Homodimeric Escherichia coli Toxin CcdB - ACS Publications

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The homodimeric E. coli toxin CcdB (Controller of Cell Division or Death B protein) folds via parallel pathways Chetana Baliga, Raghavan Varadarajan, and Nilesh Aghera Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00726 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Biochemistry

The homodimeric E. coli toxin CcdB (Controller of Cell Division or Death B protein) folds via parallel pathways

Chetana Baliga1, Raghavan Varadarajan1,2, Nilesh Aghera1*.

1

Molecular Biophysics Unit,

Indian Institute of Science, Bangalore 560 012, India.

2

Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O. Bangalore 560 004, India.

* Corresponding author: Nilesh Aghera, Telephone: +91-80-2293-2612. Fax: +91-80-23600535 Email address – [email protected]

Funding sources This work was funded by the Department of Science and Technology (DST) and Department of Biotechnology (DBT), Government of India. CB is a recipient of the Council for Scientific and Industrial Research (CSIR) fellowship, Government of India. NA was funded by an INSPIRE AORC fellowship from DST, Government of India.

1 ACS Paragon Plus Environment

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Key words CcdB, Dimeric protein, Folding, Kinetics, Parallel pathways, Toxin-Antitoxin

Abbreviations CcdB -Controller of Cell Division or Death B protein, TA system – Toxin Antitoxin system, CcdA peptide – here, residues 46-72 of CcdA, the antitoxin part of the CcdAB TA system, GdnHCl - Guanidinium Hydrochloride, CD- Circular Dichroism


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Abstract The existence of parallel pathways in the folding of proteins seems intuitive, yet remains controversial. We explore the folding kinetics of the homodimeric E. coli toxin CcdB using multiple optical probes and approaches. Kinetic studies performed as a function of protein and denaturant concentrations demonstrate that the folding of CcdB is a four-state process. The two intermediates populated during folding are present on parallel pathways. Both form by rapid association of the monomers in a diffusion limited manner and appear to be largely unstructured, as they are silent to the optical probes employed in the current study. The existence of parallel pathways is supported by the insensitivity of the amplitudes of the refolding kinetic phases to the different probes used in the study. More importantly, interrupted refolding studies and ligand binding studies clearly demonstrate that the native state forms in a bi-exponential manner, implying the presence of at least two pathways. Our studies indicate that the CcdA antitoxin binds only to the folded CcdB dimer and not to any earlier folding intermediates. Thus, despite being part of the same operon, the antitoxin does not appear to modulate the folding pathway of the toxin encoded by the downstream cistron. This study highlights the utility of ligand binding in distinguishing between sequential and parallel pathways in protein folding studies, while also providing insights into molecular interactions during folding in Type II TA systems.


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The study of the protein folding process is one approach to understand the relationship between amino acid sequence of a polypeptide chain and its three-dimensional structure. Along with folding kinetics, characterization of transiently formed intermediates and transition states may provide insights into the relation between structure, function, stability and solubility. A large number of protein folding kinetics studies have examined monomeric proteins

1-9

while

there have been fewer studies that have explored the folding of dimeric proteins10-15. The majority of proteins within the cell are oligomeric and exist as protein-protein complexes. Therefore, understanding the folding of multimeric proteins and their modulation by ligands is of biological significance. A fundamental question in protein folding is whether folding proceeds via a single, sequential pathway or if there exist multiple, parallel pathways by which an unfolded polypeptide can attain its native structure 16. The existence of parallel pathways is thought to add robustness to the folding mechanism and hasten the folding process 17, 18. Several model proteins such as RNAseA 19, 20, α-lactalbumin 21, 22, hen egg white lysozyme 23, 24, dihydrofolate reductase (DHFR) 25, 26, the alpha-subunit of Trp synthase 27, the dimeric E. coli Trp repressor 14, 28, 29, the heterodimeric luciferase monellin

11

30

, superoxide dismutase

31, 32

and the heterodimeric sweet protein

, have been shown to fold via parallel pathways. Yet, the existence of parallel

pathways has not been universally accepted. There are theories that propose that proteins fold via a single, pre-determined pathway and that the observed multiple pathways arise due to misfolding and aggregation, and are not true folding pathways33, 34. In this study, we have explored the folding pathway of the dimeric E. coli protein CcdB. CcdB (Controller of Cell Division or Death B protein), is a 101 residue, homodimeric protein encoded by the ccd operon carried on the F plasmid 35. It is a part of the ccdAB toxin-antitoxin


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(TA) system in E. coli. The CcdB protein is an inhibitor of DNA gyrase and is a potent cytotoxin 36, 37

. Gel filtration studies at neutral pH indicate that the protein exists as a homodimer at various

concentrations, and does not show any indication of a stable monomeric form 38, consistent with crystallographic observations 39 (Figure 1). Isothermal denaturation suggests two-state unfolding, over a range of protein concentrations, indicating that a stable monomeric intermediate is not populated in the equilibrium unfolding process 38. CcdB has been used extensively as a model for understanding protein sequence – structure – function relationships40, 41. A library of 1430 single site mutants, constituting 75% of all possible single site mutants of the protein, is available

41

.

Several mutants from this library have already been characterized and it has been shown that mutations at buried sites in CcdB lead to decreased stability, solubility and activity in vivo

41-44

.

Some of these mutants appear to be folding defective. Deciphering the folding pathway of Wt CcdB is a prerequisite for studying the folding defects caused by these mutations. In this work, we have characterized the refolding kinetic mechanism of dimeric CcdB in vitro, using multiple probes. CcdB appears to fold to its native state using at least two parallel pathways, where the folding on both pathways begins with the association of unfolded monomers to form largely unstructured dimeric intermediates that subsequently fold to the native state dimer. This study provides additional evidence for the existence of multiple pathways for a protein to fold from its unfolded state to its native state. It also highlights the utility of ligand binding as a tool for differentiating between sequential and parallel folding pathways.

Experimental procedures All reagents used in the experiments were purchased from Sigma and were of the highest purity grade, except Guanidinium Hydrochloride (GdnHCl), which was from USB (USA) and


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also was of the highest purity grade. All the experiments were carried out at pH 7 in 10 mM HEPES buffer. Concentration of GdnHCl was determined by refractive index measurements. The CcdA peptide (residues 46-72) was synthesized at GenScript, USA. CcdA and CcdB concentrations are expressed in terms of concentration of monomer units. Protein expression and purification. Wt CcdB was expressed from the arabinose inducible PBAD promoter, in the pBAD24 vector in the CSH501 strain of E. coli that is resistant to the toxic action of CcdB

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. A single colony was inoculated into LB medium and grown at

37°C overnight. Two litres of LB (500 mL ×4) (HiMedia) were inoculated with 1% of the primary inoculum and grown at 37 °C until the OD600 reached 0.6. Cells were then induced with 0.2% arabinose and grown at 30oC for 6 hours. Cells were harvested by centrifugation and resuspended in 1/10th volume ice-cold 10 mM HEPES buffer at pH 8 containing 10% glycerol, 1 mM EDTA and 0.5 mM PMSF. The cell suspension was lysed by sonication on ice and subsequently centrifuged at 14,000 × g. The protein was purified from the soluble fraction of the lysate either by an affinity approach or by classical chromatography. Affinity purification of Wt CcdB. The protein was purified by affinity methods using either the GyrA14 fragment (residues 363-494) coupled to Biorad Affigel-10 or the CcdA peptide (residues 46-72) coupled to Biorad Affigel-15. The manufacturer’s protocol was followed for ligand immobilisation and affinity purification. Following cell lysis by sonication, the supernatant from cells of one litre culture was added to 6 ml of ligand bound Affigel and incubated under mild rocking conditions for 2-3 hours, at 4°C. The unbound fraction was removed and the column was washed with 100mL of ice-cold Coupling Buffer (0.05 M sodium bicarbonate, pH 8.3, 0.5 M NaCl). Protein was eluted with 0.2M Glycine, pH 2.5, and 1 ml fractions were collected in 1.5 mL tubes containing 400 µL of 1.5 M Tris, pH 8.8, to neutralize


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the protein solution on elution. Fractions containing pure protein were pooled and dialysed against 10mM HEPES, pH7 and stored at -20°C. Purification of Wt CcdB by conventional chromatography. In the conventional chromatography based approach, the supernatant post cell lysis was loaded onto a Q-Sepharose ion-exchange column at 25oC. The column was washed using 10 column volumes of 10 mM HEPES buffer (pH 8) containing 50 mM NaCl. The protein was eluted by iscocratic elution using 200 mM NaCl in 10 mM HEPES buffer at pH 8. The eluted protein was buffer exchanged into 20 mM sodium acetate buffer at pH 5, and then loaded onto a SP-Sepharose column at 25oC. The column was washed with 10 column volumes of 20 mM sodium acetate buffer containing 150 mM NaCl at pH 5. Subsequently, the protein was eluted using 20 mM sodium acetate buffer, pH 5, containing 500 mM NaCl. Pure protein was buffer exchanged into 10 mM HEPES, pH 7 and stored at -20oC. The purity of the protein was confirmed by SDS-PAGE and mass spectrometry. Refolding kinetics of CcdB. CcdB was unfolded in Unfolding Buffer (4 M GdnHCl in 10 mM HEPES, pH 7), for 3 hours prior to refolding experiments. Refolding was initiated by diluting unfolded protein into native buffer, to different final GdnHCl concentrations ranging from 0.4 M to 1.6 M, at a fixed final protein concentration. Refolding studies were carried out at 25°C using a SFM-4 (Biologic) stopped-flow module. A flow cell with a path length of 1.5 mm was used. The dead time of mixing was 11.8 ms. Folding kinetic traces were monitored by measurement of the change in fluorescence at 390 ± 10 nm, using a band-pass filter (Asahi Spectra). The excitation wavelength was 280 nm and excitation slit width was 2 nm. Refolding kinetics studies were also performed at different protein concentrations, in the range of final monomer concentration from 400nM to 4 µM.


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Refolding kinetics of fluorescein labelled CcdB protein. A variant of CcdB having a single cysteine at position 92 (N92C) was labelled with fluorescein maleimide. The labelling efficiency was estimated by using mass-spectrometry to be greater than 80%. Refolding kinetic studies were performed at 10 nM protein concentration using an SFM4 stopped-flow module from Biologic. A flow cell with a path length of 2.0 mm was used, which resulted in 14.8 ms dead time for mixing. The labelled protein was excited using light of 495 nm, a bandwidth of 4 nm, and emission was monitored using a band pass filter at 540 ± 25 nm. The concentration of the labeled protein was determined by measuring the absorbance at 498 nm using a value of ε498 = 80000 M-1 cm-1 45. Unfolding kinetics of CcdB. Unfolding was initiated by mixing native CcdB (10mM HEPES, pH 7) with unfolding buffer (6M GdnHCl, 10mM HEPES, pH 7), diluted to different final GdnHCl concentrations ranging from 3 M to 5 M, at a fixed final protein concentration. Unfolding studies were carried out at 25°C using a SFM-4 (Biologic) stopped-flow module. A flow cell with a path length of 1.5 mm was used. The dead time of mixing was 11.8 ms. Unfolding kinetic traces were monitored by measurement of the change in fluorescence at 390 ± 10 nm, using a band-pass filter (Asahi Spectra). The excitation wavelength was 280 nm and excitation slit width was 2 nm. Interrupted refolding experiment. Interrupted refolding experiments were performed by manual mixing using a MOS450 optical system from Biologic, with a 7s dead time of mixing. 12 µM CcdB was unfolded in 3.2 M GdnHCl, and then refolded by diluting it to a final concentration of 1.6 M GdnHCl. The refolding process was interrupted at various times ranging from 5 to 200 s, by rapidly changing the GdnHCl concentration to 3 M. The final concentration


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of CcdB was 2 µM. The excitation wavelength was 280 nm and the unfolding kinetic traces were acquired by monitoring the intrinsic tryptophan fluorescence at 390 nm. Interrupted unfolding experiment. Interrupted unfolding experiments were performed by manual mixing, where the dead time of mixing was 7 s. Fluorescence was measured using the MOS450 optical system. The unfolding of the native CcdB (8 µM) was triggered at 3 M GdnHCl concentration. Unfolding was allowed to proceed for various times and was interrupted subsequently by triggering the refolding. This was facilitated by a jump in GdnHCl concentration to 1.6 M by dilution, using native buffer. The final concentration of CcdB during the refolding process was 2 µM. The unfolding kinetic traces were acquired by monitoring the tryptophan fluorescence at 390 nm upon excitation at 280 nm. CcdA binding studies. The studies were performed by monitoring the change in tryptophan fluorescence, induced upon binding of CcdA peptide (residues 46-72) to CcdB. The binding of CcdA was initiated by manual mixing after different time periods of CcdB refolding. The refolding of CcdB was performed at 1.5 M GdnHCl at 5 µM protein concentration and binding was initiated by adding 5µM CcdA peptide after 0s, 30s and 200s of CcdB refolding. The protein was excited using a wavelength of 280 nm, and the emission was monitored at 313±10 nm. The fluorescence was monitored using a Fluoromax-3 spectrofluorometer from Horiba Scientific and the dead time of mixing was 7 s. Global analysis of the kinetic data. To validate the kinetic model, different kinetic models were globally fitted to the refolding kinetic data at different GdnHCl concentration using MATLAB (MathWorks). The model was defined using differential equations, and was simulated using function ode23s, where it was fitted to experimental data by minimizing the RMSD between experimental traces and simulated traces using function fminsearchbnd. While fitting


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the model to the kinetic refolding data, the rate constants were floated over a broad range but were constrained such that the ratio of forward and backward rate constants agree with the experimentally determined equilibrium parameters obtained from isothermal denaturation of the CcdB dimer from the native state (N2) to the unfolded state (U)

38

. The kinetic parameters, for

which values were known from experimental work, were allowed to change over a small range around the experimental values.

Results CcdB refolds in a bi-phasic manner. The refolding kinetics of CcdB was studied over a range of denaturant concentrations from 0.4 to 1.6 M GdnHCl. The refolding process was monitored using the change in intrinsic tryptophan fluorescence at 390nm, upon excitation at 280nm. The protein emission spectra look identical when excited at 280nm or 295nm, but the magnitude of the signal is much larger at the former excitation wavelength (Figure S1a and b). The highest relative change in fluorescence occurs at higher wavelengths (Figure S1c), and therefore fluorescence emission was monitored at 390 nm. The refolding kinetic traces obtained over the entire range of GdnHCl concentration could be described by a biexponential decay equation, with one fast-phase and one slow-phase. Figure 2a shows representative refolding kinetic traces of CcdB obtained at 4 µM protein concentration. The comparison of the equilibrium amplitude with the magnitude of signal change for the refolding process at different GdnHCl concentrations (Figure 2b) indicates the absence of any burst phase process, implying that the acquired refolding kinetic traces represent the complete folding process. The gelfiltration analysis also demonstrates that the refolded protein is indeed dimeric (Figure S2). The observed rates of the fast phase show very high dependence on the denaturant concentration,


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while the observed rates of the slow phase of the refolding process show low dependence on denaturant concentration (Figure 2c). The data were fit to a straight line, the slope of which gives the refolding m-values of the two transition states. The amplitudes of both the phases show denaturant dependence, with the amplitude of the slow phase increasing at the expense of that of the fast phase, with increase in denaturant concentration (Figure 2d). At lower denaturant concentrations, the fast phase accounts for 90% of the total amplitude, whereas at higher denaturant concentrations, it accounts for less than 50% of the amplitude. Refolding kinetics is independent of protein concentration. Refolding kinetics was monitored over a range of protein concentrations to delineate the association process. The studies were performed from 400 nM to 4 µM protein concentration by monitoring the change in intrinsic tryptophan fluorescence (Figure 3). The observed rate constants of both the fast and the slow phase do not vary with changes in protein concentration. The relative amplitude of the fast phase increases marginally with increasing protein concentration, while that of the slow phase decreases. A surface cysteine mutant of CcdB (N92C, Figure 1) was labeled with the environment sensitive fluorophore fluorescein and this labeled protein was used for monitoring refolding kinetic traces at 10 nM protein concentration. The refolding kinetic traces obtained at 10 nM protein concentration are identical to those obtained at 4 µM protein concentration (Figure 2a and 4a) and they represent the complete refolding process, as is evident from the agreement between the kinetic and equilibrium amplitudes (Figure 4b). Furthermore, the observed rate constants (Figure 4c) of the fast phase and slow phase of the refolding process are identical to those obtained by monitoring tryptophan fluorescence at 4 µM protein concentration (Figure 2),


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while the amplitudes are marginally different. Thus, the observed refolding rates of CcdB are independent of protein concentration over a wide concentration range, from 10nM to 4 µM. Multiple probes give identical refolding kinetic behavior. The refolding process of CcdB was monitored using four different optical probes. The refolding kinetic traces monitored using tryptophan fluorescence (Figure 2), fluorescein fluorescence (Figure 4), far-UV CD (Figure S3) and tryptophan anisotropy (Figure S4) are identical. The refolding process observed using all four probes could be described by a two-exponential equation without any burst phase process. The values of the observed rates and the amplitudes for both the phases of the refolding process are identical for all four probes. CcdB unfolds in a single exponential manner. The unfolding kinetics of CcdB was studied over a range of denaturant concentrations from 3 to 5 M GdnHCl. The unfolding was monitored using the change in intrinsic tryptophan fluorescence at 385 nm. The unfolding kinetic traces obtained over the entire range of GdnHCl concentration could be described by a single exponential decay equation. Figure 5a shows representative unfolding kinetic traces of CcdB obtained at 4 µM protein concentration. The comparison of the equilibrium amplitude with the magnitude of signal change for the unfolding process at different GdnHCl concentrations (Figure 5b) indicates that the acquired unfolding kinetic traces represent the complete unfolding process. The log of the observed unfolding rate constant varies linearly with the denaturant concentration, and the slope of the fit gives the unfolding m-value (Figure 5c). Interrupted refolding studies. Interrupted refolding studies (double jump experiments) were performed by manual mixing to dissect out the nature of intermediates. Unfolded CcdB was refolded in the presence of 1.6 M GdnHCl and the refolding process was interrupted at different time points, ranging from 5 s to 200 s, by triggering unfolding at 3 M GdnHCl. Figure 6a shows


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representative unfolding kinetic traces obtained after different times of refolding. All the unfolding kinetic traces could be described satisfactorily by a single-exponential equation, wherein the observed unfolding rate at any time point is identical to that of the unfolding rate of the native protein. The extrapolated t = 0 point falls on the refolding trace, indicating the absence of any burst phase process (Figure 6a). The fraction of the native state populated with the time of refolding is plotted in Figure 6b. The fractional native population was calculated from the ratio of the amplitude of the unfolding process after different times of refolding, to the total amplitude of the unfolding process of the native protein. The kinetics of formation of the native state population is best described by a two-exponential equation. The values of the observed rates and amplitudes of the two phases observed in the formation of the native state are identical to those observed in the refolding process (Figure 2). Interrupted unfolding studies. Interrupted unfolding studies were also performed by a manual mixing approach, where the dead time of mixing was 7 s. Again, the studies capture the full amplitude of the process. The unfolding was initiated at 3 M GdnHCl and was interrupted after different time periods, ranging from 5 s to 100 s, by triggering refolding at 1.6 M GdnHCl. Figure 7a shows the unfolding kinetics trace and the representative refolding kinetic traces obtained upon interrupting the unfolding process after different times. All the refolding kinetic traces obtained are best described by a two-exponential equation. The extrapolated t = 0 points coincide with the unfolding kinetic trace, suggesting the absence of any burst phase process. The observed rate constants of the slow and fast phases do not change with increase in time of unfolding prior to the refolding process, and are identical to those observed for the refolding of fully unfolded proteins. However, the relative and absolute values of the fractional amplitudes of the slow and fast phase change with the time of unfolding (Figure 7b). Kinetics of rise of


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fractional amplitude of the fast and slow phase can each be described by a single-exponential process, where the observed rate for the rise of the fast phase amplitude (0.07 s-1) is greater than that obtained for the slow phase (0.02 s-1) amplitude (Figure 7b). It should be noted that both the rates are faster than the typical proline isomerization rate of ~ 0.01 – 0.005 s-146. Kinetics of CcdA-CcdB binding mimics that of CcdB refolding. Binding of CcdA to CcdB was monitored using the fluorescence signal change at 313nm, during the refolding of CcdB. The emission wavelength to monitor the fluorescence was chosen such that the change in fluorescence is minimal upon the folding of CcdB and maximum upon binding of the CcdA to CcdB (Figure S5). The binding of CcdA to both native (Figure 8, blue trace) and fully refolded CcdB (Figure 8, maroon trace, overlaps with blue trace) completes within the dead time of mixing (7 s). The binding of CcdA to CcdB was also monitored by adding CcdA after different time periods of the refolding of CcdB. The kinetics of the fluorescence change in the presence of CcdA is similar to the kinetics of CcdB folding in the absence of CcdA. When CcdA is present from the start of the refolding process, the process could be described by a two-exponential equation (Figure 8, black trace), where the rates and amplitudes of the fast and slow phase are comparable to those of the refolding process of CcdB. The addition of CcdA to CcdB was done after refolding CcdB for 30 s by which time the fast phase is complete. Under these conditions, the binding process appears single-exponential (Figure 8, purple trace), where the rate and amplitude are comparable to those of the slow phase of the refolding process of CcdB. Refolding of CcdB in the absence of CcdA under identical conditions (Figure 8, dark green trace) reflects only marginal signal change at 313nm, confirming that the signal change being monitored arises predominantly from the binding of CcdA to the refolded protein and not from the refolding process itself.


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The binding kinetics of CcdA to CcdB was also studied by BioLayer Interferometry. The refolded CcdB binds the ligand CcdA in a manner similar to the native protein incubated in the same denaturant concentration of 0.4M GdnHCl (Fig. S6, Table S1). These studies also confirm very fast binding of CcdA to CcdB (kon ~105 M-1s-1). The CcdA peptide binds CcdB across the dimer interface as seen in the crystal structure (PDB id 3hpw)47. This observation, coupled with the observation that the refolding kinetic parameters are identical in both the presence and absence of CcdA, indicate that the CcdA binding is diagnostic of a properly folded, native dimer. The refolded protein binds to CcdA, in a manner similar to the native protein, further confirming that upon refolding, the dimeric interface has been restored.

Discussion We present the folding mechanism of the homodimeric protein CcdB, which is an E. coli toxin and part of the ccdAB TA system involved in F plasmid addiction 48-52. The study provides general insights into the folding of oligomeric proteins and provides evidence to support the existence of parallel pathways in the course of folding to the native state. The study also indicates operation of a fly-casting mechanism 53-55 during the association of the monomers. CcdB folds in multiple steps. Refolding kinetics of CcdB was monitored using multiple optical probes, and the observed refolding kinetics was identical for all the probes. Tryptophan fluorescence monitored refolding kinetics of CcdB was biphasic without any burst-phase process in stopped-flow assisted mixing experiments over the entire range of GdnHCl concentration used for the studies. The biphasic folding process suggests that at least one intermediate populates during the course of folding. Refolding studies were also performed by monitoring the fluorescence of fluorescein labeled CcdB protein. Consistent with the tryptophan monitored


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refolding kinetics study, it was seen that refolding is biphasic and occurs without any burst phase process. Additionally, stopped-flow assisted far-UV CD monitored and tryptophan anisotropy monitored refolding kinetics show kinetic parameters identical to those obtained in fluorescence monitored refolding studies. All the kinetic data suggest the presence of at least one intermediate. Protein concentration dependent studies were carried out, with the aim of identifying the phase involving sub-unit association, but it was observed that both the fast and the slow phase were insensitive to changes in protein concentration, in the range of 10nM to 4µM used in this study. This suggests that the folding of CcdB is not a simple three-state process. It is possible that the association of unfolded monomers occurs early during the folding process, is very fast and silent to fluorescence. It is also possible that the association step follows monomer folding, wherein the rapid association step is masked by the slower folding process, and hence is not captured. Studies indicate that unfolded proteins have increased binding rates as compared to folded monomers, due to orientational and directional selectivity in the case of the latter 53, 54, 56. Refolding kinetics studied at 10 nM protein concentration, using the fluorescein labeled N92C variant of CcdB, are identical to the refolding kinetics observed at higher protein concentration (400 nM to 4 µM) monitored using tryptophan fluorescence. These protein concentration independent folding kinetics over this wide concentration range imply that the association might precede, rather than succeed the folding. Moreover, equilibrium studies of CcdB under a variety of conditions (pH, denaturant; data not shown) do not show any species except the folded dimer and the unfolded monomer. Studies indicate that the preference for the binding mechanism is governed by the native state topology

57-59

, and therefore, given the extensive burial of surface

area upon dimer formation (~1000Å2) in CcdB (from PDB id 3VUB), it is very unlikely that a monomer would fold independently. The argument is consistent with other reports which pointed


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out that the association of monomers can occur even in the absence of any structure in monomer60, 61. Thus, it is likely that the association of the unfolded CcdB monomers precedes their folding. The interrupted refolding experiment could not capture any intermediate. The unfolding kinetic traces obtained after different times of refolding of CcdB are monophasic without any burst-phase process, where the observed rate of unfolding is identical to that of the unfolding of native CcdB. The result indicates that the fluorescence properties of any intermediates are identical to that of the unfolded state, and hence they cannot be detected in interrupted refolding studies. It is also possible that the observed unfolding rates for the native state and the intermediates are identical and hence the intermediates cannot be detected. However, this is unlikely as there appear to be significant free energy barriers between intermediate and unfolded states. Also, since both intermediates are silent to fluorescence, it is unlikely that they arise along a sequential pathway. Multiple pathways in the folding of CcdB. The two phases of the refolding process appear to arise due to two barriers on parallel pathways rather than on a sequential pathway. The amplitudes of both the phases for refolding kinetics monitored using tryptophan fluorescence, and for the refolding kinetics monitored using fluorescein fluorescence are comparable and have identical dependence on GdnHCl. Refolding kinetics monitored using far-UV CD (Figure S3) and tryptophan anisotropy (Figure S4) also yield amplitudes and rates in agreement with the fluorescence studies. Insensitivity of the amplitudes of both the phases of refolding kinetics to different optical probes supports the idea that the observed phases arise due to parallel barriers rather than sequential barrier

62

. For a sequential pathway, it is highly improbable to have

identical amplitudes and observed rates for all four probes. This would require that both


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intermediates have identical fraction change in their properties relative to native CcdB for all four optical probes. The amplitudes of both the phases change with GdnHCl concentration, wherein the amplitude of the slow phase increases at the cost of fast phase amplitude. The fractional value of the fast phase amplitude reduces from 0.9 to 0.5. For sequential barriers, such a change in amplitude value would come about, when the rates through the barrier approach comparable values. Our data show that at the highest value of the denaturant concentration, the rates for the fast and slow phase differ by at least 5 fold. If one were to assume a sequential pathway, the change in amplitude value is not consistent with the change in relative rates of the slow and fast phase (simulation study, data not shown)

63

. Our observations can be better

described by parallel pathways, where the change in amplitude is governed by the change in stability of the intermediates and not by the change in rate of folding. We have also probed the existence of parallel pathways using ligand binding as an additional probe. Ligand binding can provide compelling evidence to differentiate between sequential and parallel folding pathways. For this approach to be fruitful, the following criteria have to be met – (a) ligand binding should be rapid, (b) the ligand selectively binds the native state of the protein with high affinity, (c) the binding can be monitored at a wavelength at which the contribution from the folding process of the protein is minimal, and (d) the presence of the ligand should not alter the folding mechanism of the protein 62. The binding of CcdB toxin to the CcdA anti-toxin (ligand) has been studied by Surface Plasmon Resonance (SPR)64 and BioLayer Interferometry (Figure S6, Table S1), and they are seen to bind with high affinity (KD ~10-9M). In refolding conditions of 0.4M GdnHCl, the on-rate is about 3x105 M-1s-1, giving an apparent t½ of 0.3s at protein and peptide concentrations of 5 µM, confirming very fast binding between CcdA-CcdB. The kinetics of this high affinity binding was also studied here by monitoring the


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fluorescence change at 313 nm (Figure 8). At this wavelength, there is a significant change in fluorescence upon binding of CcdA to CcdB, while the contribution towards the fluorescence from the folding of CcdB is very small (Figure S5). Binding kinetics of CcdA to folded CcdB is rapid and is complete within the dead time of the manual mixing experiment (Figure 8, blue trace), consistent with kinetic data from BioLayer Interferometry (BLI) (Figure S6, Table S1) 47, 64

. Thus, it was feasible to use CcdA binding as a means of elucidating the folding mechanism of

CcdB. If folding of CcdB occurs via a sequential pathway, then binding kinetics of CcdA to CcdB during its refolding should be monophasic, where the observed rate of the binding should correspond to the rate of formation of the native state. It is possible that CcdA also binds to partially folded CcdB. In such a case as well, the CcdA binding process will be monophasic and the observed rate would correspond to the formation of the respective intermediate. However, this is unlikely as CcdA binds to CcdB across the dimer interface as seen from the crystal structure (PDB id 3hpw)47. We observe the binding kinetics of CcdA to CcdB to be biphasic, wherein the observed rates and amplitudes of the fast and the slow phase of binding are similar to those observed during the refolding of CcdB. The simplest explanation is that CcdA binds to the native state which forms in a biphasic manner. This can only be explained if the native state forms via two separate pathways. Such biphasic kinetics could also arise if an off-pathway intermediate was forming during the refolding process, however, this appears to be unlikely, as the interrupted refolding experiment failed to capture any stable intermediate. Additionally, the refolding model with an off-pathway intermediate failed to fit the refolding kinetic data (Simulation study, Figure S7). Therefore, the folding of the CcdB has to have at least two parallel pathways. Thus, ligand binding is a simple and direct approach for validating the operation of the parallel pathways. There are studies which have used ligand binding and


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competitive inhibitor binding studies to characterize slow and fast phases of folding, and the intermediates that form along those pathways

20, 25, 62, 65-67

. However, this approach is not

generally used to distinguish between sequential and parallel pathways, as reported here. While it may not always be possible to discriminate between the presence of off-pathway intermediates and parallel pathways by means of ligand binding alone, this is a simple approach that provides additional evidence to support interpretations obtained by other means. Probing the role of proline isomerization in folding. The CcdB monomer has four proline residues, the cis-trans isomerization of which could interfere with its folding. Also, it is seen that the slow phase observed in CcdB refolding kinetics shows very low dependence on denaturant concentration, and could arise due to proline isomerization. However, the observed rate constant for the slow phase is higher than that reported for proline cis-trans isomerization 46. Moreover, the interrupted unfolding studies do not support the presence of any intermediate that forms or disappears at the rate that corresponds to the proline isomerization process. In the interrupted unfolding experiment, the native state unfolds and reaches the equilibrated unfolded state within 60 seconds. This is faster than the typical time-scale for the proline isomerization process

46

, although it must be noted that the kinetics of the process of proline isomerization is

known to vary, depending on the neighboring residues and the length of the polypeptide chain 6871

. Thus, while it appears unlikely that proline isomerization plays any observable role in the

folding of CcdB, this possibility remains. Hence, attempts were made to purify a mutant of CcdB where all the four proline residues had been substituted with alanines, but the mutant was seen to form inclusion bodies upon expression in CSH501 E. coli strain, and hence was not pursued for further studies. It is also known that prolines in CcdB have very low tolerance to mutation 40, 44,


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thus, other approaches might have to be explored to examine the possibility of proline isomerization during the CcdB refolding process. CcdB unfolding involves at least one intermediate. The fluorescence monitored unfolding process of CcdB can be described by a single-exponential equation, where the kinetic amplitude is in good agreement with the equilibrium amplitude (Figure 5). However, the unfolding process is not a simple two-state process. When the unfolding of CcdB was interrupted by triggering refolding after different time intervals, the refolding process could be described by a two-exponential equation, without any burst phase process. The observed rates of the two phases were identical to those seen in the refolding of the fully unfolded CcdB. The result indicates the absence of any detectable intermediate that is populated during the unfolding of the CcdB, and this matches with the observations in the unfolding studies. However, relative refolding amplitudes of the two phases in the interrupted unfolding experiment change with the time of unfolding. As the CcdB unfolds fully, these relative amplitudes eventually become identical to those observed during refolding of fully unfolded CcdB (Figure 7). At short unfolding times, the fast phase amplitude is significantly higher than the slow phase amplitude, and this gradually decreases with increase in time of unfolding. This indicates that the unfolding process is not a simple two-step process, but involves sequential structural transitions, as the species contributing to the fast phase equilibrates with the species giving rise to the slow phase. The nature of the transition is not clear and it could be possible that the unfolded state consists of two populations

72

. Also, the involvement of the proline isomerization in the unfolding process

cannot be ruled out. While there is evidence to show that the folding of the CcdB is occurring through at least two parallel pathways, there is insufficient evidence for the operation of parallel pathways during its unfolding. It is possible that only one of the pathways is favored under


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unfolding conditions, or that the kinetics of unfolding through both the pathways are comparable and hence, they cannot be differentiated. Further studies using native state HX-MS are being carried out, and might shed more light on the unfolding mechanism of CcdB. Refolding kinetic model of CcdB. The refolding kinetic data obtained at different GdnHCl concentrations and at different protein concentrations could be explained by a relatively simple mechanism shown in Scheme 1 (also see Figure 9a). In this scheme, the folding of CcdB begins with the formation of two homodimeric complexes, which appear to form in a diffusion limited manner. The complexes are silent to the optical probes used in these studies, which indicates lack of compaction, at least around the fluorophores used to monitor the process. The rapid association rates, along with the optically silent nature of the intermediates, give rise to kinetics which are independent of protein concentration (Figure 9b). These transient complexes then fold to their native state by a fast and a slow pathway, respectively. These fast and slow pathways correspond to the fast and slow phases captured in our refolding studies. The denaturant dependent changes in the amplitudes of the two phases arise due to the change in relative utilization of the fast and slow pathway. This is dictated by the difference in the dependence of the stabilities of the two intermediates on the denaturant concentration (i.e., the m-values). These two proposed dimeric intermediates on the parallel pathways could not be characterized. It is possible that they differ in their proline isomerization state. The proposed model was validated by globally fitting the model to the experimental kinetic data. The kinetic parameters obtained from the global analysis are shown in Table 1. An alternative model, with an off-pathway monomeric intermediate (Scheme S1), was also considered, but it failed to fit the refolding kinetic data (Figure S7a). Off-pathway dimeric or oligomeric intermediates were deemed unlikely as they would result in protein concentration dependent changes in amplitude,


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Biochemistry

however the models were built and tested. As anticipated, the model with an off-pathway dimeric intermediate also failed to fit the kinetic data (Scheme S2, Figure S7b). We also considered a model, where the unfolded state initially folds to two monomeric intermediates along two parallel pathways (Scheme 2), which subsequently associate in a diffusion limited manner to give the native state dimers. This model also fails to fit the data (Figure 9d). Thus, it seems most likely that the CcdB dimer folds via parallel pathways, each involving a dimeric intermediate which is optically silent. The kinetic parameters derived from the global analysis of the four-state model presented in Scheme 1 also explain the unfolding kinetics (seen in Figures 5, 6 and 7), where an apparent single exponential unfolding process arises due to destabilization of the slow pathway in unfolding conditions (Figure 9c and Table 1). From the global analysis, the unfolding rate constant and m-value obtained for the N2 If2 transition match those obtained in unfolding kinetics experiments, while the unfolding rate constant obtained for the N2 Is2 transition is lower than the experimentally obtained value. This implies that the N2 Is2 2U pathway is masked by the N2 If2 2U pathway, and thus, unfolding operates almost entirely via the latter pathway. The value of the t0 anisotropy from refolding kinetic data slightly deviates from the equilibrium value (difference is ~ 0.01) (Figure S4a). It could account for a burst phase wherein change in anisotropy arises due to association of the monomers, thus supporting the proposed model. However, this difference in tryptophan anisotropy is within the range of experimental error and hence, this is not presented as evidence for the model. The kinetic parameters derived from the global analysis of the four-state model presented in Scheme 1 also explain the anisotropy data (Figure S4b).


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The folding mechanism of CcdB sheds light on the broader aspects of the folding process of oligomeric proteins. It is suggested that folding can be multi-state, on a rugged energy landscape, which appears to have multiple trajectories to the native state

73

. The parallel

pathways we observe for CcdB folding support this idea. The study also provides insights into the nature of the association process, which is relatively well understood for intrinsically disordered proteins (IDPs). The association process in globular proteins is quite diverse and has several possibilities such as association before folding and association coupled to folding

78

11, 74, 75

, association after folding

32, 76, 77

. The CcdB dimer is a globular protein, where association

via the fly-casting mechanism suggests ease of association for the unfolded monomers and the requirement of dimerization for the acquisition of structure. Parallel versus sequential folding of protein. Historically, it was believed that the folding and unfolding of a protein occurs via a single defined pathway. Since then, this view has been challenged and there are several reports claiming the existence of multiple pathways, often based on indirect evidence11, 28, 79-83. Despite this, the existence of the parallel pathway remains controversial

33

. Several reported observations of parallel pathways have been investigated and

stated to be artifacts induced by experimental conditions33,

84-86

. However, other studies have

presented direct signatures of parallel pathways and these attest to the parallel nature of the protein folding or unfolding process

79, 80, 87

. The existence of parallel pathways is intuitive,

whereas single channel folding appears improbable. However, it is quite possible that one pathway is preferred over other pathways due to energy bias in the initial events of the folding process, and hence, masks the existence of other pathways. This report presents ligand binding as a means to detect the existence of parallel pathways in protein folding. The ligand binding


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studies along with the simulation analysis strongly supports the existence of at least two folding pathways in the folding of CcdB. Folding mechanism of CcdB and its implication in the functioning of the TA system. CcdB forms the toxin part of the ccdAB TA system. The CcdB/CcdA system is a Type II TA system, and the genes are typically organized in an operon. In the ccd operon, the gene for the antitoxin ccdA is located upstream of the ccdB gene 88, 89. The CcdA antitoxin is transcribed and translated prior to the toxin, and binds the CcdB toxin with high affinity, preventing it from binding its target, DNA Gyrase, thus enabling cell survival. There is some evidence to show that proteins encoded by genes located in an operon undergo co-translationally coupled folding 90-92. Therefore, the upstream localization of ccdA relative to ccdB might ensure that the already synthesized CcdA immediately binds to the freshly synthesized CcdB, forming a complex, thus protecting the cell from probable DNA Gyrase poisoning. The crystal structures of CcdB with various peptides derived from CcdA revealed that a C-terminal peptide of CcdA (comprising of residues 37-72) binds asymmetrically to the CcdB dimer, and the binding site extends across both the CcdB subunits 47. Our current studies suggest that the folding of the CcdB begins with the association of the monomers, followed by rearrangements in the dimeric intermediate, to form the native state dimer. This suggests that formation of the CcdA binding site is concomitant with formation of the native homodimer. It is also observed in this study that the folding kinetics of CcdB remains identical, when performed either in the absence or in the presence of the CcdA peptide (residues 46-72). This implies that in vitro, CcdA might not have a direct role in assisting the folding of CcdB. Yet, possible involvement of CcdA in assisting the folding of CcdB in vivo cannot be ruled out. Coexpression of CcdA with folding defective mutants of CcdB might shed more light on the role of


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CcdA in assisting the folding of CcdB. The proposed CcdB folding mechanism will be useful in exploring the low in vivo solubility and folding defects of such mutants. Such studies will give insights into the importance of the folding energy landscape in determining mutant fitness and help understand how a single amino acid substitution can modulate the energy landscape leading to misfolding and inactivity of the protein.

Acknowledgements Authors would like to thank Prof. Jayant B. Udgaonkar, National Centre for Biological Sciences, Bangalore, India, for his valuable inputs and for the stopped flow (SFM-4) and other facilities provided in his lab. The authors also thank the stopped flow facility at the Indian Institute of Science. Molecular graphics and analyses were performed with the UCSF Chimera package

93, 94

. Chimera is developed by the Resource for Biocomputing, Visualization, and

Informatics at the University of California, San Francisco (supported by NIGMS P41GM103311).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedure for measurement of binding affinity of CcdA to native and refolded CcdB using BioLayer Interferometry is given. Binding parameters between CcdA and CcdB are given in Table S1. Schemes S1 and S2, also used in the analysis of kinetic data are described. Figure S1 shows the fluorescence emission spectra of CcdB upon excitation at 280 and 295 nm. Figure S2 shows the Size Exclusion Chromatography profiles of native and refolded 26 ACS Paragon Plus Environment

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CcdB. Far-UV monitored refolding kinetics of CcdB are shown in Figure S3, while Anisotropy monitored refolding kinetics and its validation by global analysis are given in Figure S4. Fluorescence characterization of CcdA-CcdB complex is depicted in Figure S5. Kinetic profiles of binding of native and refolded CcdB to the ligand CcdA are given in Figure S6. Figure S7 shows the fits from global analysis of kinetic data using schemes S1 and S2 to the refolding kinetic data.


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[41] Bajaj, K., Dewan, P. C., Chakrabarti, P., Goswami, D., Barua, B., Baliga, C., and Varadarajan, R. (2008) Structural correlates of the temperature sensitive phenotype derived from saturation mutagenesis studies of CcdB, Biochemistry 47, 12964-12973. [42] Bajaj, K., Madhusudhan, M. S., Adkar, B. V., Chakrabarti, P., Ramakrishnan, C., Sali, A., and Varadarajan, R. (2007) Stereochemical criteria for prediction of the effects of proline mutations on protein stability, PLoS Comput Biol 3, e241. [43] Bajaj, K., Chakrabarti, P., and Varadarajan, R. (2005) Mutagenesis-based definitions and probes of residue burial in proteins, Proc Natl Acad Sci U S A 102, 16221-16226. [44] Tripathi, A. (2013) Determinants of stability, structure and function of the bacterial toxin, CcdB; PhD Thesis, Indian Institute of Science. [45] Sjöback, R., Nygren, J., and Kubista, M. (1995) Absorption and fluorescence properties of fluorescein, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 51, L7-L21. [46] Cook, K. H., Schmid, F. X., and Baldwin, R. L. (1979) Role of proline isomerization in folding of ribonuclease A at low temperatures, Proc Natl Acad Sci U S A 76, 6157-6161. [47] De Jonge, N., Garcia-Pino, A., Buts, L., Haesaerts, S., Charlier, D., Zangger, K., Wyns, L., De Greve, H., and Loris, R. (2009) Rejuvenation of CcdB-poisoned gyrase by an intrinsically disordered protein domain, Molecular cell 35, 154-163. [48] Jaffe, A., Ogura, T., and Hiraga, S. (1985) Effects of the ccd function of the F plasmid on bacterial growth, J Bacteriol 163, 841-849. [49] Ogura, T., and Hiraga, S. (1983) Mini-F plasmid genes that couple host cell division to plasmid proliferation, Proc Natl Acad Sci U S A 80, 4784-4788. [50] Miki, T., Yoshioka, K., and Horiuchi, T. (1984) Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA, J Mol Biol 174, 605-625. [51] Mori, H., Ogura, T., and Hiraga, S. (1984) Prophage lambda induction caused by mini-F plasmid genes, Mol Gen Genet 196, 185-193. [52] Karoui, H., Bex, F., Dreze, P., and Couturier, M. (1983) Ham22, a mini-F mutation which is lethal to host cell and promotes recA-dependent induction of lambdoid prophage, EMBO J 2, 1863-1868. [53] Shoemaker, B. A., Portman, J. J., and Wolynes, P. G. (2000) Speeding molecular recognition by using the folding funnel: the fly-casting mechanism, Proc Natl Acad Sci U S A 97, 8868-8873. [54] Huang, Y., and Liu, Z. (2009) Kinetic advantage of intrinsically disordered proteins in coupled folding-binding process: a critical assessment of the "fly-casting" mechanism, J Mol Biol 393, 11431159. [55] Trizac, E., Levy, Y., and Wolynes, P. G. (2010) Capillarity theory for the fly-casting mechanism, Proc Natl Acad Sci U S A 107, 2746-2750. [56] Yakovenko, O., Tchesnokova, V., Sokurenko, E. V., and Thomas, W. E. (2015) Inactive conformation enhances binding function in physiological conditions, P Natl Acad Sci USA 112, 98849889. [57] Levy, Y., Papoian, G. A., Onuchic, J. N., and Wolynes, P. G. (2004) Energy Landscape Analysis of Protein Dimers, Israel J Chem 44, 281-297. [58] Levy, Y., Wolynes, P. G., and Onuchic, J. N. (2004) Protein topology determines binding mechanism, Proc Natl Acad Sci U S A 101, 511-516. [59] Rumfeldt, J. A., Galvagnion, C., Vassall, K. A., and Meiering, E. M. (2008) Conformational stability and folding mechanisms of dimeric proteins, Prog Biophys Mol Biol 98, 61-84. [60] Rogers, J. M., Oleinikovas, V., Shammas, S. L., Wong, C. T., De Sancho, D., Baker, C. M., and Clarke, J. (2014) Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein, Proc Natl Acad Sci U S A 111, 15420-15425. [61] Rogers, J. M., Wong, C. T., and Clarke, J. (2014) Coupled folding and binding of the disordered protein PUMA does not require particular residual structure, J Am Chem Soc 136, 5197-5200. [62] Wallace, L. A., and Matthews, C. R. (2002) Sequential vs. parallel protein-folding mechanisms: experimental tests for complex folding reactions, Biophysical chemistry 101-102, 113-131. 30 ACS Paragon Plus Environment

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[63] Fersht, A. (1999) Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding, W. H. Freeman and Company, United States of America. [64] Tripathi, A., Dewan, P. C., Barua, B., and Varadarajan, R. (2012) Additional role for the ccd operon of F-plasmid as a transmissible persistence factor, Proc Natl Acad Sci U S A 109, 12497-12502. [65] Schmid, F. X. (1986) Fast-folding and slow-folding forms of unfolded proteins, Methods Enzymol 131, 70-82. [66] Wallace, L. A., and Matthews, C. R. (2002) Highly divergent dihydrofolate reductases conserve complex folding mechanisms, J Mol Biol 315, 193-211. [67] Heidary, D. K., O'Neill, J. C., Jr., Roy, M., and Jennings, P. A. (2000) An essential intermediate in the folding of dihydrofolate reductase, Proc Natl Acad Sci U S A 97, 5866-5870. [68] Brandts, J. F., Halvorson, H. R., and Brennan, M. (1975) Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues, Biochemistry 14, 4953-4963. [69] Salahuddin, A. (1984) Proline peptide isomerization and protein folding, J Biosci 6, 349-355. [70] Lu, K. P., Finn, G., Lee, T. H., and Nicholson, L. K. (2007) Prolyl cis-trans isomerization as a molecular timer, Nature chemical biology 3, 619-629. [71] Grathwohl, C., and Wüthrich, K. (1981) NMR studies of the rates of proline cis–trans isomerization in oligopeptides, Biopolymers 20, 2623-2633. [72] Shastry, M. C., and Udgaonkar, J. B. (1995) The folding mechanism of barstar: evidence for multiple pathways and multiple intermediates, J Mol Biol 247, 1013-1027. [73] Dill, K. A., and Chan, H. S. (1997) From Levinthal to pathways to funnels, Nat Struct Biol 4, 10-19. [74] Placek, B. J., and Gloss, L. M. (2005) Three-state kinetic folding mechanism of the H2A/H2B histone heterodimer: the N-terminal tails affect the transition state between a dimeric intermediate and the native dimer, J Mol Biol 345, 827-836. [75] Zhang, X., Kung, S., and Shan, S. O. (2008) Demonstration of a multistep mechanism for assembly of the SRP x SRP receptor complex: implications for the catalytic role of SRP RNA, J Mol Biol 381, 581-593. [76] Seifert, T., Bartholmes, P., and Jaenicke, R. (1985) Influence of cofactor pyridoxal 5'-phosphate on reversible high-pressure denaturation of isolated beta 2 dimer of tryptophan synthase bienzyme complex from Escherichia coli, Biochemistry 24, 339-345. [77] Galvagnion, C., Smith, M. T., Broom, A., Vassall, K. A., Meglei, G., Gaspar, J. A., Stathopulos, P. B., Cheyne, B., and Meiering, E. M. (2009) Folding and association of thermophilic dimeric and trimeric DsrEFH proteins: Tm0979 and Mth1491, Biochemistry 48, 2891-2906. [78] Rosengarth, A., Rosgen, J., and Hinz, H. J. (1999) Slow unfolding and refolding kinetics of the mesophilic Rop wild-type protein in the transition range, Eur J Biochem 264, 989-995. [79] Aghera, N., and Udgaonkar, J. B. (2013) The utilization of competing unfolding pathways of monellin is dictated by enthalpic barriers, Biochemistry 52, 5770-5779. [80] Wright, C. F., Lindorff-Larsen, K., Randles, L. G., and Clarke, J. (2003) Parallel protein-unfolding pathways revealed and mapped, Nat Struct Biol 10, 658-662. [81] Nguyen, H., Jager, M., Moretto, A., Gruebele, M., and Kelly, J. W. (2003) Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation, Proc Natl Acad Sci U S A 100, 3948-3953. [82] Chang, J. Y., Lu, B. Y., and Li, L. (2009) Fast and slow tracks in lysozyme folding elucidated by the technique of disulfide scrambling, The protein journal 28, 300-304. [83] Jha, S. K., Dasgupta, A., Malhotra, P., and Udgaonkar, J. B. (2011) Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry, Biochemistry 50, 30623074. [84] Roder, H., Elove, G. A., and Englander, S. W. (1988) Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR, Nature 335, 700-704.


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[85] Nawrocki, J. P., Chu, R. A., Pannell, L. K., and Bai, Y. (1999) Intermolecular aggregations are responsible for the slow kinetics observed in the folding of cytochrome c at neutral pH, J Mol Biol 293, 991-995. [86] Krishna, M. M., and Englander, S. W. (2007) A unified mechanism for protein folding: predetermined pathways with optional errors, Protein Sci 16, 449-464. [87] Aggarwal, V., Kulothungan, S. R., Balamurali, M. M., Saranya, S. R., Varadarajan, R., and Ainavarapu, S. R. (2011) Ligand-modulated parallel mechanical unfolding pathways of maltosebinding proteins, J Biol Chem 286, 28056-28065. [88] Van Melderen, L. (2002) Molecular interactions of the CcdB poison with its bacterial target, the DNA gyrase, Int J Med Microbiol 291, 537-544. [89] Engelberg-Kulka, H., and Glaser, G. (1999) Addiction modules and programmed cell death and antideath in bacterial cultures, Annu Rev Microbiol 53, 43-70. [90] Lee, S. J., Ko, J. H., Kang, H. Y., and Lee, Y. (2006) Coupled expression of MhpE aldolase and MhpF dehydrogenase in Escherichia coli, Biochem Biophys Res Commun 346, 1009-1015. [91] Basu, A., Chatterjee, S., and Das Gupta, S. K. (2004) Translational coupling to an upstream gene promotes folding of the mycobacterial plasmid pAL5000 replication protein RepB and thereby its origin binding activity, J Bacteriol 186, 335-342. [92] Tian, T., and Salis, H. M. (2015) A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons, Nucleic Acids Res. [93] Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF Chimera--a visualization system for exploratory research and analysis, J Comput Chem 25, 1605-1612. [94] Sanner, M. F., Olson, A. J., and Spehner, J. C. (1996) Reduced surface: an efficient way to compute molecular surfaces, Biopolymers 38, 305-320.


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Table 1: Kinetic parameters obtained for Scheme 1.

Equilibrium Kinetic parameters parameters Structural Unfolding

Refolding Transition

Equilibrium

Refolding rate

Unfolding m-value

constant

constant

m-value rate constant

(M-1)

(M-1)

2U ↔ If2

3.0*1010 M-1

9.9*108 M-1 s-1

-0.5

0.03 s-1

0.1

2U ↔ Is2

3.8*1011 M-1

1.0*108 M-1 s-1

-0.1

2.6*10-4 s-1

0.4

If2 ↔ N2

5.8*104

13.8 s-1

-1.37

2.4*10-4 s-1

0.76

Is2 ↔ N2

4.5*103

0.07 s-1

-0.3

1.5*10-5 s-1

0.6

Kinetic parameters were obtained by globally fitting the Scheme 1 to the refolding kinetic data obtained at different denaturant concentrations. The obtained kinetic parameters also describe the refolding kinetic data at different protein concentrations and unfolding kinetic data at different denaturant concentration. The kinetic parameters for the fitting were constrained such that each pathway satisfies the KU-N2, equilibrium constant for folding obtained from the isothermal denaturation studies. The association of unfolded monomers is inferred to be diffusion limited from the refolding data for 10nM CcdB, and constraints were set accordingly. The m-values given here, and throughout the manuscript, represent the slope of the log of the observed rate versus denaturant concentration. The m-values for the association and dissociation process presented in this table represent probable values, since there are no direct experimental measurements for the association processes.


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Scheme 1:

Scheme 1 – the proposed mechanism for refolding of the CcdB dimer. U represents the unfolded monomer. If2 and Is2 represent the dimeric fast folding and slow folding intermediates, respectively. N2 represents the native dimer.

Scheme 2:

Scheme 2 – An alternate mechanism for refolding of the CcdB dimer. U represents the unfolded monomer. 2If and 2Is represent the monomeric fast folding and slow folding intermediates, respectively. N2 represents the native dimer.


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Figure Legends

Figure 1. Structure of homodimeric CcdB (PDB id 3VUB). Asn 92, which was mutated to cysteine to generate the N92C variant, is shown in yellow. The tryptophans (W61, W99) are shown in red. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR001081).

Figure 2. Refolding kinetic studies of CcdB. (a) Biphasic refolding kinetics of Wt CcdB, with a fast and a slow phase. A few representative kinetic traces at 4µM CcdB concentration are shown. All studies were carried out at pH 7 and at 25°C. The experimental refolding kinetic traces obtained at 0.4 M, 1.0 M and 1.6 M final GdnHCl concentration (from bottom to top) are shown in black while the fits are shown in red. The inset shows an expanded view of the first 6 seconds of the refolding process. (b) Observed fast and slow kinetic phases account for the entire refolding reaction. A comparison of the observed equilibrium and kinetic amplitudes is shown. ) and infinite In the figure, data points for the equilibrium unfolding transition (), zero time ( time points ( ) during refolding are shown. The equilibrium unfolding transition was obtained under the same chemical and physical conditions using 4 µM CcdB and fluorescence was monitored on the same machine using identical settings

38

. The dashed line represents the

extrapolated unfolding baseline. (c) The dependence of the observed refolding rate constants on the denaturant concentration, of fast ( ) and slow phase ( ). The log of the rate constants varies linearly with the denaturant. The observed rate of the fast phase at zero denaturant concentration is determined to be 12.8 s-1, while that of the slow phase is 0.08 s-1. The refolding m-values of


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the transition states of the fast and the slow phases were calculated to be -1.3 M-1 and -0.32 M-1, respectively (d) The amplitudes of the fast ( ) and slow ( ) phases show complementarity, as the slow phase amplitude increases at the expense of the fast phase, with increase in GdnHCl concentration. The error bars wherever shown represent the standard deviation from three independent experiments.

Figure 3. Refolding kinetic studies of CcdB at different protein concentrations. (a) The dependence of the observed refolding rate constants, of both fast ( ) and slow phase ( ), on the protein concentration. The observed rate constants of both phases do not vary with changes in protein concentration. (b) The relative amplitude of the fast ( ) phase increases gradually with increase in protein concentration, while that of the slow phase ( ) decreases. The error bars wherever shown represent standard error from two independent experiments.

Figure 4. Denaturant dependent refolding kinetics of fluorescein labeled N92C variant of CcdB at 10 nM concentration. Refolding kinetic traces were acquired by monitoring fluorescence emission at 540 nm upon excitation at 495 nm. (a) Refolding kinetic traces (solid black lines) acquired in the presence of 0.4, 0.8, and 1.2 M GdnHCl (from bottom to top) and bi-exponential fits through the data (solid red lines). The inset shows an expanded view of the kinetic traces for the initial 6 seconds. (b) Comparison of the equilibrium and kinetic amplitudes for the refolding process. Shown are the data points for the equilibrium unfolding transition (), extrapolated zero time ( ) and extrapolated infinite time ( ) for the refolding process of fluorescein labeled N92C-CcdB. Brown dashed line represents the extrapolated equilibrium unfolding baseline. The equilibrium unfolding transition was acquired under conditions identical to that of the refolding


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kinetics studies. Panels c and d show the observed rates and amplitudes, respectively, for fast ( ) and slow phases ( ) as a function of denaturant concentration. In panel c, the log of the rate constants varies linearly with the denaturant. The slope of the fit (dashed lines) gives the refolding m-values of the transition states. The observed rate of the fast phase at zero denaturant concentration is determined to be 11.8 s-1, while that of the slow phase is 0.09 s-1. The refolding m-values of the transition states of the fast and the slow phases were calculated to be -1.2 M-1 and -0.35 M-1, respectively. Error bars, wherever shown, represent the spread of measurement from two independent experiments.

Figure 5. Unfolding kinetic studies of CcdB. (a) Unfolding of Wt CcdB follows single exponential kinetics. A few representative kinetic traces at 4 µM CcdB concentration are shown. All studies were carried out at pH 7 and at 25°C. The experimental kinetic traces are shown in black while the fits are shown in red, for unfolding traces obtained at 3, 3.5 and 4 M GdnHCl concentration (from right to left). The inset shows an expanded view of the first 10 seconds of the refolding process. (b) Observed amplitude of the unfolding process accounts for the entire unfolding reaction. A comparison of the observed equilibrium and kinetic amplitudes is shown. In the figure, data points for the equilibrium unfolding transition (), zero time ( ) and infinite time points ( ) during unfolding are shown. The dashed line represents the extrapolated folded baseline. (c) The dependence of the observed unfolding rate constant () on the denaturant concentration. The log of the rate constant varies linearly with the denaturant concentration. The slope of the fit gives the unfolding m-value. The error bars, wherever shown, represent the standard error from two independent experiments.


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Figure 6. Interrupted refolding experiment. (a) Refolding kinetic trace (dotted blue line) initiated by diluting out the GdnHCl from 3.2 M to 1.6 M at pH7. The dark pink line through the refolding trace is a least-squares fit to a two-exponential equation. The refolding kinetic trace was found to decay in two phases with observed rate constants of 0.1 s-1 and 0.02 s-1. Unfolding was initiated by increasing the GdnHCl concentration to 3 M (solid black line), by interrupting the refolding process after 5 s, 25 s, 50 s, 100 s and 150 s (left to right). The red lines represent single-exponential least squares fits to the unfolding kinetic traces. All the unfolding traces are mono-phasic, where the observed rate constant is 0.07 ± 0.01 s-1 and show no burst phase. (b) Native CcdB populates in a bi-exponential manner. Shown are the fractional populations of native CcdB with the time of refolding obtained from extrapolated t0 points of the unfolding traces. The fractional native population was obtained by normalizing amplitudes of the unfolding traces with the total amplitude of the unfolding trace of the native CcdB protein at 3 M GdnHCl. The solid blue line through the data represents a least-squares fit to a two-exponential equation. The amplitude and the observed rate constant for the fast and slow phases are identical to those observed for the refolding traces obtained in 1.6 M GdnHCl. The error bars represent the standard deviation from three separate experiments.

Figure 7. Interrupted unfolding experiment. (a) Kinetic trace of unfolding (solid black line) initiated by a jump of GdnHCl to 3 M at pH7. The line (red) through the unfolding trace is a least-squares fit to a single-exponential equation. The unfolding trace was found to rise with a rate of 0.054 ± 0.01 s-1. Kinetic traces of refolding (dotted blue lines) in 1.6 M GdnHCl obtained by interrupting the unfolding process after 5, 20, 40 and 60 s (left to right) are shown. The dark pink lines represent bi-exponential least squares fits to the refolding traces. All the refolding


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traces decay in two phases, where observed rate constants for the fast and slow phase are 0.11 ± 0.02 s-1 and 0.03 ± 0.006 s-1, respectively. (b) Fractional populations representing the fractional amplitude of the fast phase ( ) and slow phase () are plotted as a function of the unfolding time. The fractional populations were obtained by normalizing amplitudes of the fast phase and slow phase with the total amplitude of the refolding trace of the fully unfolded protein at 1.6 M GdnHCl concentration. The solid lines through the data represent a least-squares fit to a singleexponential equation. The fast and slow phase amplitudes change with observed rate constants of 0.07 s-1 and 0.021 s-1, respectively. The error bars represent the standard deviation of measurements made in three separate experiments.

Figure 8. Kinetics of binding of CcdA to CcdB during folding of CcdB. CcdA binds to native CcdB within 7 s, which is within the dead time of the manual mixing experiment (blue line). The experiment was performed by monitoring the fluorescence at 313 nm upon excitation at 280 nm. Binding of CcdA to CcdB was initiated after refolding CcdB for 0 s (black line), 30 s (purple line) and 200 s (maroon line, overlaps with the blue trace). The binding kinetic trace of CcdA to CcdB initiated after 0 s of refolding (folding and binding initiated simultaneously) fits to a twoexponential equation (red line). Binding of CcdA to CcdB was performed in the presence of 1.5 M GdnHCl concentration. The refolding kinetic trace of CcdB in absence of CcdA (dark green line) and the corresponding fit using two-exponential equation (light green line) are also shown.

Figure 9. Validation of the folding mechanism by global fitting of the folding model (Scheme 1) to refolding kinetic traces. The kinetic parameters obtained from the fit are given in Table 1. (a) Representative refolding kinetic traces (black lines) obtained at 0.4, 1 and 1.6 M GdnHCl


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(bottom to top) using a monomer protein concentration of 4 µM and fit through the data (red lines). (b) Representative experimental refolding kinetic traces (black lines) obtained at 0.4, and 4 µM monomeric CcdB concentrations (bottom to top) in the presence of 0.4 M GdnHCl. The red lines represent the simulated traces using Scheme 1 and kinetic parameters from Table 1. (c) Experimental kinetic unfolding trace (black lines) obtained at 3, 3.5 and 4 M GdnHCl concentrations and the simulated traces (red lines) for the same using Scheme 1 and parameters from Table 1. (d) Representative refolding kinetic traces (black) and fit (red) through them, using Scheme 2. The fits to Scheme 2 are inferior relative to Scheme 1 (compare a and d). In all the panels, insets show expanded views of the process during early times of refolding/ unfolding.


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.


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Figure 7.


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Figure 8.


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Figure 9.


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For Table of Contents Use Only

The homodimeric E. coli toxin CcdB (Controller of Cell Division or Death B protein) folds via parallel pathways. Chetana Baliga, Raghavan Varadarajan, Nilesh Aghera.

Graphic for the Table of Contents (ToC Graphic)


Homodimeric Escherichia coli Toxin CcdB - ACS Publications

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