Unraveling the Folding Mechanism of the Smallest Knotted Protein

Article pubs.acs.org/JPCB

Unraveling the Folding Mechanism of the Smallest Knotted Protein, MJ0366 Iren Wang,† Szu-Yu Chen,† and Shang-Te Danny Hsu*,†,‡ †

Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan Institute of Biochemical Sciences, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan

‡

S Supporting Information *

ABSTRACT: Understanding the mechanism by which polypeptide chains thread themselves into topologically knotted structures has emerged to be a challenging subject not least because of the additional complexity associated with the spontaneous and efficient knotting and folding events. While recent theoretical calculations have made significant progress in establishing the atomistic folding pathways for a number of knotted proteins, experimental data on the folding stabilities and kinetic pathways of knotted proteins has been sparse. Using MJ0366 from Methanocaldococcus jannaschii, the smallest knotted protein known to date, as a model system, we set out to systematically investigate its folding equilibrium, kinetics, and internal dynamics under native and chemically denatured states. NMR hydrogen−deuterium exchange analysis indicates that the knotted region is the most stable structural element within the novel fold. Additionally, 15N spin relaxation analysis reveals the presence of residual structures in urea-denatured MJ0366. Despite the apparent two-state equilibrium unfolding behavior during chemical denaturation, the kinetic unfolding pathway of MJ0366 involves the dissociation of the homodimeric native state into a native-like monomeric intermediate followed by unfolding into a denatured state. Our results provide comprehensive structural information regarding the folding dynamics and kinetic pathways of MJ0366, whose small size is ideal for converging experimental and theoretical findings to better understand the underlying principles of the folding of knotted proteins.

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INTRODUCTION Protein folding has been an outstanding fundamental problem over the past decades. Its association with debilitating human disorders has made it even more biomedically relevant.1,2 While experimental findings and theoretical calculations have begun to converge for a number of model protein systems that are relatively small in size, the emerging question of how a polypeptide chain manages to thread itself through a number of loops and attain a defined topologically knotted structure has put forward a new dimension to render the protein folding problem ever more complex.3 Recent surveys of the protein structure database have identified hundreds of topologically knotted proteins with highly diversified protein folds.4,5 While the majority of the knotted proteins contain a trefoil (31) knot, i.e., three projected crossings, in their backbone structures, more complex figure-of-eight (41) knots, Gordian (52) knots, and even a Stevedore’s (61) knot have been identified in protein structures.6,7 It remains elusive as to why these topological knots have evolved into integral parts of knotted protein families since no biological function has hitherto been attributed to any of these protein knots. Nonetheless, it is fascinating to realize that many of these topologically knotted proteins can refold spontaneously after chemical or thermal denaturation without the aid of auxiliary factors such as molecular chaperones.8,9 YibK from Haemophilus inf luenzae and YbeA from Escherichia coli are two of the most studied knotted proteins. © 2015 American Chemical Society

Both contain a long C-terminal helix of ca. 40 amino acids that threads through a loop to form a deep trefoil knot.10,11 Both YibK and YbeA are homodimeric in solution: dimerization and therefore correct quaternary structure is essential for cofactor binding.12 While YibK exhibits parallel folding pathways with multiple folding intermediates, YbeA exhibits a linear folding pathway with a monomeric folding intermediate.11,13−15 Remarkably, recent data have suggested that both YibK and YbeA remain topologically knotted under chemically denaturing conditions.16 It is unclear how both proteins maintain their knotted topologies without the presence of appreciable secondary and tertiary structures according to far-UV circular dichroism (CD), intrinsic fluorescence, and nuclear magnetic resonance (NMR) spectroscopy.17,18 Nevertheless, a follow-up study using a reconstituted cell-free protein synthesis system showed that both YibK and YbeA can attain their natively knotted structures de novo in the absence of molecular chaperones while addition of the chaperonin system, GroEL/ GroES, can accelerate the folding kinetics of both proteins.9 Additionally, ubiquitin C-terminal hydrolases (UCHs) are conserved cysteine proteases that contain a highly intricate Gordian (52) knot with five projected backbone crossings. Equilibrium chemical unfolding of human UCH-L3 is an Received: November 4, 2014 Revised: March 4, 2015 Published: March 5, 2015 4359

DOI: 10.1021/jp511029s J. Phys. Chem. B 2015, 119, 4359−4370

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The Journal of Physical Chemistry B

Figure 1. Structure and dynamics of MJ0366. (A) Three-dimensional structure of the dimeric MJ0366 (PDB code: 2EFV) that contains a topological trefoil knot within its backbone structure. The knotted core defined by the pKnot server (residues 17−78 shown in pale blue) is flanked by the N- (blue) and C-terminal (red) regions that are involved in the crossing (right). The simplified topological representation from the backbone of MJ0366. (B) 15N−1H HSQC spectrum of MJ0366 recorded at pH 6.0 and 310 K. (C) The far-UV CD spectrum of 10 μM MJ0366 at pH 6 and 298 K. (D) NMR analysis of the structure and dynamics of MJ0366. The TALOS+ predicted secondary structure contents, the ratio of 15N R2/R1, and the order parameters (S2) as a function of residue number are shown in descending order. Residues located in the β-strands and α-helices of the crystal structure are indicated by yellow and red rectangles, respectively. The segment that exhibits an elevated R2/R1 ratio due to diffusion anisotropy is highlighted in cyan. (E) SEC-MALS profile that confirms MJ0366 as a stable dimer in solution. The scales of calculated molecular weight and light scattering (LS) intensity are shown on the left and right axes, respectively.

provided the energy landscapes of these systems where parallel folding pathways with multiple local energy minima were identified, which correspond to well-defined folding intermediates.24,25 Additionally, many of the theoretical works suggest that the formation of a slipknot is an essential step in the knotting process.22−24 Misfolding occurs when incorrect loop threading leads to wrong knot chirality, which becomes the rate-limiting step of the folding pathways of many knotted proteins. This has led to an apparent rollover in the chevron plot of the refolding kinetics of a designed knotted protein from Helicobacter pylori, HP0242, which was also observed experimentally.26 However, there is a general lack of experimental data on the folding dynamics and kinetics of the knotted proteins, which have been subjected to theoretical calculations. In this study, we have employed a variety of biophysical tools to investigate in detail the folding equilibrium and kinetics of a hypothetical protein, MJ0366, from Methanocaldococcus jannaschii. MJ0366 adopts an α/β knot fold with a short antiparallel

apparently two-state process, but its kinetic unfolding exhibits a hyper-fluorescent species and three distinct kinetic phases can be identified by stopped-flow fluorescence measurements, which correspond to two parallel folding pathways in which two distinct folding intermediates are present.19 Unlike UCHL3, UCH-L1 and its Parkinson disease (PD)-associated mutants display well-defined folding intermediates during equilibrium unfolding by urea. The PD-associated mutations significantly accelerate the unfolding kinetics of UCH-L1 and destabilize its native structure.20 Importantly, under dilute conditions, both UCH-L1 and -L3, as well as YibK and YbeA, can refold spontaneously and efficiently in isolation in vitro.19,20 Over the years, remarkable progress has been made in theoretical calculations of the folding pathways of topologically knotted proteins. While early attempts using simplified coarsegrained simulations have failed to reproduce the efficient refolding of some of the knotted proteins unless a biased energy potential was provided,21−23 recent studies using allatom simulations on a number of small knotted proteins have 4360


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The Journal of Physical Chemistry B β-sheet and four α-helices, and has been proposed to be a family of the ribbon-helix-helix superfamily of DNA binding proteins.6,27 Importantly, it contains a trefoil knot in its backbone topology with a minimum knotted core (residues 17−78) as defined by the pKnot server28 (Figure 1). With just 92 residues in its sequence, MJ0366 is the smallest knotted protein that has been identified to date. It has also been subjected to computational simulations to delineate its folding and knotting pathways.24,25,29 Given its small size, it is an ideal system to combine experimental and computational findings to characterize the folding mechanism of a topologically knotted protein and to understand how knotting occurs in atomic detail. Our results showed that MJ0366 is a stable homodimer in solution and that the knotted structural element as well as the dimer interface are sequestered from solvent exposure in the native state. While equilibrium unfolding of MJ0366 by urea and guanidine hydrochloride (GdnHCl) reveals an apparently two-state process, dissociation of the dimeric structure into a native-like folding intermediate takes place prior to the unfolding of the secondary and tertiary structures. The onpathway folding intermediate leads to rollover in the refolding arm of the corresponding chevron plot derived from stoppedflow fluorescence measurements. Besides, the urea-denatured MJ0366 exhibits pronounced backbone 15 N transverse relaxation rates in clusters of residues that correspond to secondary structure elements in the native state, suggesting the presence of residual structures in the urea-denatured state, which may be attributed to long-range interactions between hydrophobic side chains. Together, we proposed an onpathway three-state unfolding kinetics pathway of MJ0366 as a native dimer (N2) dissociating into two monomeric MJ0366 (2I) and then starting denatured into an unfolded state (2U) as N2 ⇆ 2I ⇆ 2U.

mM imidazole with the same buffer background. The eluted fractions were pooled and incubated with TEV protease in a dialysis bag of 3.5 kDa molecular weight cutoff with a 1:100 dialysis ratio at room temperature overnight. This step can efficiently remove the z2 fusion tag from MJ0366 and remove the imidazole from the protein solution. A second His-tag based affinity purification step was carried out on the following day to separate the His-tagged z2 tag from the tag-free MJ0366. Finally, size-exclusion chromatography (SEC; HiLoad 26/60 Superdex 75, GE Healthcare Life Sciences) with 50 mM potassium phosphate (pH 6.0) and 50 mM NaCl was carried out to remove impurities to yield a purity of higher than 95% based on visual inspection of the coomassie-blue-stained sodium dodecyl sulfate (SDS) polyacrylamide gel (PAGE). The protein solution was aliquoted, flash-frozen by liquid nitrogen, and stored at −80 °C until further use. Unless otherwise specified, the SEC buffer was used for all the biophysical characterizations described herein. Far-UV CD Spectroscopy. Protein stock solution was diluted to 10−15 μM and 300 μL in volume for CD measurements using a quartz cuvette with a path length of 0.1 cm (Hellma, Germany). The far-UV CD spectra were recorded between 200 and 260 nm using a CD spectrometer (J815, JASCO, Japan). The samples were temperature-controlled at 25 °C, and the data were collected with a bandwidth of 1 nm, a data interval of 0.5 nm, and an averaging time of 1 s. The molar ellipticity (deg·cm2·dmol−1) was calculated by using the following equation: [θ] = θ/(10 × C × l × n), where θ is the CD signal in units of millidegrees, C the molar concentration in M, l the cell path length in cm, and n the number of amino acids. For equilibrium unfolding by urea or GdnHCl, 41 aliquots of MJ0366 solution were prepared containing a gradient of urea (0−8 M) or GdnHCl (0−6 M) concentrations with a linear increment step of 2.5%. A two-channel liquid dispensor (Hamilton, USA) was used to generate the 41-point denaturant gradient, and the dispensing of protein stock solution was achieved by using an electronic pipet (Eppendorf, Germany) to minimize pipetting errors. The far-UV CD spectra were collected over 220−260 nm, and the titration series were subjected to singular value decomposition (SVD) analysis using MatLab (MATLAB and Statistics Toolbox Release 2012b, The MathWorks, Inc., Natick, Massachusetts, United States) to determine the number of states associated with the equilibrium unfolding. Intrinsic Fluorescence Spectroscopy. The intrinsic fluorescence of MJ0366 originating from the phenylalanine and tyrosine residues was monitored by exciting the samples at 275 nm and recording the emission spectra between 285 and 450 nm. For chemical denaturation analysis, the same sets of samples that were used for far-UV CD spectroscopy were used for intrinsic fluorescence measurements followed by the same SVD analysis procedure. SEC-MALS. The absolute molecular weight of MJ0366 was determined as previously described8 by static light scattering (SLS) using a Wyatt Dawn Heleos II multiangle light scattering detector (Wyatt Technology) coupled to an AKTA Purifier UPC10 FPLC protein purification system using a Superdex 75 5/150 GL size-exclusion column (GE Healthcare). 1.3 mg/mL MJ0366 (10−20 μL) was applied to the size-exclusion column with a buffer containing 50 mM potassium phosphate (pH 6.0), 50 mM NaCl, and 0.02% NaN3 by a flow rate of 0.4 mL/min. BSA (2 mg/mL) was used for the system calibration as a

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MATERIALS AND METHODS Constructs Preparation, Protein Expression, and Purification. An E. coli codon usage-optimized synthetic gene was synthesized that corresponds to the DNA reading frame of MJ0366 (GenScript, USA) and subcloned into a modified pET-z2 vector between NcoI and KpnI sites, resulting in a fusion with a His-tag at the N-terminus followed by a z2domain fusion tag, a TEV protease cleavage site, and the MJ0366 reading frame. The plasmid was transformed into the BL21(DE3) E. coli strain. Unlabeled, uniformly 15N labeled, or uniformly 15N/13C labeled protein samples were expressed by growing the transformed cells in Luria−Bertani (LB) medium or M9 minimal medium containing 15NH4Cl (and 13C-glucose for uniformly 15N/13C labeling) in the presence of kanamycin (30−50 μg/mL) for antibiotics selection. The overexpression of recombinant MJ0366 was induced by the addition of 0.5 mM IPTG when the cell density reached OD600 = 0.6−0.8 followed by overnight growth at 16 °C. The cells were harvested by centrifugation using a Beckman J20XP centrifuge with a JLA 8.1000 rotor for 30 min with 6000 rpm at 4 °C and resuspended in buffer containing 50 mM potassium phosphate (pH 8.0) and 300 mM NaCl. The harvested cells were disrupted using a sonicator, and the cell debris and supernatant were separated by a second centrifugation step at 45000 × g for 30 min at 4 °C. The supernatant was loaded onto a prepacked 5 mL His-Trap HP column (GE Healthcare Life Science) followed by extensive washing using a buffer containing 15 mM imidazole to remove protein impurities and prevent nonspecific binding. Target fusion protein was eluted using 250 4361


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elongated quaternary structure, the anisotropic fitting option within Tensor2 was used to define the anisotropic diffusion tensor and to calculate the order parameters of individual amide groups. The intrinsic R2 profile of 8 M urea-denatured MJ0366 was estimated using an in-house script as described previously to account for the reduced R2 relaxation at the fraying N- and Ctermini.36,37 The widths and heights of the clusters of the enhanced R2 profile were estimated by fitting the observed profiles to Gaussian distributions in addition to the bell-shape basal R2 profile. NMR Hydrogen−Deuterium Exchange (HDX) Experiments. 500 μL of 100 μM 15N-labeled MJ0366, which was buffered in 50 mM potassium phosphate (pH 6 or pH 6.92) and 50 mM sodium chloride, was flash-frozen and lyophilized prior to the HDX experiments. 500 μL of 99.9% D2O was added to the lyophilized NMR sample and immediately transferred to a 5 mm NMR tube for NMR measurements. A series of 2D [15N−1H] HSQC spectra were recorded over 2 weeks at 25 °C and at 600 MHz. The NMR samples were incubated at 25 °C in between the NMR measurements, with intervals from hours to days. The individual peak intensities were fit to a single-exponential decay, I(t) = I0 exp(−kex·t), by using the software GraphPad Prism (version 6.0 for Mac, GraphPad Software, La Jolla, CA, USA). The observed HDX rate constants of amide protons, kex, were used to calculate the protection factor (PF), defined as PF = log(kint/kex), where kint corresponds to the intrinsic exchange rate constant. Folding Kinetics Analysis by Stopped-Flow Fluorescence Measurements. Stopped-flow fluorescence measurements were performed using an Applied Photophysics SX18 stopped-flow spectrometer as described previously.20 The folding reactions were triggered by rapidly mixing the folding or unfolding buffer to protein solution with an asymmetric mixing ratio of 10:1. The changes in total fluorescence were monitored using an excitation wavelength of 280 nm with a 320 nm cutoff filter. All experiments were performed at 25 °C using a water circulation system to maintain the sample temperature. Twenty μM of protein was buffered in 50 mM potassium phosphate (pH 6) and 50 mM NaCl with and without 6 M GdnHCl for unfolding and refolding measurements. After the 11-fold dilution, the final protein concentration was 1.8 μM. The observed reaction rates were extracted by fitting the kinetic traces to a single or double exponential function with an offset using the software package GraphPad Prism (version 6.0 for Mac, GraphPad Software, La Jolla, CA, USA). The choice of model, i.e., single of double kinetic phases, was decided using the F-test statistics by Prism. Data Analysis of Chemical-Denaturant-Induced Equilibrium Unfolding and Folding Kinetics. As the dissociation of dimeric MJ0366 into native-like monomers occurs prior to the complete unfolding of the monomeric MJ0366 and the first dissociation step does not give significant spectral changes during equilibrium unfolding analysis, we fit the equilibrium denaturation curves derived from intrinsic fluorescence and farUV CD spectroscopy by GdnHCl to a two-state unfolding equilibrium model:

control. The absolute molecular weights of individual peaks in the size-exclusion chromatogram were determined by the SLS data in conjunction with the refractive index measurements (Wyatt Optilab T-rEX, connected downstream of the LS detector). A standard value of refractive index increment, dn/dc = 0.185 mL/g, was used for the proteins, and the buffer viscosity η = 1.0257 cP at 25 °C was calculated using SEDNTERP. The value of the reference refractive index, 1.3458 RIU, was taken directly from the measurement of the Wyatt Optilab T-rEX when buffer was only passing through the reference cell. Individual viscosity and reference refractive index for buffers with various concentrations of GdnHCl were derived and applied by the same ways. NMR Assignments of Native and Urea-Denatured MJ0366. Uniformly 13C and 15N labeled MJ0366 was buffered in 50 mM potassium phosphate (pH 6.0), 100 mM NaCl, 50 mM arginine and 50 mM glutamic acid, 0.02% NaN3 with 10% (v/v) 2H2O. The addition of arginine and glutamine was necessary to maintain the protein solubility at 0.55 mM.30 All the triple resonance experiments were acquired at 310 K using an AVIII600 Bruker NMR spectrometer, equipped with a cryogenic TCI probe or an AV600 Bruker NMR spectrometer equipped with a room temperature TXI probe. For 8 M ureadenatured MJ0366, the protein solution was buffered in 50 mM potassium phosphate (pH 6.0) and 50 mM NaCl. All the triple resonance spectra were recorded at 298 K. All NMR spectra were processed using NMRPipe/Draw31 and analyzed using Sparky packages (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco, CA). The chemical shift assignments of the backbone nuclei of native MJ0366, i.e., 15N, 1 N 13 α 13 ß H , C , C , and 13C′, were derived from CBCA(CO)NH, HNCA, HN(CO)CA, CN-NOESY-HSQC, and HNCO experiments,32 while those of 8 M urea-denatured MJ0366 were obtained from HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB experiments using the iterative procedure as described previously.17,33 The NMR assignments have been deposited in the Biomolecular Magnetic Resonance Database, BMRB, under the accession code 25282. NMR 15N Spin Relaxation Dynamics Experiments. 15N spin relaxation measurements of native MJ0366 were recorded at 298 K using a Bruker AVIII 600 spectrometer equipped with a cryogenic TCI probe. For the longitudinal relaxation (R1) measurements, the recovery delays were set to 10, 100, 250, 500, 750, 1250, 1500, and 2200 ms; for the transverse relaxation (R2) measurements, the relaxation delays were set to 17, 34, 51, 68, 85, 102, and 119 ms. The longitudinal (T1) and transverse (T2) relaxation time constants were calculated by fitting the peak heights of individual cross-peaks as a function of relaxation delays using the rate analysis module within the Sparky package. Steady-state 15N−{1H} heteronuclear NOE (hetNOE) were recorded with 3 s of interscan recovery delays with and without a weak 1H saturation pulse train for the saturated and reference spectra, respectively. The residuespecific hetNOE is defined as the ratio of cross-peak intensities between the saturated and reference spectra, i.e., hetNOE = Isat/Iref. The 15N R2 relaxation rates and hetNOE of 8 M ureadenatured MJ0366 were obtained using the same experimental settings, and the used relaxation delays for measurements were set to 17, 34, 68, 102, 136, and 238 ms. Analysis of the backbone 15N relaxation dynamics of native MJ0366 was carried out using Tensor234 with default settings as described previously.35 Specifically, to account for the anisotropic diffusion of the dimeric MJ0366 due to its

KU

N⇌U

(1)

where N and U correspond to the native and unfolded states. KU is the equilibrium constant of unfolding. The observed 4362


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The Journal of Physical Chemistry B Table 1. Thermodynamic Parameters for the Fit of MJ0366 Equilibrium Unfolding Data GdnHCl

Urea

ΔGHNU2O (kcal mol−1)

mNU (kcal mol−1 M−1)

[D]50% NU (M)

ΔGHNU2O (kcal mol−1)

mNU (kcal mol−1 M−1)

[D]50% NU (M)

10.37 ± 0.90 12.04 ± 1.57 8.34 ± 0.64

3.37 ± 0.29 3.76 ± 0.49 3.11 ± 0.44

3.08 ± 0.02 3.21 ± 0.02 2.68 ± 0.31

8.47 ± 1.71 10.45 ± 1.57

1.31 ± 0.26 1.50 ± 0.23

6.48 ± 0.10 6.99 ± 0.06

Intrinsic fluorescence Far-UV CD Stopped-flow (by rate)

Table 2. Kinetic Parameters for the Folding and Unfolding of 1.8 μM MJ0366 at pH 6 and 298 K KNI (M s−1) −11

(1.16 ± 2.25) × 10

mNI (kcal mol−1 M−1)

kf,UI2 (s−1)

2 ku,IU (s−1)

mf,UI (kcal mol−1 M−1)

mu,IU (kcal mol−1 M−1)

ΔGIU2 (kcal mol−1)

−7.17 ± 0.72

1598 ± 1678

0.0012 ± 0.0003

−4.59 ± 0.46

0.69 ± 0.07

8.34 ± 0.64

HO

HO

intrinsic fluorescence or far-UV CD signal (Sobs) could be defined as Sobs = S N(1 − fU ) + SU fU

dimer dissociation into intermediate monomer (I), the subsequent unfolding into a fully unfolded state (U) is protein concentration independent and can be described as

(2)

KU =

where f N and f U are the fractional populations of the N and U states and SN and SU are the signals resulting from the N and U states, respectively. Under equilibrium conditions, the fractional populations can be presented as fN =

1 1 + KU

fU =

KU 1 + KU

(3)

fI =

(4)

(5)

(6)

KNI

H 2O (−mf,UI[GdnHCl]) H 2O (−m u,IU[GdnHCl]) k f,UI e e + k u,IU

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RESULTS Solution Structure and Folding Dynamics of MJ0366. In order to evaluate the structure of the recombinant MJ0366 in solution, we have employed solution NMR and far-UV CD spectroscopy to access its folding properties. The 15N−1H heteronuclear single quantum correlation (HSQC) spectrum of MJ0366 exhibits highly dispersed cross-peaks, confirming that the recombinant protein is well-folded in aqueous solution (Figure 1). Furthermore, the far-UV CD spectrum of recombinant MJ0366 exhibits two negative bands at 208 and 222 nm, indicating that the secondary content of MJ0366 is highly α-helical, as was shown in the crystal structure (Figure 1C). The secondary structure content of MJ0366 calculated by

(8)

In this model, the equilibrium constant of dissociation of native dimer (N2) into monomeric intermediate (2I) is defined as

KNI =

[I]2 [N2]

(13)

where q is equal to 1 + KU, mNI is the m-value of native state HO H2O unfolded into intermediate, kf,UI2 and ku,IU are the folding and unfolding rate constants, respectively, and mf,UI and mu,IU are the associated m-values. The fitting results are tabulated in Table 2.

(7)

kIU

kUI

and of a and at a

⎛ ⎞ K e(−mNI[GdnHCl]) 8PT ⎟ NI kobs = ⎜⎜ q2 + q − ⎟ (−mNI[GdnHCl]) 4PT K e ⎝ ⎠ NI

where Iobs is the detected signal intensity, I0 is the fluorescence signal of the original input, and A is the corresponding fluorescence amplitude change. To consider a rollover refolding arm in the chevron plot of MJ0366, the observed reaction rates were fit to a three-state on-pathway kinetic unfolding model in which the intermediate is monomeric:38−40 N2 HooI 2I ⇌ 2U

(11)

(12)

The observed reaction rate, kobs, is the sum of folding unfolding rates, i.e., kobs = kf + ku. With the assumption linear relationship between denaturant concentration activation free energies, the observed reaction rate kobs given concentration of GdnHCl could be expressed as

For a two-state equilibrium unfolding system, the free energy of unfolding ΔG follows a linear relationship with the denaturant concentration. The nonlinear regression of the data fitting was carried out using Prism. The MJ0366 kinetic traces of refolding and unfolding reactions were analyzed at different concentrations of GdnHCl and fit to a single-exponential kinetics equation defined as Iobs = I0 + A e( −kobst )

⎤ KNI ⎡ 8P ⎢ (1 + KU)2 + T − (1 + KU)⎥ ⎥⎦ 4PT ⎢⎣ KNI

k f = fI ·k fH2O

S N + SU e−ΔG NU / RT 1 + e−ΔG NU / RT

(10)

where PT is the total protein concentration expressed per mole of monomers. The folding rate, kf, depends on the fraction of intermediate, and can be expressed as

Therefore, eq 2 can be expressed as a function of free energies between different states and sample temperature, T: Sobs =

kIU [U] = kUI [I]

where [U] is the concentration of the fully unfolded population. We assume that the folding and unfolding process has reached equilibrium and that the fraction of the folding intermediate is time-independent. Accordingly, the fraction of the folding intermediate monomers can be presented as

The equilibrium constants can be transformed into a free energy difference between states i and j as ΔGij = −RT ln K ij

HO

(9)

where [N2] and [I] are the concentrations of the native dimer and native-like monomeric intermediate, respectively. After 4363


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was evoked for the model-free analysis, which showed that most of the secondary structure elements are highly ordered, while some degree of flexibility is present in the loop region. The first 10 residues at the N-terminus as well as the last 5 residues at the C-terminus are highly disordered with an order parameter of 0.5 or less, which explains the lack of electron density for these residues in the crystal structure. Equilibrium Unfolding of MJ0366 Monitored by Intrinsic Fluorescence and Far-UV CD Spectroscopy. To evaluate the thermal stability of MJ0366, we monitored its far-UV CD spectra from 5 to 99 °C. Clear precipitation could be observed when the sample was heated to 75 °C and above, hence precluding further analysis of the thermodynamics of thermal unfolding. A fluorescent-dye-based thermal shift assay was applied subsequently to scout for better buffer conditions that would enhance the thermal stability of MJ0366 by varying salt concentration and pH value (Figure S2, Supporting Information). The result showed that MJ0366 is sensitive to changes in the pH values: the melting temperature anticorrelates with the pH value between 5.0 and 8.0 with a maximum melting temperature of 86 °C. We therefore chose to buffer MJ0366 in 50 mM potassium phosphate (pH 6.0) and 50 mM NaCl for subsequent equilibrium and kinetic unfolding studies unless otherwise indicated. To examine the equilibrium unfolding of MJ0366 induced by urea or GdnHCl, far-UV CD and intrinsic fluorescence spectroscopy were applied to monitor the changes in secondary and tertiary structures, respectively. MJ0366 has no tryptophan in its primary sequence; hence, the intrinsic fluorescence of the four tyrosine and two phenylalanine residues was used instead even though they have relatively low fluorescence (Figure 2A). MJ0366 is highly resistant to urea-induced unfolding, as the intensity of its intrinsic fluorescence continued to increase without reaching a plateau when the concentration of urea reached 8.5 M and beyond (Figure S3A, Supporting Information). Using GdnHCl, which is a much stronger protein denaturant compared to urea, a sigmoidal unfolding curve could then be obtained with a transition point located at around 3 M. Singular value decomposition (SVD)44 of the intrinsic fluorescence and far-UV CD spectra of MJ0366 as a function of GdnHCl concentration indicates that the titration series can be sufficiently described by only two components; i.e., the chemical denaturation of MJ0366 by GdnHCl is a two-state process (Figure 2). Note that the free energy of GdnHClinduced unfolding of MJ0366 derived from far-UV CD data is 2.3 kcal mol−1 higher than that derived from intrinsic fluorescence data: both the m-value and [D]50% are higher in the case of far-UV CD data (Table 1). The intrinsic fluorescence spectra of MJ0366 displayed minimal changes in the wavelengths of the maximum emission intensity, which were 303 and 301 nm upon the addition of 8.5 M urea and 5.7 M GdnHCl, respectively. Oligomeric State of MJ0366 during Chemical Denaturation Determined by SEC-MALS. As the dimerization state of MJ0366 by SEC-MALS analysis could be determined accurately without assuming that the protein of interest is compact and globular in solution (Figure 1E), we carried out SEC-MALS analysis for MJ0366 to monitor its oligomeric state in the presence of different amounts of GdnHCl (Figure 2C). In contrast to the equilibrium unfolding by intrinsic fluorescence and far-UV CD spectroscopy, which correspond to a two-state unfolding process with a transition point at

the Jasco built-in program Multivariate SSE consists of 49.2, 10.4, 12.6, and 27.8% of α-helix, β-sheet, turns, and others, respectively, which are in good agreement with the amount of α-helix and β-sheet (48.9 and 13.0%) observed in the crystal structure (PDB code: 2EFV). As the crystal structure of MJ0366 is homodimeric, we employed size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) to determine the oligomeric state of MJ0366 in solution. The SEC-MALS analysis gives an apparent molecular mass of 22.4 ± 0.4 kDa of the highly symmetric elution peak with a monodispersity of 1.003, which corresponds to a dimeric form of MJ0366 (the theoretical molecular weight is 10.9 × 2 = 21.8 kDa according to its primary sequence; Figure 1E). To investigate the solution structure and dynamics of MJ0366 in more detail, we completed the backbone NMR assignments, and used the backbone chemical shiftsthose of 1HN, 15N, 13Cα, 13Cβ, and 13 C′ atomsas inputs to predict the secondary structure content of MJ0366 using TALOS+41 (Figure 1D). The results are highly consistent with the reported crystal structure, which is composed of two antiparallel β-strands (β1, residues 12−18; β2, residues 55−59) flanked by four α-helices (α1, residues 23−34; α2, 41−49; α3, 62−71; α4, 74−87). To examine the internal backbone dynamics of MJ0366, standard 15N spin relaxation experiments, including longitudinal (R1) and transverse (R2) relaxation rates and 15N−{1H} heteronuclear NOE,42 were collected to determine the order parameters (S2) on the ps to ns time scale. An overall rotational correlation time (τc) of 13 ns was obtained from the ratio mean R2/R1 rate, in line with the expectation for a dimeric MJ0366 assembly (Figure S1, Supporting Information). Model-free analysis43 was carried out using Tensor234 to determine the order parameters of individual backbone amide groups. It is apparent that α3 displays an elevated R2/R1 ratio compared to the rest of the protein. The enhanced R2 relaxation in this helical region can be rationalized by the anisotropic diffusion of the highly asymmetric dimeric assembly (Table 3) of which α3 is oriented along the long axis. Therefore, an anisotropic tumbling model Table 3. Anisotropic Diffusion Parameters Derived from 15N Relaxation Data of MJ0366 at 298 K number of experimental R2/R1 ratios Axial Symmetric Model Euler angles (deg) ϕ θ Rotational diffusion coefficients (107 s−1) D⊥ D∥ Anisotropy D∥/D⊥ χ2 Full Asymmetric Model Euler angles (deg) α β γ Rotational diffusion coefficients (107 s−1) Dx Dy Dz χ2

67

−88.1 ± 3.8 −47.3 ± 3.8 1.03 1.64 1.59 8.66

± 0.03 ± 0.06 ± 0.07 × 101

−32 ± 38 −87 ± 4 −47 ± 4 1.00 1.06 1.65 4.42

± 0.03 ± 0.37 ± 0.56 × 101 4364


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Figure 2. GdnHCl-induced equilibrium unfolding of MJ0366 monitored by intrinsic fluorescence and far-UV CD spectroscopy. (A) The fluorescence emission spectra of MJ0366 are color ramped from blue to red for data points recorded from 0 to 5.7 M GdnHCl. (B) GdnHCl titration series of MJ0366 monitored by far-UV CD spectroscopy. The coloring follows the same scheme as that for intrinsic fluorescence analysis. (C) SVD analysis of the two-state equilibrium unfolding process of MJ0366. The first SVD components of the intrinsic fluorescence and far-UV CD data are shown in open red circles and filled blue squares, respectively, with vertical dashed lines to indicate their respective unfolding transition points. SECMALS-derived molar masses of MJ0366 are shown in filled green circles as a function of GdnHCl concentration and overlaid with the intrinsic fluorescence and far-UV CD data points.

therefore classified the observed HDX rates into four categories as follows: • EX2:

around 3.2 M GdnHCl, the SEC-MALS analysis revealed that the dissociation of dimeric MJ0366 into monomers takes place between 1 and 2 M GdnHCl, suggesting that the monomeric intermediate remains native-like with most of the secondary and tertiary structures which only begin to unfold in the presence of 2.5 M GdnHCl and higher. Hydrogen−Deuterium Exchange (HDX) Reveals Stable Structures within the Knotted Core and Dimer Interface. In order to examine the folding dynamics of MJ0366 under native conditions, we employed NMR hydrogen−deuterium exchange (HDX) analyses at pH 6 and pH 6.92 to extract the protection factors (PFs) of individual backbone amide groups at 25 °C. About half of the 15N−1H cross-peaks fully exchanged beyond detection within the 10 min of experimental dead time, while 12 cross-peaks remained visible after 2 weeks of HDX at pH 6, which correspond to the residues located in the secondary structures, suggesting the presence of a very stable folding core structure45−47 (Figure S4A, Supporting Information). Overall, 44 PFs could be obtained from the HDX analysis at pH 6, except for Leu30 and Asn80, the cross-peak intensities of which only reduced marginally after 2 weeks of HDX, precluding reliable determination of the associated HDX rates (Figure 3). Structural mapping of the observed PFs showed that most of the highly protected residues are located in α1, α2, and α4, while Phe15 at β1 and Lys19 at the following turn structure, Arg56 and Thr59 at β2 also exhibit high PF values. These residues are either part of the knotted structure or located at the dimer interface. We next repeated the same HDX analysis at pH 6.92 to verify whether the observed HDX rates correspond to the EX1 or EX2 regime48 (Figure 3B). In the case of the EX2 regime, the HDX process takes place under the native condition as a fast pre-equilibrium, the rate of which is proportional to the concentration of the catalyst, [OH−]. Hence, the difference in observed HDX rates in logarithm scale between pH 6.0 and pH 6.92 is expected to be log kex (pH 6.92) − log kex (pH 6) = 0.92. In the case of the EX1 regime, the HDX process is limited by the opening rate of the corresponding hydrogen bond and hence the observed HDX rates are independent of the sample pH values. Nonetheless, the observed HDX rates exhibit a broad distribution between ideal EX1 and EX2 conditions. We

log kex (pH 6.92) − log kex (pH 6) > 0.7

• General EX2: 0.7 > log kex (pH 6.92) − log kex (pH 6) > 0.5

• General EX1: 0.5 > log kex (pH 6.92) − log kex (pH 6) > 0.3

• EX1: log kex (pH 6.92) − log kex (pH 6) < 0.3

According to such a classification, 13 residues are in the EX2 regime, 11 residues are in the general EX2 regime, 7 residues are in the general EX1 regime, and 5 residues are in the EX1 regime. Structural mapping shows that the EX2 and general EX2 residues are mostly solvent exposed, whereas the EX1 and general EX1 residues are more buried within the hydrophobic core, except for Lys19 (Figure 3C). Specifically, the EX1 residues Leu17 and Lys19 at the turn following β1 and Leu44 at α2 are involved in extensive contacts with the threading Cterminal α4, including the other two EX1 residues Leu78 and Leu79 as well as Asn80, which exchanges with solvent too slowly. Additionally, the general EX1 residues Phe15 at β1, Leu29 at α1, and Cys81 and Leu83 at α4 are also involved in the hydrophobic interactions within the knotted region. Finally, Asp42 and Leu44 at α2, which exhibit high PFs (similar to those of Leu78 and Leu79 at α4) and are classified as general EX1 and EX1 residues, respectively, are located at the dimer interface. The results suggest that dissociation of the native dimer may be the ratelimiting step for the local unfolding of α4, which is directly associated with knot formation. NMR Analysis of Urea-Denatured MJ0366. The intrinsic fluorescence and far-UV CD data showed that MJ0366 could be unfolded by urea with a transition point at around 6.5 M (Table 1). In order to investigate in further detail the structure and dynamics of urea-denatured MJ0366, we carried out an 4365


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increased to 8 M, the poorly dispersed resonances of the denatured state MJ0366 dominated with a few remaining native resonances (Figure 4A). Structural mapping of the chemical shift perturbations of MJ0366 in 1 M urea compared to the native state showed that residues Lys10−Ala13, Lys55, and Ser57 of the antiparallel β-sheet as well as Asn76 and Leu77 of α4 exhibit the most pronounced perturbations, which may result from their higher solvent accessible surface area that acts as the nucleus of unfolding. To gain further insights into the urea-denatured MJ0366, we completed 94.5% (87 out of 92) of the backbone resonance assignments of 8 M urea-denatured MJ0366, which exhibits no significant residual secondary structure according to the chemical-shift-based prediction (Figure 4). The 15N R2 as a function of residue number displays a bell-like baseline with four pronounced clusters of enhanced R2 rates that correspond to residues located in α1, α2, α3, and the end of α4, suggesting the presence of residual structures in these helical regions in the 8 M urea-denatured state (Figure 4). These enhanced R2 relaxation rates potentially resulted from long-range interactions of clusters of hydrophobic side chains,36,49 which are involved in the knot formation. Additionally, the 15N−{1H} hetNOE values of all residues are lower than 0.5 (Figure 4B), reflecting the highly flexible backbone structure. Notably, the regions that exhibited enhanced 15N R2 values in the helical regions and those in α2, in particular, also exhibit slightly elevated 15N−{1H} hetNOE values. Folding Kinetics of MJ0366 by Intrinsic Fluorescence. Stopped-flow fluorescence measurements were carried out to extract the folding and unfolding rates of MJ0366 in the presence of a varying amount of GdnHCl. All the unfolding and refolding kinetic traces were fit to a single exponential function to extract the observed reaction rates (kobs) (Figure S6, Supporting Information). A chevron plot is generated by plotting kobs as a function of GdnHCl concentration50 and a rollover refolding arm with a transition at around 1.5 M GdnHCl was observed, suggesting the presence of a folding intermediate during kinetic refolding (Figure 5). Generally speaking, a chevron plot with a rollover refolding arm can be fit to both on- and off-pathway three-state models and whether the observed intermediate is on- or off-pathway will require additional experimental evidence to differentiate.38,40 In the case of MJ0366, however, we observed by SEC-MALS a clear dissociation process of the native dimer into intermediate monomers between 1 and 2 M GdnHCl, while the intermediates maintain most of the secondary and tertiary structure as evidenced by the native-like far-UV CD and fluorescence spectra (Figure 2). Furthermore, the transition point of the monomer−dimer equilibrium coincides with the inflection point of the rollover refolding arm in the chevron plot (Figure 5), indicating that the rollover is associated with the monomer−dimer equilibrium. Finally, the refolding rate exhibits protein concentration dependency only at a denaturant concentration well below 1 M GdnHCl (inset of Figure 5). Taken together, there is strong evidence to suggest that the folding intermediate is indeed on-pathway rather than an offpathway product. We therefore fit the resulting chevron plot to an on-pathway three-state unfolding kinetics model with an equilibrium constant KNI and an mNI-value to describe the equilibrium between the native and intermediate states (Materials and Methods). The fitting results lead to a transition point between the intermediate and denatured state, [D]50% NU , of 2.68 ± 0.31 M GdnHCl, which is statistically the same as that

Figure 3. NMR HDX analysis of MJ0366. (A) PFs of MJ0366 amide protons as a function of residue numbers at pH 6. The positions of Leu30 and Asn80 whose PFs cannot be determined reliably are indicated in gray bars. (B) Double log plot of the HDX rate, kex, obtained at pH 6 and pH 6.92. The residues that are classified as EX2, general EX2, and general EX1 and EX1 are labeled in dark blue, blue, green, and red, respectively. The HDX rates and corresponding log values at pH 6 and pH 6.92 are listed in Table S1 (Supporting Information). (C) Topological mapping of the EX1/EX2 regime (left) and PFs (right) derived from observed HDX rates at pH 6. The EX1/ EX2 residues are in the same colors as mentioned in part B, and the PFs are colored from pink to dark blue ranging from 2.5 to 6.5. Those slow-exchanging residues (gray bars in part A) are shown with gray circles.

NMR titration experiment of MJ0366 by recording a series of 15 N−1H HSQC spectra of MJ0366 under 0, 0.2, 0.4, 0.6, 0.8, 1, 5, 6, 7, and 8 M urea to monitor the changes in the chemical shifts, and analyzed the internal backbone dynamics of MJ0366 in 8 M urea (Figure 4 and Figure S5, Supporting Information). The well-dispersed resonances of native MJ0366 persisted up to 6 M urea, which is consistent with the two-state unfolding process of the secondary and tertiary structures revealed by farUV CD and intrinsic fluorescence (Figure S3, Supporting Information). On increasing urea concentration to 7 M, a new set of resonances that correspond to unfolded MJ0366 emerged. The coexistence of native and unfolded species in 7 M urea is consistent with the two-state folding equilibrium established from the intrinsic fluorescence and far-UV CD spectroscopy (Figure 2). When the urea concentration was 4366


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Figure 4. Structure and dynamics of urea-denatured MJ0366. (A) 15N−1H HSQC spectrum of 8 M urea denatured MJ0366 recorded at pH 6.0 and 298 K. (B) The secondary structure content, 15N R2 relaxation rates, and hetNOE are shown in descending order. The secondary structure content was predicted by δ2D. The residual 15N R2 relaxation rates of 8 M urea denatured MJ0366 are shown in open purple circles. Regions that exhibit larger 15N R2 relaxation rates are fit to four separate Gaussian distributions (in solid line). The basal 15N relaxation rate profile is shown in a dashed line.

observed folding rates of MJ0366 in the presence of four different GdnHCl concentrations (0.5, 1.5, 2.5, and 4 M) as a function of final protein concentration ranging between 1.5 and 5.6 μM. As expected, the observed folding rates of MJ0366 at 1.5, 2.5, and 4 M GdnHCl are independent of protein concentration, while the refolding rates at 0.5 M GdnHCl increase on protein concentration, indicating that the refolding of MJ0366 is associated with dimerization (Figure 5). Therefore, a three-state unfolding pathway can be established on the basis of the kinetic measurements where a dimeric MJ0366 dissociates into native-like monomers that subsequently unfold into a denatured state, which contains no significant secondary structures but retains a significant amount of long-range interactions (Scheme 1). Figure 5. Chevron plot analysis of the folding kinetics of MJ0366. The observed reaction rates and corresponding amplitudes are shown in the top and bottom panels, respectively. The observed reaction rates are fit to an on-pathway intermediate three-state unfolding model. The amplitudes associated with the unfolding reactions are fitted to a twostate equilibrium unfolding model. Protein-concentration-dependent kinetic measurements were carried out at four different GdnHCl concentrations, as shown in red, blue, green, and orange vertical dashed lines, respectively. Inset: The observed folding rates of MJ0366 as a function of protein concentration with colors that correspond to the reference dashed lines for various GdnHCl concentrations.

Scheme 1. Kinetic Unfolding Pathway of MJ0366

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DISCUSSION Using MJ0366 as a model system, we systematically characterized the folding equilibrium and kinetics of this smallest trefoil knotted protein known to date. Equilibrium chemical denaturation of MJ0366 is an apparently two-state process, according to far-UV CD and intrinsic fluorescence spectroscopy (Figure 2). NMR titration by urea revealed the coexistence of the observed resonances of both native and unfolded MJ0366 in the presence of 7 M urea further supporting the two-state unfolding behavior of MJ0366 (Figure S5, Supporting Information). The NMR HDX results showed that MJ0366 contains a very stable dimeric structure with a knotted core formed by hydrophobic interactions, involving the C-terminal α4 and the dimer interface (Figure 3). While most

derived from the equilibrium unfolding analysis using intrinsic fluorescence ([D]50% NU = 3.08 ± 0.02 M GdnHCl; Table 1). Although the nonlinear regression obtained high uncertainties associated with the equilibrium constant KNI and the refolding HO rate kf,UI2 due to their convoluted contributions to the refolding arm of the chevron plot, the remaining kinetic parameters, H2O namely, m-values and unfolding rate ku,IU , could be determined with high confidence. To verify the association between the rollover with the dissociation of the dimeric MJ0366, we determined the 4367


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knotted proteins.24,25,29 Slipknotting is the rate-limiting step, which involves backtracking to overcome kinetic traps due to the formation of slipknots of wrong chirality. Importantly, the simulations also showed that the deepness of the C-terminal segment in knotting is correlated with the efficiency of the folding rates. Extension of the C-termini of MJ0366 by five amino acids to account for the missing electron density of these residues in the crystal structure significantly reduced the observed folding efficiency in silico.29 An unbiased all-atom explicit-solvent simulation29 demonstrated that similar native contacts responsible for threading the slipknot through the loop could be observed as in the structure-based simulations,24 but additionally observed non-native salt bridges would compete with the native salt bridges during threading of the slipknot and increase the energetic roughness of the energy landscape. When taking the last five C-terminal residues into consideration, the three charged amino acids (Glu88, Glu90, and Arg91) might form as the kinetic traps with other charged residues while the slipknot is threading into the loop; e.g., it was proposed that Glu34-Arg91 forms as a salt bridge to stall the side-chain of Arg91 fully crossing the loop, and the replacement of the non-native salt bridge of Glu34-Arg45 helps the movement of Arg91 eventually threading across the loop.29 In the simulation studies, the knotted proteins with a smaller size like MJ0366 can achieve a higher success rate in refolding through a slipknot pathway while deep trefoil knotted proteins such as YbeA and YibK show less comparable success, as a longer slipknot does not favor threading the loop. While in silico simulations have provided a plausible folding mechanism that may be generalized for knotted proteins, much needed experimental data such as Φ-value analysis could shed light on the nature of the folding pathways of these interesting folding systems, particularly with regard to the contributions of individual residues along the folding pathways and the formation of slipknots and the eventuality of the native knotted conformations. Although our current results have not been able to fully address the question of how and when the protein knot is formed, they should help pave the way toward a comprehensive atomistic understanding of the folding mechanism of these smallest knotted proteins.

of the residues undergo HDX under equilibrium (EX2 regime), few exhibit slow HDX rates and are under kinetic control by the opening rates of the associated hydrogen bonds (EX1 regime). These EX1 residues are involved in extensive hydrophobic contacts with the C-terminal α4, and at the dimer interface, suggesting that the dimer interface may be involved in knot formation, reminiscent of the case of YibK.12 Additionally, although the secondary chemical shift of 8 M urea-denatured MJ0366 suggested little residual secondary structure content, the observed clusters of 15N R2 (Figure 4) indicated the presence of long-range interactions within the highly disordered polypeptide chains. It remains to be established as to whether these long-range interactions are involved in the knot formation. Interestingly, the SEC-MALS analysis of MJ0366 in the presence of different concentrations of GdnHCl clearly showed that dimeric MJ0366 starts to dissociate into monomeric forms between 1 and 2 M GdnHCl (Figure 2). Together with the results of far-UV CD and intrinsic fluorescence spectroscopy, we propose that the monomeric MJ0366 is a native-like intermediate with most of the secondary and tertiary structures and then unfolds subsequently. The folding kinetics of MJ0366 as determined by stoppedflow fluorescence measurements reveal a well-defined chevron plot of the observed folding rates as a function of GdnHCl concentrations. A rollover in the refolding arm around 1.5 M GdnHCl suggests the presence of an intermediate (Figure 5). Indeed, the refolding rate of MJ0366 at 0.5 M GdnHCl is clearly protein-concentration-dependent (inset, Figure 5): the apparent refolding rate increased by 53% from a protein concentration of 1.5 to 5.6 μM, providing clear evidence of the contribution of dimer dissociation to the rollover refolding arm. Furthermore, this is consistent with the observation by SECMALS measurements (Figure 2C). Accordingly, we propose an on-pathway three-state unfolding pathway model for MJ0366, as shown in Scheme 1, which shows a linear unfolding pathway with a monomeric native-like intermediate. In comparison with the other two trefoil knotted proteins, YibK and YbeA, the equilibrium unfolding of MJ0366 involves the same dimer dissociation from the native state to the intermediate, which is native-like structures.11,13 Unlike YibK and YbeA, however, the stopped-flow fluorescence measurements of MJ0366 reveal a single chevron trace with a rollover refolding arm, while multiple kinetic phases were observed for YibK and YbeA with all the unfolding and refolding arms being linear.11,14 While MJ0366 shows a linear folding pathway as HO does YbeA, its folding rate (kf 2 = 1598 s−1) is 4 orders of magnitude faster than that of YbeA (kHf 2O = 0.2 s−1). In contrast, their unfolding rates are on a similar scale (kHu 2O = 0.0012 s−1 for MJ0366 and kHu 2O = 0.002 s−1 for YbeA).11 Compared to YibK, which exhibits parallel pathways with multiple folding intermediates, the two observed folding rates kHf 2O (130 and 15 s−1, respectively) are also significantly slower than that of MJ0366; furthermore, their unfolding rates kHu 2O (0.3 and 0.015 s−1, respectively) are slightly faster than that of MJ0366.14 Such a difference in the observed folding and unfolding rates may be associated with the depth of the knotted element: YibK and YbeA contain a deeply knotted C-terminal helix of about 40 residues, while MJ0366 contains a shallower knotted Cterminal helix of less than 10 residues (Figure 1). Indeed, recent molecular dynamics simulations on knotted proteins have proposed that the formation of a slipknot prior to the completion of folding pathways is a common feature for

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ASSOCIATED CONTENT

S Supporting Information *

NMR HDX rate constants, backbone 15N relaxation dynamics data, thermal stability analysis under different buffer conditions, urea-induced equilibrium unfolding of MJ0366 monitored by far-UV CD, intrinsic fluorescence, and NMR spectroscopy, and exemplar stopped-flow kinetics traces. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +886-2-27855696. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Council (100-2113-M-001-031-MY2 and 101-2627-M-001-004) and Academia Sinica, Taiwan. S.-T.D.H. was supported by a Career Development Award (CDA-00025/2010-C) from the International Human Frontier Science Program. The NMR spectra 4368


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were obtained at the Core Facility for Protein Structural Analysis, supported by the National Core Facility Program for Biotechnology, Taiwan. We thank Dr. Meng-Ru Ho of the Biophysics Core Facility of the Institute of Biological Chemistry, Academia Sinica, Taiwan, for providing technical assistance and support with data of the far-UV CD experiments.

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