Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins
1Departments of Chemistry, Yale University, Bass 322, 266 Whitney Avenue, New Haven, CT 06520-8114, USA 2Molecular Biophysics and Biochemistry, Yale University, Bass 322, 266 Whitney Avenue, New Haven, CT 06520-8114, USA 3Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, UK 4Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
3 To whom correspondence should be addressed. E-mail: lynne.regan{at}yale.edu
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The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k0f = 29 s–1) and unfolds (k0u = 6 x 10–7 s–1) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.
Keywords: four-helix bundle/homologues/protein folding/protein stability/RNA-binding
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Introduction |
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Proteins fold with differing mechanisms and a wide range of time constants that vary from microseconds to hours. Theoretical studies have implicated factors such as protein size (Wolynes, 1997
), topology (Gross, 1996), proline content and thermodynamic stability (Sali, 1994 #901; Wright et al., 2004) as factors which may influence the large range of kinetic behaviours observed. It has also been suggested that for single domain proteins, there is a significant correlation between the rate of folding and the topological complexity (contact order) of the protein (Plaxco et al., 1998; Ivankov et al., 2003; Dobson, 2003).
Kinetic and thermodynamic studies have been performed on several families of homologous proteins to understand better the role a protein's sequence plays in acquiring the properties of a particular fold. These include studies on the all β-protein families of the fibronectin-like modules (Clarke et al., 1997; Plaxco et al., 1997), the intracellular lipid binding protein family (Burns et al., 1998), SH3 domains (Martinez and Serrano, 1999; Riddle et al., 1999), the all 
-helical acyl-CoA binding proteins (Kragelund et al., 1996) and the helical immunity proteins, Im7 and Im9 (Ferguson et al., 1999), and the mixed /β proteins acylphosphatase and ADA2 h (Chiti et al., 1999). In some cases, all members of a family have been shown to fold with a similar mechanism (Kragelund et al., 1996; Martinez and Serrano, 1999; Riddle et al., 1999) whereas in others, different family members have been proposed to fold via significantly different mechanisms (Burns et al., 1998; Ferguson et al., 1999).
Here, we present a study of the thermodynamic and kinetic properties of the small (63 amino acids per monomer) homodimeric, four-helix bundle protein Rop and its homologues. Despite its regular structure and simple topology, Rop has interesting kinetic properties: it folds and unfolds much more slowly than would be expected for a protein of its size that contains neither prolines nor disulphide bonds (Munson et al., 1996, 1997; Nagi and Regan, 1997; Nagi et al., 1999). Rop takes seconds to minutes to fold and hours to days to unfold. In contrast, other small dimeric proteins such as P22 Arc repressor, trp aporepressor and small leucine zippers fold in milliseconds or less and unfold in seconds to minutes (Gittelman et al., 1990; Milla and Sauer, 1994; Wendt et al., 1995; Zitzewitz et al., 1995; Sauer, 1996).
To explore further the basis of the unusual folding kinetics of Rop we identified its natural homologues in a BLAST search of the non-redundant protein database (Fig. 1) (Altschul et al., 1990). Rop is encoded by the Escherichia coli plasmid ColE1. All of the homologue sequences also map to extrachromosomal locations in gram negative bacteria, belonging to the Enterobacteriaceae family (Woese, 1990). The Rop homologues are found on plasmid DNA in Enterobacter cloacae (EC), Yersinia pestis (YP), Klebsiella oxytoca (KO), Enterobacter agglomerans (EA) and Proteus vulgaris (PV). We refer to the homologues by the initials denoted in brackets. Of the proteins identified, EA and PV are the least homologous to Rop, with 51% and 48% identity to Rop, respectively. The experimental work presented here was performed on EA (Mikiewicz et al., 1997) and PV (Calvin Koons and Blumenthal, 1995) and their RNA-binding, thermodynamic and kinetic properties are discussed in relation to each other and to Rop.
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RNA-binding activity
The function of Rop is to regulate the copy number of ColE1 plasmids in E.coli. Regulation is achieved by binding to a complex of two complementary RNA molecules, RNA I and RNA II (Polisky, 1988), and the binding of Rop facilitates the formation of the RNA I–RNA II complex. The two RNA molecules interact through their complementary loops to form a ‘kiss complex’ (Marino et al., 1995; Lee et al., 1998). It has been proposed that Rop recognizes the overall kiss complex structure, rather than specific elements in the RNA sequence (Comolli et al., 1998). In vitro, the binding of Rop to the kiss complex can be detected in an electromobility shift assay with a truncated RNA kiss complex (Gregorian, 1995; Predki et al., 1995) (Fig. 2). The kiss complex sequences used in the in vitro binding assay of Rop are shown in Fig. 2A along with the equivalent sequences identified in the regulatory regions of plasmid DNA from EA (Mikiewicz et al., 1997) and PV (Calvin Koons and Blumenthal, 1995). A comparison of these RNA sequences suggests that EA and PV have RNAs that potentially adopt a kiss complex structure similar to that recognized by Rop.
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Alanine scanning mutagenesis studies in Rop identified five residues as critical for RNA binding (Predki et al., 1995
). These residues are Lys3, Asn10, Phe14, Gln18, Lys25 in helix 1 and the complementary set of residues in helix 1', which form a narrow stripe down the positively charged 1/1' face of the protein (Fig. 1B). A comparison of the RNA-binding residue positions of Rop with the homologues demonstrates that these residues are identical across all of the homologues except for EA where conservative Lys to Arg substitutions occur at positions 3 and 25. Because of the similar predicted secondary structures for the different RNAs, and the conservation of residues required for RNA binding, we predicted that EA and PV also function by binding regulatory RNA molecules in a similar fashion to Rop. To test the ability of EA and PV to bind RNA, an electromobility shift assay of EA, PV and Rop with the RNA sequences recognized by Rop, was performed. All three proteins bind the RNA complex resulting in a lower mobility complex compared with the free kiss complex (Fig. 2B). The similar RNA-binding properties of Rop and its homologues suggest that the relative orientation of the helices in all three proteins is similar, and that they likely share the same topology. However, the affinity of EA and PV for Rop's RNA is lower than that for Rop, suggesting that their sequences may be optimized to bind their individual cognate kiss complexes.
For the equilibrium studies, CD spectroscopy was used to compare the secondary structure in the three proteins. Rop, EA and PV are all highly helical (Fig. 3A and Table I). Thermal and chemical denaturation transitions were followed by monitoring the loss of helical secondary structure. The equilibrium stability of all three proteins is significantly modulated by the concentration of NaCl: Protein stability increases with increasing NaCl concentration (data not shown). The studies discussed below are all performed at 200 mM NaCl, unless explicitly stated otherwise. The thermal stability of the proteins, in the order of increasing Tm is PV<Rop<EA (Fig. 3A inset and Table I). The relative stability (
G) by GuHCl denaturation also increases in the order PV<Rop<EA (Fig. 3B and Table I).
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Folding mechanism for Rop
In addition to CD, fluorescence spectroscopy also provides a sensitive technique by which to monitor changes in structure. Each monomer of Rop has a single fluorophore, Tyr49, which is buried within the folded protein. The fluorescence intensity of Tyr49 changes depending on its chemical environment, and its fluorescence intensity can therefore be used as a probe of tertiary structure to monitor folding and unfolding. EA also has a Tyr at position 49 and therefore it is possible to use fluorescence to monitor its folding and unfolding as well. PV however has a histidine residue at this position and therefore it was necessary to monitor its folding and unfolding kinetics exclusively by stopped-flow CD.
Detailed kinetic refolding studies have led to the proposal that Rop folds through a dimeric intermediate (Munson et al., 1997; Nagi et al., 1999; Brockwell and Redford, 2007). At low GuHCl concentrations, the dimeric intermediate is stable and biphasic folding kinetics are observed. The fast rate is strongly dependent on protein concentration, whereas the slow rate has little dependence on protein concentration. This observation suggests that the fast phase involves a bimolecular association leading to the formation of the dimeric intermediate. The slow phase is modeled as a structural rearrangement of the dimeric intermediate to yield the native protein.
At high concentrations of GuHCl, however, the dimeric intermediate is destabilized and therefore the observed kinetics are slow and monophasic.
The unfolding kinetics for Rop are monophasic at all concentrations of GuHCl, with no detectable unfolding intermediates.
We investigated the refolding of EA by both stopped-flow CD and stopped-flow fluorescence spectroscopy. EA follows biphasic refolding kinetics up to 3 M GuHCl (Fig. 4A and B), becoming monophasic at concentrations of 3.5 M GuHCl and higher (Fig. 4C and D). When refolding the protein at low GuHCl concentrations (0–1.5 M), the fast phase is too rapid to be detected, whereas refolding at concentrations greater than 1.5 M GuHCl, results in a detectable fast phase. The refolding data obtained from CD and fluorescence are coincident with each other (within error), indicating that the acquisition of secondary and tertiary structure is coincident. The refolding rate constant at 0 M GuHCl for EA is estimated to be 
15 times faster than Rop (Table II).
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PV refolding monitored by stopped-flow CD follows biphasic refolding kinetics until 1.0 M GuHCl, and when it is refolded at higher concentrations of GuHCl, its refolding is monophasic (Fig. 5A and B). The refolding rates for PV are significantly slower than those for EA, and consequently there is very little missing amplitude even under conditions where this protein refolds the fastest, that is, when refolding to the lowest concentrations of GuHCl. When the refolding rate constants for the fast and slow phases of PV are calculated at 0 M GuHCl, they are slower than those of Rop (Fig. 6B and Table II).
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Hence, the order in increasing refolding rates for the proteins is PV<Rop<<EA.
Protein concentration dependence for the refolding of EA and PV
To investigate whether the rate determining step in the folding process is unimolecular or multimolecular, we studied the dependence of the refolding rates for EA and PV on protein concentration. The plot of protein concentration versus the rate constant, k, shows that the fast phases for both EA and PV have a significant dependence on protein concentration, whereas the slow phases have a much lower dependence on protein concentration (Fig. 7). This observation is similar to the behaviour seen for Rop suggesting that the fast phase in EA and PV may represent the formation of a dimeric intermediate, as proposed for Rop (Munson et al., 1996; Nagi et al.,1999).
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The protein concentration dependence of the refolding rates for EA measured by CD and fluorescence correlate well with each other (Fig. 7A and B). A comparison of the theoretical diffusion limited folding rate obtained by assuming that a doubling of the protein concentration results in a doubling of the refolding rate with those obtained experimentally for EA and PV (Fig. 7) demonstrates that the structure formation for both of these proteins is determined predominantly by the bimolecular collision event.
Salt concentration dependence for the refolding of EA and PV
It was observed that the equilibrium stabilities of Rop, EA and PV are dependent on NaCl concentration—increasing NaCl concentration stabilizes the protein. Therefore, we probed the effect of increasing NaCl concentrations on the refolding rates for both proteins. The fast phase for both proteins has a significant dependence on NaCl concentration, whereby increasing NaCl concentration increases its rate. The rate of the slow phase is independent of NaCl concentration (Fig. 8). These observations suggest that an increase in the NaCl concentration facilitates the formation of the dimeric intermediate (see Discussion).
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Unfolding of EA and PV
The unfolding kinetics of Rop are monophasic at all concentrations of GuHCl, and there are no detectable intermediates along the unfolding pathway. Rop unfolds extremely slowly, with a rate constant at 0 M GuHCl of 6 x 10–7 s–1. The unfolding of EA and PV are also monophasic at all concentrations of GuHCl. The slow unfolding of EA can be monitored by manual mixing. The rate of unfolding for EA when extrapolated to 0 M GuHCl is 6 x 10–6 s–1 (Fig. 9, Table II). The unfolding rate of PV is faster than those for Rop and EA and was therefore monitored by stopped flow methods. The rate constant for PV when extrapolated to 0 M GuHCl is 9.9 x 10–3 s–1 (Fig. 9, Table II). Hence, the unfolding of EA is 10 times faster than that of Rop and the unfolding of PV is 1.7 x 104-fold faster than that of Rop (Table II).
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Discussion |
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We present a comparative study of a group of homologous four-helix bundle proteins. The high-resolution structure of one of these homologues, Rop, is known (Banner et al., 1987
; Eberle et al., 1990). The other two homologues, EA and PV, are also highly helical and bind to the RNA complex that is recognized by Rop. This suggests that all three proteins share the same topology and similar in vivo functions. The stabilities and folding rates of all three proteins are directly correlated. The rank order of stability for the three proteins is PV<Rop<EA. The rank order of folding rates is PV<Rop<<EA and the rank order of unfolding rates is Rop<EA<<PV. It is therefore apparent that PV is destabilized relative to Rop and EA by a combination of factors: its folding rate is decreased and its unfolding rate is increased. EA is stabilized relative to Rop because of a faster folding rate which more than compensates for the 10-fold increase in its unfolding rate.
The effect of systematically increasing the length of the loop connection between helix 1 and 2 in Rop on its stability and folding/unfolding kinetics has been previously studied (Nagi and Regan, 1997; Nagi et al., 1999). Rop has a tight two-residue connection (Asp–Ala) between its helices. This sequence was systematically replaced to give 10 variants with between 1 and 10 glycine residues. It was observed that as loop length was increased, equilibrium stability of the protein decreased, the folding rate decreased and the unfolding rate increased. PV has a loop length of five amino acids (when aligned as shown in Fig. 1A) compared with two amino acids for Rop and EA and thus provided a natural protein in which to investigate the effect of increasing loop length on stability and kinetics. The trends of the kinetic and thermodynamic properties of PV mirror those of the longer loop variants of Rop (Nagi and Regan, 1997; Nagi et al., 1999). The folding rate of PV is decreased relative to EA and Rop, presumably due to a stabilization of the unfolded ensemble resulting from the greater entropy allowed in the longer loop and/or an increase in the energy of the transition state relative to EA and Rop caused by constraining the longer loop in the helix-turn-helix monomer. The increase in unfolding rate is likely due to the decrease in stability of the native state of PV relative to Rop and EA.
Rop has also been used as a model protein to study the effects of systematically repacking the hydrophobic core with combinations of aliphatic residues (Munson et al., 1994, 1996). The Rop core is very regular and is composed of eight layers with each helix of the four-helix bundle contributing one residue per layer (Fig. 1C). The repacked cores are composed entirely of two hydrophobic residues (e.g. alanine and leucine) packed in regular layers and have more ‘idealized’ cores compared with wild-type Rop which has a number of polar residues in its core (Fig. 1A). In particular, when Rop's core is repacked with Ala at the ‘a’ position, and Leu at the ‘d’ position of the hydrophobic core for all layers except for second and seventh layers where the packing is reversed (Ala2Leu2-8-rev), it results in a protein with increased stability compared with wild-type Rop while retaining the RNA-binding ability of the protein. EA is more similar in its hydrophobic core to the idealized variant Ala2Leu2-8-rev, and EA's second and seventh layers are identical to it. Its properties parallel what is observed with the repacked Rop core variants where each additional repacked layer increases the equilibrium stability of the variants, while increasing both the folding and unfolding rates (Munson et al., 1996, 1997). The enhanced stability of EA along with its faster folding and unfolding rates suggests that the transition state is stabilized for EA relative to Rop. This is perhaps due to the presence of more favourable core packing interactions in its transition state compared with that of Rop. Although we discuss the properties of the homologues in terms of their core packing interactions and the length of loop connections between elements of secondary structure, it is important to emphasize that other differences in the sequences may also play a significant role.
The dependence of the refolding rates on protein concentration is similar for Rop, EA and PV. This suggests that they all follow a similar folding mechanism that proceeds through the formation of a dimeric intermediate. For EA and PV, the fast phase has a significant dependence on NaCl concentration, such that higher NaCl concentrations increase its rate. The rate of the slow phase is independent of NaCl concentration (Fig. 8). The dependence on NaCl concentration of the fast phase for refolding suggests that increasing NaCl concentration facilitates the formation of the dimeric intermediate, possibly by facilitating the hydrophobic collapse step or by shielding the repulsion of the negative charge as the 2/2' face forms.
Our results from this study on a dimeric protein family are similar to the results from a number of studies on monomeric protein families (Kragelund et al., 1996; Clarke, 1997; Plaxco et al., 1997; Martinez and Serrano, 1999; Riddle et al., 1999; Chiti et al., 1999). We have demonstrated that even though members of a homologous protein family may have differing thermodynamic and kinetic properties, they all follow a similar folding and unfolding mechanism.
We have shown that this group of Rop homologues provide a representative set in which to relate the equilibrium stabilities to the kinetic properties of a family of protein variants. The properties of the homologues are discussed in relation to the properties of the designed proteins in which Rop was used as a model system. Studies on families of homologous proteins clearly demonstrate how small changes at the sequence level can dramatically influence the kinetics and stability of a protein while maintaining the overall three-dimensional fold and function.
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Database search to identify the Rop homologues
The BLAST search was performed using the entire sequence of wild-type Rop protein and searching against a non-redundant GenBank CDS translations + PDB + Swiss Prot + PIR + PRF database at www.ncbi.nlm.nih.gov/BLAST/.
GuHCl, ultra pure, was purchased from American Bioanalytical (Natick, MA, USA).
Cloning and protein purification
The genes encoding Rop, EA and PV sequences were constructed into the vector pMR103 (Munson et al., 1994) using overlapping DNA templates in a polymerase chain reaction. A glycine residue was incorporated as the residue following the initiator methionine in order to ensure uniform processing of the N-terminal methionine by N-terminal methionine aminopeptidase (Smith et al., 1995). The sequences of the genes were confirmed by dideoxynucleotide sequencing. All proteins were expressed and purified as described previously for Rop, using anion exchange chromatography (Munson et al., 1994, 1996). The purified proteins were concentrated using CentriPrep 3.5 (Amicon) concentrators and dialyzed against 100 mM Na-phosphate (pH 7), 200 mM NaCl (phosphate buffer), for use in experiments. The identity of the proteins was confirmed by amino acid analysis and MALDI mass spectrometry.
All CD experiments were performed on an AVIV 62DS spectropolarimeter (Aviv Instruments, Lakewood, NJ, USA). Experiments were performed using 20 µM dimeric protein in phosphate buffer, in a 2 mm pathlength cuvette, unless explicitly stated. The protein concentration determined initially by amino acid analysis was used to calculate the MRE of the proteins. To determine the protein concentration for use in experiments, the CD ellipticity at 222 nm was measured and the MRE was used to calculate the protein concentration. We estimate the error in protein concentration determination to be approximately ± 5%. Far-UV wavelength scans were obtained from 260 to 200 nm with an averaging time of 5 s. Protein unfolding was monitored by following change in ellipticity at 222 nm either as a function of temperature or GuHCl concentration in phosphate buffer supplemented with 1 mM DTT. The thermal denaturation was performed in increments of 1°C, with a signal averaging time of 1 min and an equilibration time at each temperature of 1 min.
To ensure complete equilibration for the chemical denaturation, samples were incubated in varying concentrations of phosphate buffered GuHCl for points along the equilibrium denaturation curve, for 48 h. The concentrations of the stock GuHCl and samples used in denaturation experiment were determined by measuring the refractive index with an Atago R5000 refractometer (Atago, Japan) (Pace, 1986).
The GuHCl denaturation curves were analyzed to derive the free energy and m-values. To obtain these values, the baselines were corrected manually and the Kd values were obtained using Kd = 2CT(FU)2/(FN), where CT = total concentration of dimeric protein, FU = fraction unfolded and FN = fraction native. Using these Kd values, the G at each concentration of GuHCl was calculated using the relationship G = –RTlnKd. In order to calculate the free energy value in the absence of denaturant, the graph of G versus GuHCl is fit to a straight line. The intercept of this line is G0 and the slope of this line gives the m-value.
The kinetics of refolding were followed by stopped-flow CD and stopped-flow fluorescence. The unfolding kinetics of PV were followed by stopped-flow CD and the unfolding kinetics of EA were monitored by CD using manual mixing methods. EA and GuHCl were mixed to initiate unfolding. In manual mixing experiments, typically 20–25 s elapsed between the mixing of solutions, transfer of the solution to a cuvette and transfer of the cuvette to the CD instrument. The loss of secondary structure for EA was monitored as a function of time at 222 nm, with an averaging time and time constant of 3 s.
Prior to performing the refolding experiments, PV was denatured in 4 M GuHCl and EA was denatured in 6.5 M GuHCl in phosphate buffer, supplemented with 10 mM DTT. All the kinetic experiments were performed in the presence of 10 mM DTT to ensure complete reduction of the cysteine residues in the proteins. The samples were equilibrated overnight in denaturant. Unfolding and refolding experiments were carried out using a Bio-Logic (Claix, France) SFM3 mixer with a cell path length of 1.5 mm and a Bio-Logic PMS 400 detection system. The theoretical dead time based on the flow rate was 5.4 ms. To enable the CD and fluorescence signals to be monitored simultaneously, the excitation wavelength was set at 220 nm and the fluorescence emission was detected at 305 nm using a monochromator set at the emission wavelength. To improve the signal-to-noise ratio of the fluorescence experiment, EA refolding was repeated under exactly the same conditions but by exciting at 275 nm. Three syringes were used in the experiments: for refolding, the first contained GuHCl buffered in phosphate at the highest desired concentration of GuHCl, the second contained native phosphate buffer and the third contained the unfolded protein at x10 the final concentration. To monitor the unfolding for PV, the three syringes were as follows: the first one contained GuHCl buffered in phosphate buffer, the second contained native phosphate buffer and the third one contained protein in native phosphate buffer at x10 the final concentration. In general, varying volumes of the components in syringes 1 and 2 were mixed to get the desired final concentration of GuHCl while maintaining a constant protein concentration of 20 µM dimer, except in experiments that probed protein concentration. Data acquisition was performed using two time bases (every 2 ms for the first 4 s and thereafter every 10 ms) in order to record a higher number of points in the initial times following mixing. Hence, the signals show a higher noise level, because they are less filtered, at the start of the kinetic profiles.
Stopped-flow CD and manual mixing CD data were fit to either a single or double exponential growth or decay function.
For the growth function,
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the signal at time t = , k, k1 and k2 the rates of the single phase or of the fast and slow phases, respectively, and A, A1 and A2 the signal amplitudes associated with those phases. To determine whether the data fit better to the single exponential or double exponential, fits to both were performed and when an obvious improvement in residual plots was observed, two exponentials were used. Data sets are the average between 5 and 8 separate mixing events. The average rate constants obtained at different GuHCl concentrations were used to determine the rate constants, k0, in the absence of denaturant by linear extrapolation of the plot of lnk versus [GuHCl] to 0 M GuHCl concentration. To generate this line for the proteins at least five different concentrations of GuHCl were used.
The unfolding data were used to plot lnku versus [GuHCl] for at least five different GuHCl concentrations and a k0u was obtained by extrapolation to 0 M GuHCl. The standard deviation for the rate constants derived from individual fits is approximately ± 1% and the standard deviation for the average rate constants is approximately ± 3%. Taking into account the error in individual rate constants, along with the uncertainties in protein and denaturant concentrations, we estimate that the reported rate constants have an associated error of ± 15%.
RNA I and RNA II were generated by T7 RNA polymerase transcription of DNA templates. The RNA I and RNA II transcripts were heated separately to 85°C for 2 min and then quickly cooled on ice for 5 min to facilitate the formation of the kiss complex preferentially over the duplex. RNA I and RNA II were then mixed and the protein and incubation buffer (final concentration of 25 mM tris-borate, 5 mM MgCl2 and 100 mM NaCl) were added and incubated on ice for 30 min. Reactions were loaded onto an 18% (36:1 crosslink) native polyacrylamide gel in TBM buffer (25 mM tris-borate, 5 mM MgCl2), and run at 4°C and 100 V to separate the kiss complex from the protein–RNA complex. Bands were visualized by staining with ethidium bromide.
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National Institute of Health (GM 49146-01A1); NATO Collaborative Travel Grant. The work was also supported by the Oxford Centre for Molecular Sciences, with funding from the UK BBSRC, EPSRC and MRC.
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5 Present address: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
6 Present address: Stanford University, School of Medicine, Stanford CA94306
Abbreviations: GuHCl, Guanidine hydrochloride; DTT, dithiothreitol; UV, ultraviolet; CD spectroscopy, circular dichroism spectroscopy.
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We would like to thank the members of the Regan laboratory for critical reading of the manuscript.
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Received December 6, 2007; revised December 6, 2007; accepted December 11, 2007.
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