Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement
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- Nabuurs, S.M., de Kort, B.J., Westphal, A.H. et al. Eur Biophys J (2010) 39: 689. doi:10.1007/s00249-009-0556-4
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Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin.
KeywordsUnfolded protein Flavodoxin α–β Parallel protein Paramagnetic relaxation enhancement MTSL CMTSL
Understanding the molecular mechanisms of protein folding is one of the fundamental challenges of structural biology. Proteins initially fold from disordered unfolded states. Transient structures in unfolded proteins are important in elucidation of the molecular details of initiation of protein folding. Early observations of residual structure in unfolded proteins prompted the suggestion that native-like structure is present in the denatured state (Gillespie and Shortle 1997; Yi et al. 2000; Lietzow et al. 2002) and that this residual structure biases the subsequent conformational search toward the native conformation (Yi et al. 2000; Daggett and Fersht 2003). However, evidence of non-native structure has been found in the unfolded states of few proteins (Kristjansdottir et al. 2005; Platt et al. 2005; Reed et al. 2006; Marsh et al. 2007).
Recently, non-native secondary structure and non-native hydrophobic interactions have been observed in the unfolded state of a 179-residue flavodoxin from Azotobacter vinelandii (Nabuurs et al. 2008), which is the protein of interest in the study presented here. Remarkably, structure formation in unfolded flavodoxin does not direct folding to the native state, but instead causes formation of a misfolded off-pathway intermediate (Nabuurs et al. 2009a).
Both denaturant-induced equilibrium and kinetic (un)folding of flavodoxin and apoflavodoxin (i.e., flavodoxin without FMN) have been characterized using guanidine hydrochloride (GuHCl) as denaturant (van Mierlo et al. 1998; van Mierlo and Steensma 2000; Bollen et al. 2004, 2005, 2006; Bollen and van Mierlo 2005). The folding data show that apoflavodoxin autonomously folds to its native state, which is structurally identical with that of flavodoxin except that residues in the flavin-binding region of the apo protein have considerable dynamics (Steensma et al. 1998; Steensma and van Mierlo 1998). Binding of FMN to native apoflavodoxin is the last step in flavodoxin folding.
Apoflavodoxin kinetic folding involves an energy landscape with two folding intermediates and is described by: Ioff ↔ unfolded apoflavodoxin ↔ Ion ↔ native apoflavodoxin (Bollen et al. 2004). Intermediate Ion lies on the productive route from unfolded to native protein, is highly unstable and is, therefore, not observed during denaturant-induced equilibrium unfolding of apoflavodoxin. Consequently, GuHCl-induced equilibrium unfolding of apoflavodoxin is described by: Ioff ↔ unfolded apoflavodoxin ↔ native apoflavodoxin (Bollen et al. 2004). Approximately 90% of folding molecules fold via molten globule-like off-pathway intermediate Ioff, which is a relatively stable species that needs to unfold to produce native protein and thus acts as a trap (Bollen et al. 2004). Elevated protein concentrations (van Mierlo et al. 2000) and molecular crowding (Engel et al. 2008) cause severe aggregation of this species. The formation of an off-pathway species is typical for proteins with a flavodoxin-like topology (Bollen and van Mierlo 2005). An off-pathway intermediate is experimentally observed for all other α–β parallel proteins of which the kinetic folding has been investigated, i.e., apoflavodoxin from Anabaena (Fernandez-Recio et al. 2001), CheY (Kathuria et al. 2008), cutinase (Otzen et al. 2007), and UMP/CMP kinase (Lorenz and Reinstein 2008).
To better understand why the off-pathway intermediate is formed during flavodoxin folding, GuHCl-unfolded apoflavodoxin has been characterized at the residue-level using heteronuclear NMR spectroscopy (Nabuurs et al. 2008, 2009a). Secondary shifts analysis and investigation of 1H–15N relaxation rates reveal four structured elements that transiently exist in unfolded apoflavodoxin. These transiently ordered regions have restricted flexibility on the (sub)nanosecond timescale and comprise residues Ala-41–Gly-53, Glu-72–Gly-83, Gln-99–Ala-122, and Thr-160–Gly-176 (Nabuurs et al. 2008). These regions match with regions of large average area buried upon folding (AABUF), which correlates with hydrophobicity (Rose and Roy 1980) and corresponds to sequence-dependent dynamic variations due to hydrophobic interactions in unfolded proteins (Schwarzinger et al. 2002; Le Duff et al. 2006).
Restricted flexibility in unfolded apoflavodoxin is due to transient helix formation and local and non-local hydrophobic interactions (Nabuurs et al. 2008, 2009a). On reducing the denaturant concentration, the four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule (Nabuurs et al. 2008, 2009a). As a consequence, the molten globule has a totally different topology compared with native apoflavodoxin: it is helical and contains no β-sheet (Nabuurs et al. 2009b). Structure formation within virtually all parts of unfolded apoflavodoxin precedes folding to the molten globule state. This folding transition is non-cooperative and involves a series of distinct transitions (Nabuurs et al. 2009a). Part of Ioff remains random coil down to a GuHCl concentration of 1.58 M (i.e., residues Lys-13 to Val-36).
Wild-type flavodoxin contains a single cysteine at position 69. This cysteine is poorly accessible to solvent in the holo form of the protein, because of the presence of FMN. However, in apoflavodoxin it is solvent accessible and can be used to attach a spin-label to the protein. Another variant of apoflavodoxin with a single cysteine was made for the purpose of the study presented here. In the latter variant, Gln-48, which resides in the non-native α-helix that is formed in unfolded apoflavodoxin (Nabuurs et al. 2008), is replaced by cysteine. In native apoflavodoxin Gln-48 and Cys-69 are positioned at opposite sites of the protein (Fig. 1).
Here, we show that attachment of a hydrophobic spin-label leads to hydrophobic interactions between the spin-label and various residues of an unfolded protein. Such perturbation of an unfolded protein has not been reported previously, despite the common use of spin-labels to characterise unfolded proteins. Still, even though this perturbation exists, valid information about residual structure in unfolded apoflavodoxin is obtained.
Materials and methods
Site-directed mutagenesis was used to replace the single cysteine in wild-type A. vinelandii (strain ATCC 478) flavodoxin II (i.e., WT flavodoxin) by alanine (i.e., C69A flavodoxin, which is largely similar to WT flavodoxin (Steensma et al. 1996; van Mierlo et al. 1998)). In addition, glutamine at position 48 was replaced by cysteine, resulting in the double mutant C69A-Q48C flavodoxin, hereafter named Q48C flavodoxin.
Uniformly 15N-labelled Q48C and C69A flavodoxins were obtained from transformed E. coli cells grown on 15N-labelled algae medium (Silantes, Germany). Uniformly 15N-labelled WT flavodoxin was obtained from transformed E. coli cells grown on 15N-labelled minimal medium. All protein variants were purified as described elsewhere (van Mierlo et al. 1998).
Unfolded apoflavodoxin was obtained by denaturing flavodoxin in 6 M GuHCl. Subsequently, FMN was removed via gel filtration in 5 M GuHCl. WT and Q48C protein variants were labelled with MTSL or CMTSL (Toronto Research Chemicals, Toronto, Canada) by adding a 3:1 molar ratio of spin-label to unfolded protein at room temperature. After 2 h, free spin-label was separated from labelled protein by gel filtration. Spin-labelled WT and Q48C apoflavodoxin are referred to as WT(C)MTSL and Q48C(C)MTSL apoflavodoxin, respectively.
All NMR samples contained about 0.3–0.5 mM apoflavodoxin, 10% D2O, and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) as internal chemical shift reference. To avoid covalent dimerisation of protein molecules that are not spin-labelled, a sufficient amount of DTT was present in samples of WT and Q48C apoflavodoxin.
The buffer used in all experiments was 100 mM potassium pyrophosphate (KPPi), pH 6.0, and refractometry was used to verify GuHCl concentration (Nozaki 1972).
Gradient enhanced 1H–15N HSQC spectra were recorded on a Bruker Avance 700 MHz machine. Sample temperature was kept at 25°C. In the 1H dimension of the 1H–15N HSQC experiments, 2048 complex data points were acquired, whereas in the indirect 15N dimension 360 complex data points were collected. Spectral widths were 6010 and 1750 Hz in t2 and t1, respectively, and the number of scans was 16. All NMR experiments performed with spin-labelled protein in the paramagnetic state were repeated using spin-labelled protein in the diamagnetic state. Diamagnetic protein was obtained by adding 5 μl concentrated stock solution of ascorbic acid to the NMR samples containing paramagnetic protein (the resulting dilution of the NMR sample was less than 1%). This addition resulted in a threefold molar excess of ascorbic acid compared with protein.
Thermal unfolding of C69A apoflavodoxin, Q48C apoflavodoxin, and Q48CMTSL apoflavodoxin was followed by fluorescence emission. Protein unfolding was achieved by increasing the temperature in a 1.5-ml stirred quartz cuvette (path-length 0.4 cm) from 20 to 70°C at a rate of 0.5°/min. Temperature was measured in the cuvette by using an internal probe. The excitation wavelength used was 280 nm, and fluorescence emission was recorded at 340 nm. Excitation and emission slits were set at 5 nm. In thermal unfolding experiments, protein was in 100 mM KPPi, pH 6.0, and protein concentration ranged between 4 and 6 μM.
Thermally induced equilibrium unfolding data
Results and discussion
Introducing a cysteine at position 48 and subsequent labelling with MTSL both reduce the thermal midpoint of unfolding of apoflavodoxin
Introducing a cysteine at position 48 changes the dynamic features of unfolded apoflavodoxin
Comparison of chemical shifts of cross peaks in 1H–15N HSQC spectra of Q48C and C69A apoflavodoxin in 3.4 M GuHCl previously showed that replacement of a glutamine by a cysteine at position 48 leads to chemical shift changes in unfolded apoflavodoxin. These chemical shift changes indicate long-range non-native interactions between transiently formed helices in unfolded apoflavodoxin (Nabuurs et al. 2008).
Unfolded Q48C apoflavodoxin in 6.0 M GuHCl behaves as a random coil; subsequent attachment of MTSL causes the protein to become more ordered
Far-UV CD data and transverse relaxation rates (Nabuurs et al. 2008) show that C69A apoflavodoxin in 6.0 M GuHCl behaves as a random coil. The Q48C variant of apoflavodoxin is also a random coil in 6.0 M GuHCl. This random coil behaviour is concluded because the 1H–15N HSQC spectra of both Q48C and C69A apoflavodoxin at this concentration denaturant are very similar regarding both cross peak positions and cross peak intensities (data not shown).
In conclusion, attachment of MTSL to unfolded Q48C apoflavodoxin in 6.0 M GuHCl causes the protein to behave in a more ordered fashion than random coil apoflavodoxin.
Use of PRE experiments
Magnetic interaction between an unpaired electron in a paramagnetic nitroxide spin-label and a nearby proton causes broadening of the corresponding 1H NMR signal because of the increased transverse relaxation rate of the proton involved (Gillespie and Shortle 1997). This relaxation rate has an r−6-dependence on electron–proton distance and thus enables detection of long-range interactions in proteins. Consequently, ratios of cross peak intensities (Ipara/Idia) extracted from two redox state-dependent 1H–15N HSQC spectra of the spin-labelled protein enable estimation of distances between the spin-label and affected protons in the protein (Teilum et al. 2002). Because of the ubiquitous backbone fluctuations in unfolded apoflavodoxin (Nabuurs et al. 2008), no attempt is made to extract quantitative distances from the data. Rather, a trend in PRE as a function of primary structure can be observed, as shown in the following discussion.
PRE shows that WTMTSL apoflavodoxin in 6.0 M GuHCl does not behave as a random coil, because of hydrophobic interactions between MTSL and unfolded protein
In contrast with the above observation, PRE data of Q48CMTSL apoflavodoxin (Fig. 6b) show no evidence of an interaction between Cys-48 and Ala-69. Note however, as discussed in a previous section, that attachment of MTSL to unfolded Q48C apoflavodoxin in 6.0 M GuHCl causes the protein to behave more ordered than random coil apoflavodoxin. The nitroxide radical of MTSL attached to Cys-48 does not broaden resonances of residues in the vicinity of Ala-69 (the cross peak of the backbone amide of Ala-69 suffers from severe overlap and is thus not assigned). The interaction between the MTSL spin-label attached to Cys-69 and region Ser-40–Leu-62 of unfolded apoflavodoxin in 6.0 M GuHCl is thus due to hydrophobic interactions of the label with residues in this region. Similar hydrophobic interactions with MTSL spin-label were also observed in dimerization studies of ARNT PAS-B (Card et al. 2005).
In both WTCMTSL and Q48CCMTSL apoflavodoxin unfolded in 3.4 M GuHCl the spin-label interacts with four transiently structured regions
To reduce hydrophobic interactions in unfolded apoflavodoxin due to MTSL, another spin-label, i.e., CMTSL (Card et al. 2005), which is more hydrophilic than MTSL, was chosen as paramagnetic relaxation agent. Compared with MTSL, CMTSL contains an additional amide group in the linker between protein and the proxyl ring (Fig. 2), thereby introducing a degree of polarity into an otherwise hydrophobic compound.
In Fig. 7c and d PRE data of WTCMTSL and Q48CCMTSL apoflavodoxin in 3.4 M GuHCl are shown. In both WTCMTSL and Q48CCMTSL apoflavodoxin the CMTSL spin-label interacts with various regions of the unfolded protein. In particular, interactions are observed between the spin-label and the four transiently structured regions in unfolded apoflavodoxin in 3.4 M GuHCl, strengthening our previous findings (Nabuurs et al. 2008) that non-native contacts do, indeed, exist between transiently ordered structured elements in unfolded apoflavodoxin.
In Q48CCMTSL apoflavodoxin unfolded in 3.4 M GuHCl, the spin-label interacts with residues spread over the entire sequence of the protein. Residues 40 to 54 and Leu-78 stand out, as the corresponding backbone amide resonances are broadened beyond detection in the paramagnetic state of the unfolded protein. These residues will be discussed in the next section. In addition, resonances of residues 82 to 129 are more severely broadened than the resonances of most of the residues of unfolded Q48CCMTSL apoflavodoxin.
The attached spin-label induces the presence of two distinct states in unfolded apoflavodoxin
A plausible explanation of the observed doubling of cross peaks is that the attached spin-label induces the presence of two distinct states in unfolded apoflavodoxin. In one of these states the spin-label is in the proximity of the above-mentioned residues whereas in the other state it is not. Both folding states are in slow exchange with one another on the NMR chemical shift time scale, because two separate, sharp cross peaks are observed per backbone amide of the residues discussed.
Leu-78 is the only residue that is not sequentially close to the CMTSL-label at position 48, but nevertheless gives rise to two backbone amide cross peaks. Indeed, chemical shift deviations upon replacing residue 48 show that interactions between Cys-48 and Leu-78 must exist in unfolded apoflavodoxin (Nabuurs et al. 2008). Residual structure that is neither an α-helix nor a β-sheet is found in the region Glu-72–Gly-83 of unfolded apoflavodoxin (Nabuurs et al. 2008). These observations are further support for the existence of persistent hydrophobic interactions between Leu-78 and the CMTSL spin-label in one of the two distinct states within unfolded Q48CCMTSL apoflavodoxin. The tertiary interaction between residues 48 and 78 in unfolded apoflavodoxin, as revealed by PRE experiments, must be non-native. The latter is concluded because complete disappearance of the backbone amide cross peak of Leu-78 implies that the distance between residues 48 and 78 must be much shorter than the 17.28 Å distance between the corresponding Cα atoms in native flavodoxin (calculated using the X-ray structure of A. vinelandii flavodoxin (Alagaratnam et al. 2005)).
Introducing cysteine residues in (unfolded) proteins to enable labelling with probes is used frequently (Fanucci and Cafiso 2006; Clore et al. 2007). To detect long-range interactions within (natively) unfolded proteins, for example apomyoglobin (Lietzow et al. 2002), α-synuclein (Bertoncini et al. 2005), ACBP (Teilum et al. 2002), and N-PGK (Cliff et al. 2009), MTSL is widely used as a nitroxide spin-label. However, the results presented here show that in an unfolded protein covalent attachment of a spin-label to cysteine can introduce hydrophobic interactions between the hydrophobic spin-label and various amino acid residues. These interactions alter the hydrophobic free energy (Baldwin 2005) of the unfolded state (and possibly of the off-pathway intermediate) and, as a consequence, the free energy difference between native apoflavodoxin and non-native protein molecules diminishes. Indeed, covalent attachment of a MTSL-label can alter the stability of the protein involved, as is shown here for native apoflavodoxin and as is reported for ACBP variants (Teilum et al. 2002). Remarkably, in contrast with unfolded apoflavodoxin, no hydrophobic interactions, either short-range or long-range, with the MTSL-label have been reported for the unfolded proteins mentioned. Neither has doubling of cross peaks of these spin-labelled unfolded proteins been reported.
Native apoflavodoxin is an α–β parallel protein and contains in its core many hydrophobic residues that are shielded from the solvent. It is possible that in unfolded protein these residues are susceptible to hydrophobic interactions with the hydrophobic nitroxide spin-labels used. These interactions can give rise to the existence of distinct states in the unfolded protein, as this work shows. Despite the difficulties mentioned, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin.
The Netherlands Organization for Scientific Research supported this work. NMR spectra were recorded at the Utrecht Facility for High-Resolution NMR, The Netherlands.
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