi and Shortle 2003). More recently, however, this interpretation has been questioned because it has been shown that nonzero residual dipolar couplings of denatured states can also arise from chain-like molecules without a defined three-dimensional (3D) spatial organization (Louhivuori et al. 2003). The topology of the native state is a major determinant of the folding rate (Clementi et al. 2000; Makarov et al. 2002; Miller et al. 2002), and if native-state topologies are indeed present in the unfolded states of these two proteins, it would imply that the entropic cost of bringing two regions of the polypeptide chain together so that they can interact is borne by the unfolded state itself.

Local structural features such as propensity of the polypeptide backbone to populate preferentially the α or β region of ϕ, ψ space, or to exist in a random coil conformation can be derived from chemical shifts (Wishart and Sykes 1994). Here, we report NMR data on backbone conformational propensities in the unfolded state of barstar in 8 M urea (pH 6.5) at 25°C. Barstar is an 89-amino-acid residue, single-domain α/β protein. Its native structure has been solved by NMR, and consists of three parallel helices packed against a three-stranded parallel β-sheet and a fourth more poorly defined helix that connects the second β-strand and third major helix (Fig. 1 ▶; Lubienski et al. 1994). The refolding of barstar from high denaturant concentrations has been characterized extensively, and exhibits multistate kinetics (Schreiber and Fersht 1993; Agashe et al. 1995; Shastry and Udgaonkar 1995; Bhuyan and Udgaonkar 1999; Killick et al. 1999; Sridevi et al. 2000). It is therefore important to get structural information about the unfolded state of the protein to understand the early events of folding. In this study of the unfolded state of barstar in 8 M urea, secondary chemical shifts indicate that segments that are helical in the native protein do not show any preference for helical structure in the unfolded state. Residues in β-strand 3, however, show nonnative helical conformational preferences. The implications of these observations for the folding of barstar are discussed.

Figure 1.

Figure 1.

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NMR structure of barstar (PDB ID 1BTA; Lubienski et al. 1994).

Results and Discussion

Backbone resonance assignments of barstar in 8 M urea

Structural characterization by NMR is often hampered in unfolded and partially folded states of proteins because of the poor dispersion of amide and carbon resonances in these forms. Although this problem is overcome in favorable cases by carrying out standard triple resonance and NOESY experiments on a 15N- and 13C-labeled protein sample (Meekhof and Freund 1999; Schwarzinger et al. 2002), these experiments were not successful in obtaining sequential assignments of guanidine-unfolded HIV-protease (Bhavesh et al. 2001). The novel 3D NMR experimental procedures of HNN and HN(C)N provide a new protocol for the sequential assignment of both folded and unfolded forms of proteins (Panchal et al. 2001). These methods use better 15N dispersion along two of the three dimensions in the 3D triple resonance experiments and have been used successfully to assign backbone amide resonances in unfolded HIV-protease (Bhavesh et al. 2001) and to study the aggregated molten globule state of barstar at pH 3 (Juneja et al. 2002).

The complete HN and 15N backbone resonance assignments of barstar unfolded in 8 M urea at pH 6.5 and 25°C were obtained by 3D HNN and HN(C)N experiments. Figure 2 ▶ shows an illustrative sequential walk through the F1(15N)–F3(HN) planes for the stretch Gly 81 to Glu 76 in the HNN spectrum of the unfolded protein. All the back-bone amide assignments obtained for unfolded barstar in 8 M urea at pH 6.5 are indicated in the 1H–15N HSQC spectrum in Figure 3 ▶. It is satisfying to note that despite the small chemical shift dispersion (the chemical shifts of HN are within a range of 0.7 ppm), as is expected for a disordered state, we were able to obtain complete assignments. Following these assignments, 13Cα, 13Cβ, and 13CO resonances were assigned from CBCANH (Grzesiek and Bax 1992a), CBCA(CO)NH (Grzesiek and Bax 1992b), and HNCO (Kay et al. 1990) triple resonance experiments in a straightforward manner. All the assigned chemical shifts have been listed in Supplemental Table 1. They have also been deposited in the BMRB database (accession no. 6227).

Figure 2.

Figure 2.

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Illustrative sequential walk through F1–F3 planes in the HNN spectrum of barstar in 8 M urea at pH 6.5 and 25°C. Sequential connectivities are indicated for the Gly 81 to Glu 76 stretch in the protein primary sequence. Strips from the spectra at the appropriate HN chemical shifts are shown. Black and red contours are positive and negative peaks, respectively. The distinct Gly 81 plane in the HNN spectrum served as a starting triplet fixed point to obtain sequential connectivities. The F2 (15N) chemical shift is indicated at the top of each strip of the HNN spectrum (for details, see Bhavesh et al. 2001 and Panchal et al. 2001).

Figure 3.

Figure 3.

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1H–15N HSQC spectrum of barstar in 8 M urea at pH 6.5 and 25°C. The HN and 15N backbone assignments are indicated.

Secondary chemical shifts

In native protein structures, the 13Cα resonances in α-helices are shifted downfield typically by an average of 3.1 ± 1.0 ppm, and the 13Cα resonances in β-sheets are shifted upfield typically by −1.5 ± 1.2 ppm (Dyson and Wright 2001). In contrast, in unfolded states of proteins, which are highly dynamic, the observed NMR parameters are a population-weighted average of all structures in the conformational ensemble; consequently, the observed secondary shifts are considerably smaller. Nevertheless, deviations of chemical shifts from random coil values reflect the relative population of dihedral angles in the α and β regions of conformational space (Dyson and Wright 2001). Chemical shifts are the only NMR parameters that can detect even small conformational propensities of the protein backbone in disordered states of proteins, which cannot be detected by NOEs or coupling constants.

Among the various secondary chemical shifts, those of Hα, 13Cα, and 13CO are diagnostic of structural information in the polypeptide chain (Dyson and Wright 2001, 2002). Here we have used 13Cαand 13CO secondary shifts for barstar in 8 M urea at pH 6.5 and 25°C, and these are shown in Figure 4 ▶. Positive secondary shifts for 13Cα and 13CO indicate a preference for ϕ, ψ angles in the helical conformation, whereas negative secondary shifts indicate a preference for ϕ, ψ angles in the β-sheet conformation. The secondary chemical shifts of barstar unfolded in 8 M urea are small because of rapid conformational averaging, as expected for a disordered state of a protein (Dyson and Wright 2001, 2002). Several residues in the urea-unfolded state of barstar that form helices in the native structure (Fig. 1 ▶), however, show definite positive deviations of the 13Cα chemical shifts (Fig. 4A ▶), indicating a propensity of the polypeptide backbone toward populating the helical region of ϕ, ψ space. In fact, some residues show positive deviations of ≥0.6 ppm. These include Ile 13, Ser 14, His 17, Lys 21, and Lys 22, which belong to helix 1 in native barstar; Cys 40, which belongs to helix 2; Gln 58 to helix 3; and Val 70, which belongs to helix 4 (Lubienski et al. 1994). Ala 25 at the C terminus of helix 1 and Glu 57 and Gly 81, which are at the N and C termini of helices 3 and 4, respectively, also show ≥0.6-ppm secondary shifts. On the other hand, there are a few residues in the segments that are helical in the native structure, which show negative secondary shifts indicating a propensity toward an extended or β-strand conformation in the urea-unfolded protein. In helix 1 (Ile 13–Leu 24), five residues have negative 13Cα secondary shifts; Asp 15 has an upfield shift of 0.5 ppm, whereas the other four residues have ≤0.3-ppm upfield chemical shifts. Most of the residues with positive deviations of 13Cα resonances in helix 1, however, show ≥0.6-ppm downfield shifts. In helix 2 (Leu 34–Leu 41), four residues show negative and four residues show upward 13Cα secondary shifts between 0.3 and 0.5 ppm except Cys 40, which has a positive secondary shift of >0.6 ppm. In helix 3 (Gln 58–Gln 61), two residues show very small negative 13Cα secondary shifts. In helix 4 (Ala 67–Ala 79), five residues have negative 13Cα secondary shifts; Glu 68 shows a 0.5-ppm negative shift, and the other four residues show ≤0.4-ppm negative shifts. Most of the residues in helix 4 have positive 13Cα secondary shifts of ≥0.4 ppm.

Figure 4.

Figure 4.

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Secondary chemical shifts for (A) Cα and (B) CO resonances in barstar in 8 M urea at pH 6.5 and 25°C. The chemical shifts have been corrected for contributions from the local amino acid sequence (Schwarzinger et al. 2001). Reference random coil shifts used are those determined for the peptide Ac-GGXGG-NH2 in 8 M urea at pH 2.3 (Schwarzinger et al. 2000). The ϕ, ψ backbone dihedral angles for native barstar are plotted against the primary sequence in C and D, respectively. The native secondary structural elements are indicated at the top in the figure.

The deviations from random coil values for the 13CO resonances of barstar in 8 M urea are small. These secondary shifts, in general, follow the pattern of 13Cα secondary shifts, as seen in Figure 4B ▶. There appears to be a clustering of positive secondary shifts in the regions of the polypeptide that form helices 1 and 2 in the native state, again indicating a propensity of the main-chain backbone of these residues for a helical conformation. Residue Leu 37 in helix 2, however, shows a negative 13CO secondary shift of ~0.5 ppm. Residues corresponding to helices 3 and 4 in native barstar show a distribution of positive and negative shifts suggesting conformational averaging in these regions in the unfolded state.

The analysis of the chemical shifts of sequence-corrected 13Cα and 13CO resonances in urea-unfolded barstar suggests that many residues that form helices in the native protein preferentially adopt ϕ, ψ angles corresponding to the helical region of conformational space even in the unfolded state. These residues are, however, separated by residues showing negative 13Cα and 13CO secondary shifts. In a helix, hydrogen bonds are formed between residues i and i + 4, and it is the enthalpy of the hydrogen bonds that compensates for the loss of entropy that occurs when residues are fixed into a helical conformation. In the absence of the stabilizing H-bonds, as in the case of a single amino acid or two to three consecutive amino acids showing positive 13Cα and 13CO secondary shifts, it is too costly, entropy-wise, to form the first turn of a helix. Thus, it is unlikely that any residual native-like helical structure exists in the urea-unfolded state, in those segments that form helices in native barstar. For the acid-unfolded state of apomyoglobin and the low pH state of acyl-coenzyme A binding protein, residual helical structure was indicated by stretches of consecutive residues showing positive 13Cα and 13CO secondary shifts in regions corresponding to some of the native helices (Yao et al. 2001; Thomsen et al. 2002).

In the regions of urea-unfolded barstar corresponding to β-strands 1 and 2, 13Cα and 13CO secondary shifts indicate rapid conformational averaging between the α and β regions of ϕ, ψ space so that the polypeptide chain in these stretches of the protein does not appear to preferentially populate either region in the unfolded state. Most residues corresponding to β-strands 1 and 2 in the native protein have secondary shifts close to random coil values, as well as a distribution of positive and negative shifts. In β-strand 2, Leu 51 has a positive 13Cα shift of >0.5 ppm, but Val 50 and Glu 52 show negative 13Cα shifts of ~0.5 ppm. Interestingly, most of the residues that belong to β-strand 3 (Ile 84–Ser 89) in native barstar have positive 13Cα secondary shifts of ≥0.5 ppm, suggesting that these show nonnative helical conformational propensities in the unfolded state. This is confirmed by the observation of positive 13CO secondary shifts for all residues corresponding to β-strand 3, with Ile 87 and Ser 89 showing downfield shifts of ≥0.5 ppm.

Comparison of 8 M urea-unfolded barstar and cold denatured barstar

In an earlier NMR study of the cold denatured state of the C40/82A mutant form of barstar in 3 M urea and 5°C, chemical shift analysis supported by NOE data provided evidence for native-like residual helical structure in the segments corresponding to helices 1 and 2 in the native structure (Wong et al. 1996). Although our results also indicate that several residues in the helical regions of native barstar show helical conformational propensities in its urea-unfolded state, these are juxtaposed by residues showing propensities toward an extended conformation. As discussed above, it is therefore very unlikely that these segments form helices in the urea-unfolded state. It may be noted that as shown in Figure 4 ▶, C and D, there is some degree of variation observed in the ψ torsion angles in native barstar as well, in that some residues in helices 1 and 3 show positive values. Residues that form helices are, in general, characterized by negative values of ψ and those that form β-strands by positive values of ψ.

Wong et al. (1996) also observed nonnative helical residual structure in part of the second β-strand together with the loop connecting it to helix 3, and suggested that this may exist because of the formation of a hydrophobic cluster involving Trp 53 and Phe 56. In this study of the unfolded form in 8 M urea, residues Leu 51 and Trp 53–Glu 57 in this region along with Gln 58 in helix 3 show helical conformational propensities: Leu 51, Gln 55, Glu 57, and Gln 58 have positive 13Cα shifts of ≥0.6 ppm. This suggests that non-native residual helical structure may exist in this region even in urea-unfolded barstar. Rearrangement of this stretch of the polypeptide chain may occur during folding to enable spatial positioning of the native topology.

In addition, it is observed in this study that residues in the native β-strand 3 segment appear to show a preference for populating a nonnative helical conformation in the 8 M urea-unfolded form. In contrast, the C-terminal segment from Asn 65 to Ser 89, which corresponds to helix 4 and β-strand 3 in folded barstar, shows a random coil conformation in the cold-denatured protein (Wong et al. 1996). The differences in random versus nonrandom behavior of some segments of the backbone between the cold-denatured state and the urea-unfolded state of barstar probably arise because of the different methods of denaturation. Whereas cold denaturation has an effect on the enthalpy and entropy of the protein (Privalov 1990), denaturation by urea occurs as a consequence of altered solvent properties (Schiffer and Dotsch 1996). Urea changes properties such as viscosity, dielectric constant, and surface tension of the solution, which would have an effect on the folding and unfolding free energies (Graziano 2001). In future work, it will be necessary to obtain NOE data to confirm the analysis of secondary shifts, and the existence of residual native or nonnative structure in the urea-unfolded state of the protein.

Implications for the folding of barstar

Helices 1 and 2 have been suggested to be the initiation sites for barstar folding from the cold-denatured state (Wong et al. 1996). In contrast, it appears unlikely that these helices serve as initiation sites for barstar folding from the 8 M urea-unfolded state, because none of the helical regions of native barstar appear to have a preference for helical conformations in the 8 M urea-unfolded state. It is not surprising, therefore, that during the folding of barstar, the formation of specific secondary structure is seen to occur only after the initial collapse of the polypeptide chain (Agashe et al. 1995; Rami and Udgaonkar 2002; Pradeep and Udgaonkar 2004). The NMR results presented here suggest that the segment that forms β-strand 3 in the folded protein has some preference for nonnative helical structure in the unfolded state, but it is not known, at present, whether any of the intermediates on the folding pathway of barstar possess nonnative structure. The folding of barstar has been shown to occur via multiple competing pathways (Shastry and Udgaonkar 1995; Bhuyan and Udgaonkar 1999; Sridevi et al. 2004), and it remains to be seen if conformers with different native and nonnative structures in the unfolded state fold via different routes.

Materials and methods

Protein purification and NMR sample preparation

The barstar gene in pMT316 plasmid was expressed in MM294 Escherichia coli cells and purified as described earlier (Khurana and Udgaonkar 1994). For uniform labeling of the protein either singly with 15N or doubly with 15N and 13C, bacteria were grown in M9 minimal media containing 15NH4Cl and 13C-glucose as the only sources of nitrogen and carbon, respectively, as described (Juneja et al. 2002). The protein used for all experiments was >95% pure.

The sample for NMR experiments was prepared by dissolving lyophilized protein in 20 mM sodium phosphate buffer containing 8 M urea (pH 6.5) to a final concentration of 1.1 mM.

NMR spectroscopy

All NMR spectra were collected on a 600 MHz Varian Unity Plus spectrometer at 25°C. Typical spectral widths for all 2D and 3D experiments recorded were 7002.8 Hz for 1H, 1460 Hz for 15N, 13,500 Hz for 13Cα β, and 2500 Hz for 13CO. The 1H and 15N carrier frequencies were set at 4.71 ppm (water) and 120 ppm, respectively. The 13C carrier frequency was set at 56.0 ppm for HNN, HN(C)N, 45.0 ppm for CBCANH and CBCA(CO)NH, and 174.0 ppm for HNCO. In all spectra, 1H, 13C, and 15N chemical shifts were referenced, respectively, to HDO (4.71 ppm at 32°C), indirectly to 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), and to trimethylsilyl sodium propionate (TSP).

The 2D 1H–15N HSQC spectrum was recorded with 512 complex t1 increments, 8192 t2 points, and four scans for each fid. The 3D HNN spectrum was recorded with the following parameters: 32 complex points along t1 (15N) and t2 (15N) and 1024 complex points along t3 (HN), 16 scans for each fid, and TN = TC = 14 msec. The 3D HN(C)N spectrum of the same sample was recorded using parameters identical to those used for the HNN experiment. The TCC delay was set to 4.5 msec. The acquisition time for the two experiments was ~26 h each. CBCANH and CBCA(CO)NH spectra were acquired with 1024 (HN), 32 (15N), and 80 (13C) complex points, and an HNCO spectrum was acquired with 1024 (HN), 32 (15N), and 50 (13C) complex points.

Secondary chemical shifts

The deviations of specific chemical shifts (secondary shifts) from their random coil values are highly sensitive to conformational preferences of the protein backbone (Wishart and Sykes 1994). Because the chemical shifts are influenced both by neighboring amino acids and local backbone structure, it was important to correct these for contributions from the local amino acid sequence (Schwarzinger et al. 2000). The random coil values used in the secondary shift analysis (Schwarzinger et al. 2000) were corrected using sequence-dependent correction factors determined for a set of Ac-GGXGG-NH2 peptides in 8 M urea at pH 2.3 (Schwarzinger et al. 2001). Deviations in specific chemical shifts were then calculated by subtracting the corrected random coil values from the measured chemical shifts for all the residues in urea-unfolded barstar.

Acknowledgments

All NMR spectra have been recorded in the National High-Field NMR facility at the Tata Institute of Fundamental Research, Mumbai. This work was funded by TIFR and by the Wellcome Trust.

Abbreviations

  • NMR, nuclear magnetic resonance

  • HSQC, hetero-nuclear single-quantum coherence

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04805204.

Supplemental material: see www.proteinscience.org

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