Mechanical Softening of a Small Ubiquitin-Related Modifier Protein

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Mechanical Softening of a Small Ubiquitin-related Modifier Protein (SUMO2) Due to Temperature Induced Flexibility at the Core Shrabasti Bhattacharya, and Sri Rama Koti Ainavarapu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b06844 • Publication Date (Web): 11 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

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The Journal of Physical Chemistry

Mechanical Softening of a Small Ubiquitin-Related Modifier Protein (SUMO2) due to Temperature Induced Flexibility at the Core

Shrabasti Bhattacharya and Sri Rama Koti Ainavarapu* Department of Chemical Sciences, Tata Institute of Fundamental Research Dr Homi Bhabha Road, Colaba, Mumbai 400005 *Author for correspondence: Sri Rama Koti Ainavarapu; Email: [email protected] Telephone: +91 22 22782790; Fax: +91 22 22804610 ABBREVIATIONS: SUMO2, Small ubiquitin-related modifier; SMFS, Single-molecule force spectroscopy

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Abstract Despite the growing interest in the thermal softening of proteins, the mechanistic details of it are far from understood. β-grasp proteins have globular shape with compact structure and they are mechanically resilient. The β-clamp or mechanical clamp in them formed by the interactions between the terminal β strands is generally associated with the protein mechanical resistance. Although previous studies showed that temperature can perturb the protein mechanical stability, the structural changes leading to the lowered mechanical resistance are not known. Here, we investigated the temperature-dependent mechanical stability of small ubiquitinrelated modifier 2 (SUMO2) using single-molecule force spectroscopy (SMFS) and the corresponding conformational changes using ensemble experiments. SMFS studies on the polyprotein of SUMO2 estimate a decrease in the spring constant of the protein from 4.50 N/m to 1.35 N/m upon increasing the temperature from 5-45 0C. Interestingly, near-UV circular dichroism spectroscopy reveals a decrease in tertiary structure content while the overall secondary structure of the protein remains unchanged. Steady-state fluorescence and quenching studies on SUMO2 with a tryptophan mutation at the core (F60W) show that the nonpolar environment of the tryptophan is unchanged and the protein core is inaccessible to the bulk solvent, in the same temperature range. We attribute the thermal softening observed in AFM experiments to the reduction in tertiary structure of SUMO2. Our results provide evidence for the importance of the intramolecular interactions at the protein core along with the β- or mechanical clamp in providing the mechanical resistance as well as in modulating the protein stiffness.


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Introduction Protein structure, stability and dynamics are interdependent and they are important for protein biological function.1-3 Understanding the relation between protein stability, folding and the conformational dynamics is central in elucidating protein function.4 The folding and conformational dynamics of protein are dictated by the underlying protein energy landscape.5,6 The energy landscape of proteins is sometimes complex, with intermediates between the native state and the unfolded state.7,8 Structural characterization of these intermediates is important in understanding the folding and stability of proteins. Intermediate states are often populated depending on perturbing conditions such as chemical denaturants, pH and temperature.9 In the past, many methods have been used to access intermediate states between the native and unfolded states on the pathways of folding and unfolding.10,11 The intermediates which are structurally related to the native state having similar secondary structures but different tertiary structure are of particular interest.12,13 Such intermediates have been found in many globular proteins like α-Lactalbumin14, Carbonic Anhydrase15 and Bovine Growth Hormone16, where these proteins exhibit a stable equilibrium intermediate when perturbants such as an acid or chemical denaturant are added. The protein molecules in such an intermediate state are nearly as compact as the native protein, possessing native-like secondary structure but with an increase in conformational fluctuations. In addition, the side chains of amino acids in such states experience an overall ‘averaged’ isotropic environment due to decrease in van der Waals interactions.17,18 The characterization of such conformationally flexible intermediates are important as these are very close to the native state in terms of energy and conformational flexibility compared to the other intermediates which are far distant on the protein energy landscape. 3 ACS Paragon Plus Environment


These flexible intermediates fall between typical globular structured proteins and intrinsically disordered proteins, with tertiary structure being affected but secondary structure remaining intact compared to the corresponding native structures.19 The flexibility of proteins can be estimated by single-molecule force spectroscopy (SMFS) experiments, which characterize the unfolding energy landscape of proteins through forced unfolding of proteins.20-22 SMFS gives a direct quantification of the mechanical flexibility/spring constant of proteins by defining an order parameter ∆xu, the distance to the unfolding transition state from the native state, and therefore an estimate about the local curvature of the landscape of protein unfolding.23-25 Previous studies showed that protein structures which are highly flexible have a shallow unfolding potential as measured in SMFS experiments.26,27 Recently, there has been growing interest in probing the thermal softening of proteins using SMFS.28-34 Temperature has been shown to induce softening or increase flexibility in proteins.30,34 For small globular proteins, it has been hypothesized that the thermal softening might be due to a temperature-dependent gradual shift in the nature of interactions responsible for the protein mechanical stability, from hydrogen bonding networks to hydrophobic interactions and ionic clusters.30,34 It has also been suggested that the temperature might induce conformational expansion of mechanical clamp that primarily provides mechanical resistance.33 Temperature dependent protein dynamics local to the mechanical clamp has also been thought to affect the protein stiffness.32 Despite these previous reports, it remains to be understood what changes in the protein structure and dynamics manifest thermal softening. Hence, a detailed investigation of probing the temperature dependent changes in the protein structure that are responsible for the varied mechanical response is required. Temperature induced protein conformational changes which lead to increased protein flexibility are yet to be characterized. It is not known to what extent temperature perturbs protein structure


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to make it flexible without losing all mechanical resistance. Temperature dependent changes in the secondary or tertiary structure and the corresponding changes in the solvent accessibility of interior of protein leading to thermal softening are yet to be ascertained. Here we probe the temperature dependent conformational changes of a small ubiquitinrelated modifier 2 (SUMO2) protein to ascertain the mechanistic details of protein thermal softening. Protein sequence and relevant structural details of SUMO2 are given in Figure 1. SUMO2 has a ubiquitin-fold topology with five β-strands wrapped around an α-helix.35 The ubiquitin-like β-grasp proteins have a β-clamp or mechanical clamp geometry where the interactions between the terminal β-strands (β1-β5) provide mechanical resistance against stretching, as shown for SUMO2 in Figure 1B.21,36-38 We measured the mechanical properties of SUMO2 by SMFS and complemented them with ensemble structural studies to elucidate the corresponding temperature-dependent protein conformational changes. We implemented circular dichroism, steady-state fluorescence and fluorescence quenching methods to characterize the structural details of SUMO2 at different temperatures. We show that the conformational flexibility of the protein core increased without any discernible change in its secondary structure as well as accessibility of the core to the external solvent molecules in the temperature range 545 0C. We therefore hypothesize that the increase in conformational flexibility of the protein correlates to reduced mechanical resistance and thereby leading to the thermal softening of SUMO2.



Experimental methods Protein expression and purification. The genes of human (SUMO2)8 were synthesized by previously established protocol.21 The octameric protein (SUMO2)8, were overexpressed in the BL21 DE3 strain of E. coli by inducing with 1 mM IPTG (isopropylthio-D-1thiogalactopyranoside) for 6 h after the OD600 of the cell culture has reached 0.6. The harvested cells were suspended in sodium phosphate buffered saline (PBS) (pH 7.4) containing 0.1 mM PMSF (phenylmethanesulfonyl fluoride) and other protease inhibitors like Leupeptin and Pepstatin (~0.1 mg both). The cells were lysed by sonication, centrifuged, and the supernatant was applied to a column of Ni-NTA-coated agarose beads. The beads were washed with PBS containing 20 mM imidazole and the proteins containing N-terminal His-tag were eluted with PBS containing 250 mM imidazole at pH 7.4. They were further purified by size exclusion chromatography using a superdex200 column (Amersham Biosciences). The purity of proteins was checked on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. All purified proteins were stored at 4 0C in PBS adjusted to pH 7.4. SUMO2 was already cloned in pQE80L vector between BamH1 and Kpn1. It was then mutated by site-directed mutagenesis to SUMO2F60W and the resulting gene was checked by DNA sequencing. SUMO2 and SUMO2F60W proteins were expressed and purified using the same procedure as that of (SUMO2)8 but they were purified by superdex75 column (Amersham Biosciences) owing to their smaller size. Both N-acetyl-L-Tryptophanamide (NATA) and Acrylamide were purchased from Sigma-Aldrich. Ni-NTA beads were purchased from Qiagen. Single-molecule force spectroscopy (SMFS). Temperature controlled SMFS measurements were carried out on a commercial atomic force microscope (AFM) (NanoWizard III from JPK, Berlin, Germany). The sample stage is connected to the JPK heating-cooling 6 ACS Paragon Plus Environment

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module which maintains temperature by a circulating water bath with temperature resolution of 0.1 0C. Gold-coated reflective cantilevers with a silicon-nitride tip with spring constants ~40 pN/nm were purchased from Bruker, USA. Calibration of cantilevers was done using the equipartition theorem before each pulling experiment.39 In a typical pulling experiment, a 100µl sample was placed on a gold-coated glass coverslip and the cantilever was pushed on to the substrate surface until a contact force of 2 nN was obtained. The pulling velocity was varied from 100-4000 nm/sec. For each temperature, the mounted cantilever was incubated in the protein solution for half an hour before acquiring force-versus-extension (FX) data. SMFS data analysis. In the data analysis, force-versus-extension (FX) traces containing at least 5 forces peaks of protein unfolding were considered. The FX traces were fitted to the worm-like chain (WLC) model of polymer elasticity40 using Eq. 1 given below: =

1 −

− +

Equation 1

where F, x, p, Lc, kB, and T denote the force, the end-to-end molecular extension, the persistence length, the contour length, the Boltzmann constant, and the absolute temperature, respectively. The persistence length values used for fitting varied between 0.3-0.7 nm. This range of persistence length values found to best fit the FX traces. Spontaneous unfolding rate constant (ku0) and distance to the unfolding transition state (∆ ∆xu). ku0 and ∆xu were estimated using Monte Carlo simulations as described by Oberhauser et al.41 In brief, the dependence of unfolding force on pulling velocity was modeled assuming two state Markov process.20,42 Initially all the domains in a polyprotein chain were assumed to be folded (Nf) and were pulled at a given velocity and the force was calculated using



WLC model as described above. The probability of unfolding (Pu) of any of the domains is given by Pu=Nfk(F)∆t, where k(F) = ku0 eF∆xu/kBT. The unfolding event and force at which it occurred was recorded when the probability Pu, was higher than a random number between 0 and 1. The procedure was repeated till all the domains in the polyprotein chain unfolded and the unfolding force histograms were made. The values of ku0 and ∆xu were varied such that the unfolding force histograms from simulations matched with those obtained experimentally at different pulling velocities. Transition state energy barrier and spring constant of the unfolding potential. ku0 and ∆xu are calculated using Monte Carlo simulations as described above. The transition state activation energy was calculated using the Arrhenius equation as in Eq. 2 and the spring constant was calculated using harmonic approximation given by Eq. 3 as described earlier by Schlierf and Rief.30 ‡ = − . ! % =

&' ‡

&" (

# "

$

Equation 2

Equation 3

where ∆G‡ is the activation energy, kA is Arrhenius frequency factor (109) and ks is the spring constant for the deformation along the N-C termini pulling direction. Steady state fluorescence spectroscopy. Fluorescence spectra were collected on a Fluoromax3 spectrofluorometer (Horiba Jobin Yvon). For fluorescence measurements, the protein samples were excited at 295 nm and emission spectra were collected from 310 to 450 nm, with a scan speed 0.1 nm/sec, an excitation bandwidth of 1.5 nm, and an emission


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bandwidth of 1.5 nm. Measurements were made using protein concentrations of 8 µM. Each data was averaged over 3 scans. Circular dichroism (CD) studies. CD measurements were carried out on a Jasco J-810 (Jasco, Tokyo, Japan) spectropolarimeter. CD spectra for the secondary structure region (195260 nm) were recorded using 25 µM SUMO2 and ~ 28 µM SUMO2F60W in a quartz cuvette of 1 mm path length. The CD spectra in the tertiary structure region (260-320 nm) were recorded using 1mM for SUMO2 and 200µM for SUMO2F60W protein in a quartz cuvette of 1 cm path length. Each spectrum both in near and far UV was recorded as the average of three wavelength scans. Both far and near UV spectra were corrected by subtracting buffer spectra. Fluorescence quenching studies. Fluorescence quenching experiments were carried out using Acrylamide as Trp fluorescence quencher and the quenching data of SUMO2F60W was fitted to the following Stern-Volmer equation given below:43 )# )

= 1 + *+, -./

Equation 4

where F0 is the fluorescence intensity of the protein in the absence of quencher and F is the fluorescence in the presence of quencher at a concentration [Q]. The fluorescence emission was measured at 332 nm by exciting the Trp at 295 nm. The slope of the Stern-Volmer (K 12 ) plot gives an extent of how much quenching occurred.43 32 µM protein was used for quenching experiments.



Results Temperature dependent SMFS experiments on (SUMO2)8. We have used polyprotein engineering combined with SMFS experiments to probe the temperature dependent mechanical unfolding properties of SUMO2 (Figure 2A). We performed pulling experiments on the polyprotein (SUMO2)8, where individual proteins are linked through N-C termini, as a function of temperature from 5 - 45 0C. Polyproteins provide characteristic sawtooth patterns that serve as fingerprints in SMFS experiments. The experimental details are given in Experimental methods section. A characteristic force-versus-extension (FX) trace of (SUMO2)8 from SMFS experiments performed at 5 0C is shown in Figure 2B. The FX trace shows a sawtooth pattern of force peaks indicating the sequential unfolding of individual units in the polyprotein. As shown in Figure 1, SUMO2 consists of 93 residues, with the first 17 residues form a featureless Nterminus while the subsequent 76 residues form a β-grasp structure similar to Ubiquitin.35 However this unstructured terminus does not provide any mechanical resistance while stretching and it unravels prior to the unfolding of the mechanically resistant β-grasp structure of SUMO2.21 The force peaks of the FX traces were fitted to the worm-like chain (WLC) model of polymer elasticity to extract the unfolding contour lengths (∆Lc) of SUMO2.40 The ∆Lc ~24 nm between adjacent force peaks matches quite well with the theoretically calculated contour length of 24.5 nm (=[no. of amino acids, 76]*0.36 nm/aa – [folded protein length 2.8 nm, from PDB 1WM3]).21 In the FX trace of Figure 2B, the last force peak at ~250 pN is due to the detachment of the protein either from the tip or from the substrate. The temperature dependent SMFS experimental data show that the unfolding force values decrease with an increase in temperature, which suggests that the protein becomes less mechanically resistant (Figure 3A). The unfolding force histograms of (SUMO2)8 in the temperature range from 5 - 45 0C are shown in Figure 3B. 10 ACS Paragon Plus Environment

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Moreover, the protein exhibited a two-state all-or-none unfolding in the entire temperature range of 5 - 45 0C. To get a better insight into the temperature dependent effect of unfolding force of (SUMO2)8 we performed SMFS experiments by varying the pulling velocity in the range 1004000 nm/sec. To extract the unfolding energy landscape parameters, we performed Monte Carlo simulations assuming a two-state unfolding process where the native state and unfolded states are separated by a single transition state with an activation barrier.42 The details of the simulations are provided in Experimental methods section. The simulated and experimental data are shown in Figure 3C. We obtained parameters such as barrier height, spontaneous rate of unfolding (ku0), distance to the unfolding transition state or the unfolding potential width (∆xu) from fitting the data to Monte Carlo simulation.42 We find that ku0 does not depend on temperature instead it is the ∆xu that increases by nearly 3 times with an increase in temperature from 5 - 45 0C and hence lower unfolding force is required to unfold the protein at higher temperatures. We compute the directional spring constant (ks) of the protein along the N-C pulling direction, from the ∆xu and the activation barrier ∆G# (described in Experimental methods) as suggested previously by Schlierf and Rief.30 We found that the spring constant changes from 4.50 N/m to 1.35 N/m when the temperature changes from 5 0C to 45 0C, respectively (Table 1 and Figure 3D). This indicates that the protein becomes softer or malleable with an increase in temperature. A reduction in ks also suggests that the intramolecular interactions making the protein rigid at lower temperatures are either disrupted or their strength is weakened at higher temperatures making the protein more flexible. Secondary structure of SUMO2 is unchanged in temperature range 5-45 0C. We investigated the temperature dependent structural properties of SUMO2 using CD spectroscopy 11 ACS Paragon Plus Environment


by monitoring the protein secondary structure in the far UV region, and data is shown in Figure 4A. The observed CD spectra with minima at 208 and 222 nm are in accordance with that expected for ubiquitin-like proteins in their native state.44 We observe that there is no change in the secondary structure of SUMO2 in this temperature range (Table S1, Supporting Information). This is in agreement with the earlier reported melting temperature of SUMO2 (Tm ~72 0C).45 Hence, SUMO2 is thermally stable in our tested temperature range 5-45 0C. This is in contrast with what was observed for Spectrin, a helical protein, where the protein exhibited temperature dependent helix-to-coil transition in its secondary structure, which was attributed to its temperature dependent mechanical properties.28 Tertiary structure of SUMO2 is gradually melted in temperature range 5-45 0C. We further investigated the changes in the tertiary structure of SUMO2, which has aromatic residues Tyr47, Phe32, Phe60 and Phe62 (Figure 1C). Tyr47 is present in α helix and absorbs at ~280nm, whereas Phe residues are present in β2 (Phe32) and β3 (Phe60 and Phe62) and they mostly absorb in the region 55 0C that the λemmax starts red shifting towards ~360 nm

suggesting that the environment around Trp60 is becoming polar due to the solvent penetration of the bulk water. Fluorescence quenching by Acrylamide suggests a sequestered Trp60 from the bulk solvent in SUMO2F60W. We further probed the accessibility of Trp60 in SUMO2F60W mutant to small molecules in the bulk by Acrylamide quenching (Figure 6C).53,54 Fluorescence quenching experiments provide a direct quantitative measure of the accessibility of fluorophore in a protein 14 ACS Paragon Plus Environment

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to the quencher molecules in the bulk.55,56 We compared the quenching results with those of NATA as this provides the maximum limit of quenching of a fully solvated Trp60 (Table 2). The Stern-Volmer quenching constant (KSV) of NATA is found to be 28 ± 2 M-1 at 25 0C whereas the protein has a quenching constant of ~2.5 M-1 in the temperature range 5 - 45 0C, which is about an order of magnitude less than NATA. This indicates that the Trp60 is indeed inaccessible to the quencher and the protein core remains inaccessible from 5-45 0C.

Discussion This study has been designed to probe the mechanistic origin of temperature induced flexibility in SUMO2 protein. From SMFS experiments, we observe a reduction in mechanical strength of the protein from 5-45 0C. This reflects a shift in the position of the transition state (∆xu) from 0.22 nm to 0.41 nm, and thus a 3-fold reduction in the directional spring constant (ks) of the protein along the N-C termini. On the other hand, previous studies by Popa et al on ubiquitin showed a relatively smaller shift in ∆xu (0.20-0.27 nm) with increase in temperature in the range 5-45 0C.33 Ubiquitin showed only a 35% increase in ∆xu whereas SUMO2 in our study exhibits a much larger shift by 95% increase in ∆xu. Although the stiffness of SUMO2 at 5 0C is comparable to that of Ubiquitin at room temperature, with an increase in temperature to 450 C the spring constant of SUMO2 decreases by 3 times. Previous studies by Kotamarthi et al on the mechanical unfolding of Ubiquitin and SUMO2 showed that the differences in the intramolecular contacts might be the reason for the difference in their stiffness at room temperature.21 Similarly, I27 protein showed a 31% increase in ∆xu from 2-30 0C.32 The authors attribute this change in stiffness of I27 to both the hydrogen bonds in the mechanical clamp region and other interactions in the vicinity. However proline mutations at the mechanical clamp in I27 elucidated that the mechanical stability is dependent on the clamp structure.57 Also, a previous study on I27 showed 15 ACS Paragon Plus Environment


a decrease in the free energy barrier for unfolding with an increase in temperature.58 DDFLN4 and CSP showed a considerable increase in ∆xu upon heating and this was attributed to the hydrophobic effect playing more pronounced role at higher temperatures than hydrogen bonds.30,34 Fibronectin type III domain of Projectin on the other hand showed a marginal increase in the unfolding rate constant with an increase in temperature.31 Temperature dependent unfolding of Spectrin showed reduction in the unfolding forces and this was attributed to the melting of the linkers joining the helices.28 Despite all the above studies, experimental evidence for the temperature dependent changes in the protein structure which lead to varied mechanical response has remained elusive. Our study shows that SUMO2 requires lower unfolding forces with an increase in temperature. The question arises as to which part of the protein is perturbed by temperature. Therefore, we performed ensemble measurements on SUMO2 and SUMO2F60W to investigate the change in protein structure globally as well as at the protein core by far and near-UV CD and fluorescence in the same range of temperature in which AFM experiments were performed. We observe that secondary structure remains unchanged from 5-45 0C. However there is 12% loss in tertiary structure when probed at 268 nm which we attribute to near UV CD signal from Phe residues in SUMO2. Further investigation shows that Phe60 has maximum number of contacts when compared to Phe32 and Phe62 and hence it is possible that Phe60 would show more pronounced temperature response than the other two Phe residues. The reduction in asymmetry from 5-45 0C therefore will originate mostly from the loosening of core packing around Phe60. We also probed the solvent accessibility at the core from 5-45 0C by replacing Phe60 with tryptophan, which is a polarity sensitive probe. The Trp mutant, SUMO2F60W has near UV CD signal originating mostly from Trp60 which has highest absorption co-efficient amongst other 16 ACS Paragon Plus Environment

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aromatic amino acids. Probing near UV CD signal shows 13% loss in asymmetry from 5-45 0C in the case of SUMO2F60W, which is similar to 12% loss observed in the case of the wild type protein. Fluorescence experiments on SUMO2F60W monitoring the λemmax and Acrylamide quenching constant show that solvent accessibility to the core around Phe60 remains unchanged from 5-45 0C and the protein core is still nonpolar or ‘dry’ at 45 0C. Such evidences for partial unlocking of packing interactions of side chains leading to 15-20% loss in near UV CD signal have been observed in case of Barstar and Monellin in presence of GdHCl.59,60 In both these reports the secondary structure and fluorescence emission maxima (λemmax) is unaffected when this reduction in tertiary structure content has occurred leading to formation of ‘dry molten globule’ states, strikingly similar to what we observe for SUMO2 and SUMO2F60W. This reduction in tertiary structure content indicates towards formation of a flexible core with less efficient side chain packing has also been previously observed for other systems such as Rnase A, Villin headpiece.61 This further indicates that one of the reasons for the reduction in mechanical stiffness of SUMO2 might originate from the loss of interactions at the core. Dependence of mechanical stability on the core has been observed for Fibronectin and Protein L.62 The composition of the protein core is important for the mechanical stability as described previously for Fibronectin and Tenascin, where the hydrophobic core of Fibronectin was replaced by Tenascin’s core to make Fibronectin as mechanically strong as Tenascin.63 Interestingly, Protein L belongs to the same structural class as Ubiquitin and SUMO2, and it was shown that that the mechanical clamp as well as other hydrophobic interactions determine its mechanical strength.62 SUMO2 offers a paradigm system for β-grasp proteins and here we investigated the temperature dependent response of the core along with its mechanical stability. Temperature aids 17 ACS Paragon Plus Environment


in increasing the dynamics at the protein core leading to higher conformational flexibility which when acted upon by mechanical perturbation leads to lower unfolding forces. Here, our systematic studies reveal that fine tuning of the core flexibility with temperature perturbation could be used to predict modulation of the protein flexibility, by accessing their ‘metastable’ states at elevated temperatures which at low temperature cannot be accessed making it relatively rigid.

Conclusions Combining single-molecule measurements with ensemble experimental techniques would give rich information than either alone would provide. Here, we observe a significant loss in protein stiffness for SUMO2 upon heating and its directional spring constant decreases by 3 times in the temperature range 5-45 0C. One of the reasons for decrease in stiffness might be due to the loss of tertiary structure leading to conformationally flexible core without changing any secondary structure, and also retaining the overall β-grasp topology. These results suggest that the protein mechanical stability depends on the interactions at the core in addition to the mechanical-clamp geometry between the termini. This study also elucidates the molecular origin of protein flexibility at higher temperatures or temperature-dependent protein malleability. This also suggests that the protein flexibility can be increased without exposing the residues at the core of the protein to the bulk solvent. Over all these results further enhance our understanding of protein mechanical stability and malleability. In future experiments, it would be interesting to see if the molecular origin of protein malleability put forward here for the β-grasp proteins would also be applicable to the β-sandwich proteins, as these two topologies are by far the


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mechanically strongest of all other protein structure space and also have similar origins of mechanical stability such as mechanical clamp.

Acknowledgements The authors acknowledge Dr. Hemachandra Kotamarthi for providing the clone of SUMO2 octamer, Anju Yadav and Siddhi Inchanalkar for providing the SUMO2F60W clone. We also thank Madhuri Kallianpur for fluorescence experiments and Prof. Shyamalava Mazumdar, Bharat Kansara along with Abhijit Mondal for CD measurements in initial stage of the project. We are grateful to TIFR and DAE for funding.

Supporting Information The supporting material contains chemical denaturation data (Figure S1), and temperature dependent CD data (Figure S2), secondary structural estimates (Table S1), residue contact information (Table S2), and AFM experimental data (Table S3). This information is available free of charge via the Internet at http://pubs.acs.org/.



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Figure legends Figure 1. Sequence and structural details of SUMO2.A. The protein sequence of SUMO2. Nterminal 17 residue flexible linker is highlighted (bold, black). Tyr47, Phe32, Phe60 and Phe62 are also highlighted. Mechanical clamp is between strands β1 and β5. B. The mechanical clamp comprising β1 and β5 strands along with the inter-strand H-bonding is highlighted. C. Tyr47, Phe32 and Phe60 at the core, and Phe62 at the interface are shown in stick model. Structure of SUMO2 is taken from PDB ID 1WM3.

Figure 2. AFM-based SMFS experiments on (SUMO2)8. A. Schematic of temperature controlled AFM used for the experiments showing a zoom-in single polyprotein chain containing 8 domains of SUMO2. B. The sawtooth force-versus-extension (FX) unfolding pattern for the polyprotein (SUMO2)8 (red) with WLC fits (blue) for each protein domain using a persistence length of ~0.4 nm and an unfolding contour length of ~24 nm. The force-extension trace was collected at a pulling velocity of 400 nm/sec and at 5 0C.

Figure 3. Temperature dependent mechanical unfolding of (SUMO2)8. A. FX traces of (SUMO2)8 obtained at 1000nm/sec in the temperature range 5-45 0C. B. Pulling velocity dependent unfolding force histograms of (SUMO2)8. (see also Table S3 in Supporting Information for more details) C. Average unfolding forces along with their standard deviations at different temperatures. Solid lines are fits from Monte Carlo simulations. D. Directional spring constant, ks (•) and distance to the unfolding transition state, ∆xu () are plotted from 5-45 0C.



Figure 4. A. Temperature dependent far UV CD of SUMO2 from 5-45 0C. B. A characteristic near UV CD spectrum of SUMO2 acquired at 5 0C. The four bands of the spectrum were attributed to Phe (262 and 268 nm) residues and Tyr47 (278 and 285nm). C. Ellipticity of Phe residues and Tyr47 is plotted as a function of temperature from 5-45 0C.

Figure 5. A. Characteristic near UV CD spectrum obtained for SUMO2F60W at 5 0C. The high absorption coefficient of Trp60 overshadows signal from Phe residues and Tyr47. B. Near UV CD shown as a function of temperature from 5-45 0C. The trend followed by Trp60 in SUMO2F60W is similar to Phe60 in SUMO2.

Figure 6. Fluorescence spectroscopy as an indicator of the solvent accessibility around tryptophan. A. Normalized steady state fluorescence spectra of SUMO2F60W compared with Nacetyl-L-Tryptophanamide (NATA). The blue shifted emission maxima of SUMO2F60W indicate the Trp60 to be in a nonpolar environment. B. The wavelength corresponding to the emission maxima as a function of temperature. C. Acrylamide quenching was performed in the temperature range 5–55 0C.


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Figure 1



Figure 2


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Figure 3

200 150 100

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0.9 268nm (Phenylalanine) 278nm (Tyrosine)

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Figure 5

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Figure 6

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Tables Table 1. Temperature dependent mechanical stability, kinetic parameters, and elasticity of SUMO2 Temperature (0C) 5 15 35 45

Unfolding force* (pN) 207 ± 28 (N = 321) 167 ± 24 (N = 266) 145 ± 20 (N = 149) 110 ± 19 (N = 172)

∆LC* (nm) 22.6 ± 0.9 22.8 ± 0.8 22.9 ± 0.8 24.3 ± 0.8

∆xu (nm) 0.22 ± 0.01 0.27 ± 0.01 0.32 ± 0.02 0.41 ± 0.01

ks ku 0 ∆G# -1 (kBT) (N/m) (s ) 0.003 ± 0.001 25.0 4.50 0.003 ± 0.001 25.6 2.90 0.004 ± 0.002 27.0 2.20 0.006 ± 0.001 27.5 1.35

* The unfolding force and ∆LC values are for the pulling velocity 1000 nm/sec. Errors are SD.

Table 2. Stern-Volmer quenching constant for SUMO2F60W with Acrylamide as quencher Temperature KSV 0 ( C) (M-1) 5 2.5 ± 0.5 15 2.3 ± 0.2 25 2.1 ± 0.1 NATA (28.0 ± 2.0)* 35 1.4 ± 0.2 45 2.8 ± 0.4 55 5.0 ± 0.2 * The date in the parenthesis is for NATA at 25 0C with Acrylamide as quencher. KSV values, mean ± SD.



Notes The authors declare no competing financial interest.


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TOC


Mechanical Softening of a Small Ubiquitin-Related Modifier Protein

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