Residual structure and dynamics in DMSO-d6 denatured Dynein Light Chain protein

Biochimie 94 (2012) 231e241

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Research paper

Residual structure and dynamics in DMSO-d6 denatured Dynein Light Chain protein Swagata Chakraborty a, P.M. Krishna Mohan a, b, Ramakrishna V. Hosur a, c, * a

Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India Department of Chemistry & Chemical Biology, Rutgers University, 610, Taylor Road, Piscataway, NJ 08854, USA c UM-DAE Centre for Excellence in Basic Sciences, Mumbai University Campus, Kalina, Santa Cruz, Mumbai 400098, India b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 June 2011 Accepted 25 October 2011 Available online 4 November 2011

Structural and motional features in the denatured state of a protein dictate the early folding events starting from that state and these features vary depending upon the nature of the denaturant used. Here, we have attempted to decipher the early events in the folding of Dynein Light Chain protein (DLC8), starting from DMSO-d6 denatured state. Multinuclear NMR experiments were used to obtain the full spectral assignment. The HSQC spectrum shows the presence of two sets of peaks for the residues Met 1, Ser 2, Arg 4, Ala 11, Met 17, Thr 26, Lys 44, Tyr 50, Asn 51, Trp 54, His 55, Val 58, Gly 59, Ser 64, Tyr 65, His 68, Phe 86, Lys 87 indicating the presence of slow conformational transition in the heterogeneous ensemble. Analysis of residual structural propensities with secondary 13C chemical shifts, 3J(HN
Ha) coupling constants and 1H-1H NOE revealed the presence of local preferences which encompass both native and non-native like structures. The spectral density calculations, as obtained from measured R1, R2 and 1H-15N steady state NOE values provide insights into the backbone dynamics on the milli to picosecond timescale. The segment Ser 14 e His 55 exhibits slow motions on the milli- to microsecond timescale arising from conformational exchange. The presence of native like structural preference, as well as conformational exchange classifies the above segment as the nucleation site of folding. Based on the observations, we propose here, the probable hierarchy of folding of DLC8 on dilution of denaturant: the two helices are formed first followed by the formation of b2 and b5. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Denatured state Dynein Light Chain Dimethyl sulfoxide Residual structure Conformational exchange Folding initiation site

1. Introduction A newly synthesized polypeptide chain inside a cell exists in some unknown denatured state, the characteristics of which depend on the local environment. In vivo, newly formed polypeptides can be folded or assisted by chaperons and the conformational search space is thereby greatly reduced. Clearly, the denatured states represent the starting points from where the protein will fold to its native state and thereby have a significant influence on the folding pathway of a protein [1e3]. The denatured state of a protein is an ensemble consisting of species, ranging from

Abbreviations: DLC8, Dynein Light Chain; HSQC, Hetero Nuclear Single Quantum Coherence; TOCSY, Total Correlation Spectroscopy; NOE, Nuclear Overhauser Effect; CPMG, Carr Purcell Meiboom Gill; DMSO, Dimethyl Sulfoxide; Gdn-HCl, Guanidine Hydrochloride; SDS, Sodium Dodecyl Sulfate; DTT, Dithiothreitol. * Corresponding author. Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India. Tel.: þ91 22 2278 2488; fax: þ91 22 2280 4610. E-mail address: [email protected] (R.V. Hosur). 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.10.013

expanded to highly compact in nature, that are stabilized by different kinds of native and non-native interactions. Though it is really hard to predict whether residual structural motifs exist or not when a polypeptide is being formed inside cells, however, in vitro, the individual species or conformational states of the denatured ensembles exhibit different degrees of structural preferences [4e6], which can be related to the local preferences within a heterogeneous group of conformers [7]. The conformational preferences across the polypeptide chain in the denatured state have significant influence on the path a particular molecule adopts for folding in the ensemble. The presence of persistent structural elements may assist the early events in folding by reducing the accessible conformational space and thereby guiding the protein toward its native conformation. It is also believed that the presence of slow motions in certain segments along the polypeptide chain helps to identify potential folding nucleation sites. Moreover, a thorough understanding of these states is important in order to comprehend the characteristics of intrinsically unstructured functional proteins [8,9]. It has been found by various groups of researchers that even in very strong denaturant concentrations, the protein though fully

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denatured, remains partially unfolded [7,10e12]. Partial unfolding, in turn has been recognized as a significant step in fibrillation which leads to neurodegenerative diseases. The strong correlation between fibrillation and unfolding in case of Prion proteins, thereby further enhances the importance of study of the denatured states [13]. Hence, the detailed structural and dynamics characterization of the denatured equilibrium states have the potential to make a significant contribution toward elucidation of the initial folding events [14e17]. Since we do not have direct access on the unfolded polypeptide chains in vivo, the characterization of denatured states, in vitro is used as a technique to understand the biophysical properties of the starting point of protein folding in the folding funnel landscape. All in vitro studies with different environmental conditions aim to sample some subsets of the conformational space, hoping that some of these may be populated in the in vivo conditions and we try to gain insights into the numerous folding trajectories. In vitro, denatured states are produced using different chemical denaturants [12,18e25], like urea, Gdn-HCl, SDS, DMSO etc. These denaturants facilitate the unfolding of proteins by interfering with the molecular interactions which stabilize the native form of the protein. Though chemical denaturation studies of proteins in aqueous solvents like urea, guanidine-HCL are most common, it can be easily conceived that they can sample only a subset of the conformational space available to the protein chain. It has been found that the characteristics of the denatured state, as well as the folding trajectories of a protein in the two aqueous solvents, urea and Gdn-HCl, also vary to a great extent. Thus other environments such as those created by organic solvents like DMSO are also valuable and are, indeed, extensively used to get greater insights into protein folding as these provide insights into factors contributing to the stability of proteins and folding pathways with different starting preferences in the unfolded state [26e29]. Moreover, protein structures are easily disturbed by solvent perturbation, making denaturation by organic solvents worth detailed studies. The detailed characterization of the structure, function and dynamics of Dynein Light Chain protein (DLC8), the smallest subunit of the motor protein Cytoplasmic Dynein have been extensively done by various groups of researchers [30e54]. As reported, DLC8 exists as a homodimer at physiological pH [31,55] and is functional only in its dimeric form. The structure of the dimer revealed that it contains two helices and five b strands [38,56]. However at lower pH, the monomeric form of the protein is prevalent [55,57,58] and is known to contain two helices and four b strands (b3 is not present in the monomer) [59]. The global unfolding features of DLC8 in urea and Gdn-HCl, as obtained from equilibrium unfolding studies with the help of optical spectroscopic methods reveal complex transition mechanisms involving accumulation of intermediates [43,47,60]. It is proposed that while unfolding the dimeric protein first dissociates into the monomers which then unfold. However, little is known about the folding trajectory of the protein which requires a complete kinetic characterization of the intermediates in the folding pathway starting from a denatured state. Presently, for DLC8, we used different denaturants like urea, Gdn-HCl and DMSO to create the denatured ensemble of the protein. However, the HSQC spectrum of the protein was found to be changing with time in the denaturing concentrations of urea, while it tends to aggregate in the charged denaturant Gdn-HCl. But the denatured state created by Dimethyl Sulfoxide (DMSO) was stable for detailed NMR investigations and thus we report here, in detail, NMR characterization of the DMSOdenatured state of DLC8. Secondary chemical shifts, 3J(HN
Ha) coupling constants, 1H-1H NOEs and 15N relaxation experiments were used as probes to throw light on the structural preferences

and the motional restrictions in the denatured ensemble of DLC8. These, in turn, throw light on the initial events that occur during folding of DLC8 on denaturant dilution. 2. Materials and methods 2.1. Protein expression and purification DLC8 was expressed and purified as described elsewhere [61]. The protein solution containing 20 mM Tris, 200 mM NaCl and 2mM DTT at pH 7 was lyophilized. DMSO-d6 was added to the lyophilized sample followed by w3% (vol/vol) deuterated Trifluoro acetic acid and the protein was allowed to equilibrate in DMSO for some time. The pH was measured with a pH microelectrode to ensure that pH of the sample is approximately around 2.5. The sample was equilibrated for 24 hours before the start of the experiment. 2.2. NMR data acquisition NMR experiments were performed both on Varian Inova (600 MHz) spectrometer and on Bruker Avance (800 MHz) spectrometer at 45  C. A 1H-15N HSQC spectrum was recorded at the end of each set of experiments and compared with the one recorded at the start to check the stability of the protein sample. No change was seen in the series of HSQC spectra indicating that the sample was stable under the conditions used and it had reached equilibrium before the start of the experiment. During the course of all the experiments, the protein samples were found to be stable and did not precipitate or degrade with time. A variety of standard triple resonance experiments were recorded for backbone resonance assignment HNN, CBCANH, CBCA(CO)NH, HNCO, and HN(CA)CO were used for sequential walk through the polypeptide chain. 15N edited TOCSY-HSQC and NOESY-HSQC were used for assigning the spin systems and confirming the sequential connectivities. For all the experiments, standard experimental parameters were used. Along 15N and 13C0 dimensions, 80 and 96 complex points were used. 128 complex points were used along the aliphatic carbon dimension. 15N edited TOCSY-HSQC and NOESY-HSQC were recorded with mixing times of 80 ms and 150 ms, respectively. 96 complex increments were used along the indirect 1H dimension. DSS was used as a reference to calibrate 1H, 13C and 15N chemical shifts in the standard way by monitoring the chemical shift of the weak water peak. For the two dimensional relaxation experiments, 2048 and 512 complex points were acquired with a spectral width of 9 ppm and 22 ppm along the t2 and t1 dimensions, respectively. 3 J(HN-Ha) coupling constants were measured from high resolution HSQC spectrum which was recorded with 16384 and 1024 complex points with a spectral width of 9 ppm and 22 ppm along the t2 and t1 dimensions respectively, as well as from a 3D HNHA spectrum. The relaxation measurements (R1, R2 and 1H-15N NOE) were carried out using the pulse sequences described by Farrow et al. [62]. 15N transverse relaxation rates (R2) were measured with CPMG delays 10, 30, 50, 70, 90, 110, 130, 150, 170, 190 ms and 15N longitudinal relaxation rates (R1) were measured with 10, 50, 120, 220, 350, 500, 700, 900 ms inversion recovery delays. Steady state 1H-15N heteronuclear NOE measurements were carried out with a proton saturation time of 2.5 s and a relaxation delay of 2.5 s. 2.3. NMR data analysis All the data were processed using FELIX and analyzed using FELIX (Accerlys Software Inc., San Diego, CA) and CARA [63]. Prior to Fourier transformation and zero filling, the data was apodized with a sine-squared weighting function, shifted by 60 in both the dimensions for 2D experiments and by 90 in all the dimensions for

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3D experiments. After zero filling and Fourier transformation, the final matrix had 1024, 256 and 256 points along the F3, F2 and F1 dimensions, respectively, and the 2D HSQC experiments had 4096, 1024 points, respectively, along the F2 and F1 dimensions. The decays of cross peak intensities for the residues were fitted to the exponential decay model given by the equation, I(t) ¼ B exp (-R1,2 t) to extract the transverse and longitudinal relaxation rate constants. Steady state 1H-15N NOE was calculated as a ratio of intensities of the peaks with and without proton saturation. The errors in the NOEs were obtained using the root mean square value of the background noise as described by Farrow et al. [62].

2.4. Spectral density and correlation times For spherical molecule, the rotational diffusion is described by a correlation function

GðsÞ ¼ Gð0Þexpð
s=sc Þ ¼ B2loc expð
s=sc Þ ¼ B2loc gðsÞ Bloc(t) is a random function of time due to thermal motion and sc is called correlation time, g(s) is reduced correlation function which is independent of the local field. The Fourier transformation of the reduced correlation function yields the reduced spectral density function. FT

gðsÞ !

2sc 1 þ u2 s2c

The reduced spectral densities as functions of R1, R2 and NOE are as follows:

Jð0Þ ¼

  3 1 3 
R1 þ R2
RnOe
2 5 2 3d2 þ c2

JðuN Þ ¼

JðuH Þ ¼

  1 7 R
R 1 nOe 5 3d2 þ c2

where,

gN gH

RnOe ¼ ½εnOe
1 $R1 $

εnOe is the NOE enhancement; factor;



c ¼

uN sk
st



pffiffiffi 3

;

m0 is the permeability of free space; h is Planck’s constant; gH, gN are

the gyromagnetic ratios of 1H and 15N, respectively; rNH is the NeH bond length and is assumed to be 1.02 Å; uH and uN are the Larmor frequencies of 1H and 15N, respectively; ðsk
st Þ is the CSA of 15N spin and is assumed to be
160 ppm. The constant c2 takes the value w1.25  109 (rad/s)2 and w2.25  109 (rad/s)2 at 600 and 800 MHz magnetic fields, respectively .The constant d2 is equal to 1.35  109 (rad/s)2 and is independent of field strength. The errors in the spectral densities were calculated by the propagation of error analysis. Next, exchange contribution to R2 was calculated from the spectral densities calculated at two fields using the equation [64].

Rex ¼ u2N $

Jð0Þ800 MHz
Jð0Þ600 MHz

l800 MHz u2N;800 MHz
l600 MHz u2N;600 MHz

Here,

uN ¼ 81:1 MHz: l ¼ 3=2 3d2 þ c2



3. Results 3.1. Resonance assignment of DLC8 in DMSO Generally, the 1H-15N HSQC spectrum which shows one cross peak for each non-proline residue in a protein, serves as a fingerprint of the conformational state of the protein. The HSQC spectrum of DLC8 in deuterated DMSO at 45  C is shown in Fig. 1. The narrow chemical shift dispersion of the resonances in the 1H dimension, (as compared to w 4.5 ppm dispersion observed in the HSQC spectrum of native DLC8 dimer) indicates that DLC8 is denatured under the experimental conditions. Using HNN, [65] sequential walk along the backbone assignments for 80% of non-proline residues were obtained. An illustrative sequential walk through the segment Gly 63 e His 68 is shown in Fig. 2A, and the summary of sequential assignment is indicated along the sequence of DLC8 in Fig. 2B where the stretches of the residues connected with HNN walk are indicated in grey; Glycines acting as the checkpoints are indicated in green whereas Proline which is the endpoint is indicated in red. Because of very weak intensities of some peaks, we were not able to identify the sequential connectivities in certain stretches using HNN. It turned out that most of these weak peaks belonged to Lysines. In order to identify the Lysine peaks we recorded HSQC spectrum of specifically 15N Lysine labeled protein. Finally, the missing assignments were obtained using other NMR experiments like CBCANH, CBCA(CO)NH, HNCO, and HN(CA)CO. 15N edited TOCSY-HSQC yielded the chemical shifts of the side chain protons and was used to confirm the spin systems. The assignments of the peaks in the HSQC spectrum are shown by residue label annotations in Fig. 1. The detailed lists of assigned chemical shifts have been deposited in BMRB under the accession number 17692. 3.2. Slow conformational exchange in the denatured ensemble

1 ½RnOe  5d2

m h 
3  r ; d ¼ 0 gH gN 2p NH 4p

233



The denatured state ensemble is heterogeneous in nature, but the different conformers in the ensemble inter-convert rapidly on the NMR chemical shift timescale and thus, typically, only one set of peak is observed in the NMR spectrum in accordance with the amino acid sequence of the polypeptide chain. However, in the case of DLC8, the HSQC spectrum in DMSO displays additional peaks of low intensity which indicates the presence of multiple species in slow exchange. These multiple sets of cross peaks are represented by primed and unprimed residue labels for the residues Met 1, Ser 2, Arg 4, Ala 11, Met 17, Thr 26, Lys 44, Tyr 50, Asn 51, Trp 54, His 55, Val 58, Gly 59, Ser 64, Tyr 65, His 68, Phe 86, Lys 87 in Fig. 1. The sequential connectivities of the two sets of peaks of Met 1 and Ser 2 to Asp 3 are demonstrated in Fig. 3. We would like to point out here that several residues showing two sets of peaks lie in the vicinity of the Pro 52. In order to ascertain whether this heterogeneity arises from the cis-trans isomerism of the peptide bond preceding the Proline residue we analyzed the chemical shift of Cb and Cg resonances obtained from 3D (H)C(CH)(CO)NH TOCSY experiment. The chemical shift difference between the 13Cb and the 13Cg nuclei for Pro 52 was found to be 4.6 ppm (Fig. S1) which indicates that the Proline peptide bond is present solely in trans conformation and cis-trans isomerization is not the cause for the observed chemical shift heterogeneity. The populations of the two sets of peaks for the above residues were also measured from the simple formula PA/ A0 ¼ IA/A0 /(IA þ IA0 ), where IA and IA0 represent the intensities of unprimed and primed peaks in the annotated spectrum (Fig. 1), respectively. The % populations of these peaks are tabulated in

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Fig. 1. 1H-15N HSQC spectrum of DLC8 in DMSO-d6 at 45  C. Residue-specific assignment for each peak is annotated by residue labels on the 1H-15N HSQC spectrum of DLC8 in DMSO-d6 at 45  C. A blow up of the boxed region in the 1H-15N HSQC spectrum is shown on the right. The alternate sets of peaks in the HSQC spectrum for the residues are indicated by primed and unprimed annotations.

supplementary information (Table S4). The wide variations in the relative distribution of populations in the two states for these residues signify that the observed heterogeneity arises from a multitude of local states. The sources of heterogeneity, however,

Fig. 2. Sequential walk with HNN. A) Sequential walk through the F1eF3 planes of HNN spectrum of DLC8 in DMSO-d6 at 45  C. Sequential connectivities are shown for Gly 63 to His 68 stretch. The red and green contours indicate positive and negative peaks respectively. The distinct Gly plane serves as the start point in the sequential assignment. B) Summary of sequential assignment obtained from HNN walk along the primary sequence of DLC8 marked in grey. Gly shown in green worked as start/check point; Pro shown in red worked as break point. The structural elements in the native state of the folded DLC8 are depicted on top of the sequence. Arrows represent bstrands and cylinders represent a-helices. The empty arrow indicates b-strand 3 which is present in the dimer but absent in the monomer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Connectivities of alternate sets of peaks. Sequential walk through the F1eF3 planes of HNN spectrum of DLC8 in DMSO-d6 at 45  C to show the sequential connectivities of the sets of peaks belonging to the residues Met 1 and Ser 2 to Asp 3. The primed residues indicate peaks belonging to minor population. As can be noted both the sets of peaks connect to Asp3. The red and green contours indicate positive and negative peaks respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

S. Chakraborty et al. / Biochimie 94 (2012) 231e241

cannot be ascertained accurately. The motional characteristics (R2 and 1H-15N NOE) of these alternate sets of peaks of the residues mentioned above are also different compared to those belonging to the major population, thereby indicating differential conformational flexibility in the two conformations. 3.3. Structural propensities in the rapidly inter-converting conformational ensemble Denatured states are not simply random coils [66], but often possess regions of inhibited flexibility and “residual structure” [7] which relates to local preferences within a heterogeneous distribution of conformers. The structural preferences of the denatured state can provide insight into early folding events [1,2,67]. We have calculated secondary carbon chemical shifts and 3J(HN-Ha) coupling constants and measured inter-residue nuclear Overhauser entrancements to search for non-random behavior in the DMSOdenatured state of DLC8. 3.3.1. Secondary 13C chemical shifts Chemical shift deviations of 13CO, 13Ca and 13Cb from random coil values provide insights into the structural preferences present in the polypeptide chain. These chemical shifts are predominantly determined by backbone conformation, and their deviations from the random coil values are reliable indicators of residual structure. Positive deviations of 13CO, 13Ca and negative deviations of 13Cb indicate alpha helical preference, while the reverse indicate beta preferences. We calculated the deviations of the individual carbon chemical shifts of the residues from random coil values of amino acids reported in DMSO [68] and the values are plotted in Fig. 4AeC.

235

Though the individual deviations of Ca and CO chemical shifts are not very high in most part of the polypeptide chain indicating random coil like preferences, higher than average positive deviations in certain stretches Ala 6 e Ile 8, Ala 11 e Gln 18, Ala 21 e Glu 30 and Ile 34 e Tyr 41 indicate some preference toward alpha helical structure in the DMSO-denatured state. However, it is evident from the comparatively small values of these deviations that no persistent structure is present. DLC8 in its native state in both monomeric and dimeric form has two a helices in the segments 15e30 and 35e50. So it appears that the DMSOdenatured chain exhibits mostly native preferences in the segments corresponding to the helices in the native state. The presence of native structural preferences may actually decrease the conformational space sampled by the denatured polypeptide chain to fold back to its native state on dilution of the denaturing conditions. 3.3.2. 3J(HN-Ha) coupling constant The three bond 3J(HN-Ha) coupling constants represent the V torsion angle of the backbone and can provide insight into characteristics of the backbone conformation. The J values range from 3 to 5 Hz for a-helix, 8e11 Hz for b-structure and 6e8 Hz for a random coil [69]. The Jrandom values for any given residue seem to depend also on the nature of the preceding residue along the chain: aromatic side chains (class L) or other residue types (class S) [70], reflecting the steric and electrostatic basis for torsional angle propensities [71,72]. The deviations of the observed coupling constants from the sequence-dependent random coil values, (JobseJrc), called secondary coupling constants throw valuable light on the secondary structural propensities along the polypeptide

Fig. 4. Residual structural propensities in the denatured state of DLC8 in DMSO-d6 at 45  C. Residue-wise plots of secondary chemical shifts as obtained from dobs
dran for (A) CO, (B) Ca and (C) Cb. The horizontal lines indicate average values of the individual deviations. (D) The deviations of coupling constants 3J(HN-Ha) from random coil values plotted against residue numbers, as obtained from 3D HNHA spectrum of denatured DLC8. The upper and the lower horizontal lines are drawn at 0.5 Hz for the positive values, and
1 Hz for negative deviations, respectively, which indicate the accuracy of secondary coupling constant estimation. The cylinders and the arrows on top of the graphs indicate the alpha helices and the beta strands, respectively, in the native state of the folded dimer. (E) The selected region of 3D NOESY-HSQC spectrum for the side chain protons of the stretch Gly 63 e Thr 67 to show the presence of both daN (i,i þ 1), dbN (i,i þ 1) and dNN (i,i þ 1) connectivities which indicate the heterogeneous nature of the ensemble. (F) Selected region of 3D TOCSY-HSQC spectrum of DLC8 in DMSO-d6 at 45  C showing the Ha regions of all the glycines present in the polypeptide sequence. The two Ha protons of Gly 59 are showing different chemical shifts indicating that it is present in some structured region.

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chain. Positive secondary coupling constants indicate b-propensities while negative deviations indicate the presence of nascent helical or turn-like structure. The coupling constants were obtained from the HNHA spectrum [73] as well as from the splittings of the peaks in the high resolution HSQC (Fig. S2A). The accuracy of secondary coupling constant estimation is w0.5 Hz for the positive values, but
Scross ¼
tan2 ð2pJ xÞ; Sdia where x ¼ 13.05 ms. Here we could measure values for many more residues than could be done from the high resolution HSQC spectrum. However, for the residues where data was available from both methods we found that the values were in agreement within 0.5 Hz; the coupling values obtained from HNHA were lower. The residue-wise values of the 3J(HN-Ha) coupling constants is tabulated as Supplementary information (Table S5) and the residuespecific values are demonstrated in Fig. 4D. The negative Jsec values in the segment Met 13-His 55 again confirm the presence of alpha helical preferences in this segment. The remaining fragment of the polypeptide chain showed varying degrees of splitting indicating random coil like preferences. However, the contiguous stretch from Val 81- Ser 88 shows significant positive secondary coupling constants signifying the presence of some extended bstrand like propensities in this segment which corresponds to the b5 strand of the native DLC8. The b5 strand is embedded in the hydrophobic core and hence is highly protected, as has been ascertained from Hydrogen exchange studies [42]. These residual structures may act as nucleation sites for the folding of the protein. 3.3.3. Topological fluctuations in the denatured ensemble NOE has r
6 distance dependence and thereby acts as a sensitive indicator of residual structure in denatured states [74]. 1H-1H NOEs exhibit specific patterns for different secondary structures: bstructures are characterized by strong daN (i,i þ 1) connectivities, whereas a-helices are characterized by strong dNN (i,i þ 1) NOEs [75] and medium range NOEs, from daN (i,i þ 2) and daN (i,i þ 3). However, they are also influenced by the mobility of the internuclear vector. In unfolded proteins the greater flexibility of the chain makes the NOEs weak, and the above selectivity with regard to secondary structure is lost due to heterogeneous populations in the ensemble. However, a higher population of specific structures may result in some kind of preference in the NOE patterns. In the DMSO-denatured state, a large segment of the polypeptide chain of DLC8 shows strong daN (i,i þ 1) connectivities indicating the presence of perceptible a- propensities in those segments. These include the stretches Ser 2 e Asp 3, Ala 6 e Asp 12, Glu 16 e Met 17, Asp 20 e Cys 24, Asp 47 e Tyr 50 and Trp 54 e Lys 87. Moderately strong dNN (i,i þ 1) connectivities are present in the segments Met 1 e Ala 6, Ala 11 e Ser 14, Gln 19 e Gln 27, Tyr 32 e Ala 39, Ile 42 e Tyr 50, Trp 54 e Ile 74 and Gln 80- Lys 87. 3D NOESY-HSQC spectral region for the stretch of residues from Gly 63 e Thr 67 is illustrated in Fig. 4E and the detailed list of the NOE connectivities are provided in Table S6. The simultaneous presence of moderately strong daN (i,i þ 1) and dNN (i,i þ 1) connectivities in certain segments of the protein points toward the heterogeneous nature of the ensemble. This sort of simultaneous preferences for both alpha

and beta structural propensities is only observed by NOE and no other means due to the fact that NOE can filter out the population showing structural preferences in the background of major population showing no connectivities. From the above observations it is evident that DLC8 polypeptide chain in DMSO does not possess any stable secondary structures, but has some propensity for formation of transient structures. It is important to mention here that Glycine residues increase the backbone flexibility in unfolded and denatured proteins [6]. In dynamically flexible polypeptide chains, the two Ha protons of glycine show degenerate chemical shifts. However, if the glycines occupy some structured regions, this degeneracy is broken due to the difference in their electronic environments. It is important to note that Ha peaks of Glycine 59 in denatured DLC8 displayed two cross peaks in a 15N edited TOCSY-HSQC (Fig. 4F) and NOESY-HSQC spectra, thereby indicating that Gly 59 is likely to occupy some structured region in the polypeptide chain. This is further consolidated by the presence of transient structures in the segments adjacent to Glycine 59. 3.4. Backbone dynamics in DMSO-denatured DLC8 As is evident from the characterization of the structural propensities, the DLC8 polypeptide chain is not completely flexible but exhibits residual structural preferences in the denatured ensemble. So it is natural to expect variations in the motional characteristics along the length of the chain. In order to probe the motional behavior of the polypeptide chain in DMSO, we carried out 15N R1, R2 and heteronuclear 1H-15N NOE measurements at both 600 MHz and 800 MHz spectrometers, which provide insights into the motions over a wide range of timescales [76e78]. The NOEs are very sensitive to picosecond timescale motions and the R1 values are sensitive to both low and high frequency motions (nanosecondto-picosecond timescale motions). R2 has major contributions from slow milli- to microsecond timescale motions which include conformational transitions. The residue-wise plots of the R1, R2 and 1H-15N NOE as obtained at 800 MHz and 600 MHz spectrometers are shown in Fig. 5(AeC) and Fig. S3(AeC), respectively. We see that R1 remains mostly similar across the polypeptide chain, average being 1.68 s
1  0.15 s
1 at 800 MHz and 1.58 s
1  0.05 s
1 at 600 MHz. However, considerable sequence-wise variation was observed in the transverse relaxation rates which varied from 3.35  0.2 s
1 to 22.5  3.3 s
1 (average being 10.32 s
1  0.63 s
1) at 800 MHz (Fig. 5B) and from 2.33  0.02 s
1 to 12.02  1.26 s
1 (average being 6.35 s
1  0.21 s
1) at 600 MHz (Fig. S3A). This indicates nonuniform flexibility along the polypeptide chain. It is interesting to note that the segment Ser 14 e His 55 shows considerably high R2 values indicating the presence of slow timescale motions in this stretch. Since the 1H-15N NOE values of this stretch do not show any distinct increase in the values, it can be assumed that the increase in R2 result from conformational exchange. This is consistent with the presence of residual structural propensities in this segment as discussed earlier. 1H-15N NOE values span from
0.12  0.01 s
1 to 0.83  0.16 s
1, with the average being 0.53  0.04 s
1 at 800 MHz spectrometer frequency (demonstrated in Fig. 5C) and
1.61  0.02 s
1 to 0.77  0.28 s
1 on 600 MHz spectrometer (Fig. S3C). The NOE values though become considerably low at the C-terminal end, they remain almost identical (w0.5e0.6 on 800 MHz spectrometer) throughout the remaining part of the polypeptide chain. This suggests that pico-second timescale motions which involve the N-H bond rotations remain almost alike throughout the polypeptide chain, excepting at the C-terminal end. However, it is important to point out that the relatively higher average value of 1H-15N NOE (0.5e0.6 at 800 MHz) in the DMSO-

S. Chakraborty et al. / Biochimie 94 (2012) 231e241

237

Fig. 5. Relaxation parameters and spectral density functions. Plot of relaxation parameters obtained from 800 MHz versus the residue number for DMSO-d6 denatured DLC8 at 45  C. (A) 15N R1 (longitudinal relaxation rate), (B) 15N R2 (transverse relaxation rate), (C) 1H-15N heteronuclear NOE. Calculated values of spectral density functions from relaxation data at 800 MHz: (D) J(uH), (E) J(uN), and (F) J(0) versus residue numbers. The helices and the beta strands in the native DLC8 are indicated by cylinders and arrows respectively on top of the graphs. The empty arrow indicates b-strand 3 which is present in the dimer but absent in the monomer. The black bars on top of R2 and J(0) plots indicate regions with large contribution from slow motions in the micro to milli-second range.

denatured ensemble as compared to what is typically observed in a denaturant such as Gdn-HCl may suggest the possibility of more residual structure in the DMSO-denatured state. As mentioned in the earlier section, the motional characteristics of two sets of peaks of the residues exhibiting alternate conformations are different, eg, for Ser 64, R2 ¼ 8.59 s
1 and 1H-15N NOE ¼ 0.41 whereas for Ser’64, R2 ¼ 1.72 s
1 and NOE ¼
0.65, thereby indicating differential conformational flexibility in the two conformations. 3.5. Spectral density analysis and conformational exchange Reduced spectral density analysis based on 15N R1, R2 and heteronuclear 1H-15N NOE is the best suited method for studying motions at residue level in denatured proteins. The spectral density function, J(u), represents the varied timescales of motion experienced by individual N-H vectors. The values of J(uH) probing the highest frequency motions is largely dictated by heteronuclear

NOE, whereas the value of J(uN) is largely determined by R1. Fig. 5D and E depict that the J(uH) and J(uN), as calculated from the 15N R1, R2 and heteronuclear 1H-15N NOE values on 800 MHz spectrometer remain similar throughout the polypeptide chain excepting for the sharp rise in the value of J(uH) at the two termini, which may be attributed to the additional flexibility of the two ends. The above trend is expected as the polymer theory predicts a bell-shaped profile for the dynamics of a linear peptide, with increased flexibility at the termini [79]. However, intramolecular association and degree of denaturation [71] also influence the backbone dynamics of the denatured state. The spectral density function J(0) has major contribution from R2 and reflects slower (milli-second to microsecond) timescale motions resulting from conformational exchange (Rex). Regions with above-average J(0) values may represent areas of the protein that are involved in early folding events. The residuewise plot of J(0) (Fig. 5F) indicates that the denatured DLC8 polypeptide chain has certain degrees of non-random segmental motion. The higher values of J(0) in the stretch Ser 14 e His 55,

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suggest that these residues may occupy some ordered regions of the polypeptide chain and thereby experience large contributions from increased slow timescale motions in the msems timescale. The spectral density functions J(uH), J(uN) and J(0) obtained at a field strength of 600 MHz, as depicted in Fig. S3(DeF) also exhibit similar trends. Averaged R2/R1 ratios are used to approximately determine the global correlation time of a protein. However, the residues which have major contributions from fast internal mobility will exhibit decrease in the R2/R1 ratio whereas residues that undergo chemical exchange have increased R2/R1 ratios [80]. The residue-wise plot of R2/R1 ratio depicted in Fig. 6A fall in line with the R2 trend suggesting that the observed increase in R2 values of the residues is substantially affected by the conformational exchange on the msems timescale. As we have already mentioned, we find increased R2 values in the segment Ser 14 e His 55 of the denatured polypeptide chain. Now, transverse relaxation rates are determined by two factors; R2 ¼ R2,int þ Rex, where R2,int is the intrinsic rate, R2 is the observed rate and Rex is the contribution from slow exchange arising from chemical exchange. Slow topological fluctuations in the denatured ensemble occurring on the ms-ms timescale can be assessed by Rex. The residue-wise Rex have been calculated using the spectral densities at two field strengths (600 MHz and 800 MHz) and are shown in Fig. 6B. Significant variation in the contributions from slow conformational exchange to the relaxation rates is observed at different residue sites, the most conspicuous contributions occur for the residues Ser 14, Gln 18, Ala 21, Val 22, Leu 29, Ile 38, Lys 44,

Tyr 54 and His 55. We would like to point out here that the coexistence of very high Rex values close to very low ones, as observed in this case is very much possible since the contribution of Rex at any site not only depends on the exchange rate itself but also on the chemical shift difference between the exchanging sites. Moreover, if the conformational exchange is local in nature then adjacent residues may exhibit different exchange rates, thereby showing wide variation in the values of Rex. The positions of the above residues are marked on the folded monomeric DLC8 (PDB ID: 1RHW) in Fig. 6C (generated using Pymol) [81]. This indicates the presence of partially ordered structures in this segment which undergoes exchange with the unstructured form of the polypeptide chain. 4. Discussion 4.1. Implications to initial folding events The residual structural propensities and regions of slow timescale motions due to conformational exchange as observed in the denatured state of a protein throw light on the initial folding events and have a significant influence in directing the folding pathway [82e86]. We employed heteronuclear NMR as the probe to extensively investigate the structural preferences and the differential segmental motions arising from the topological preferences in the denatured state of DLC8 to recognize the early determinants of folding. Native and non-native structural propensities define the intrinsic preferences and topologies of the denatured ensemble

Fig. 6. Conformational exchange in the DMSO-d6 denatured state of DLC8. Residue-wise plots of (A) R2/R1 with black bars representing areas of significant non-random structures and (B) Rex values at (uN) 81.1 MHz as calculated from spectral density functions J(0) at two field strengths, 600 and 800 MHz. The horizontal line indicates the average value (8.21 s
1). The residues showing conspicuously high Rex are mostly located in the segment Ser 14 e His 55. The native secondary structure is shown on the top of the graphs, where cylinders and arrows represent helices and b-strands, respectively. (C) The residues exhibiting high Rex are marked on the native monomeric structure of DLC8 (PDB ID: 1RHW).

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respectively. Native like interactions in the denatured states can limit the conformational search space during folding. On the other hand non-native like interactions could create energy barriers that hamper protein folding. We observe that the DLC8 polypeptide chain in the DMSO-denatured state has preferences for both alpha helical and beta strand like structures. The helical propensities observed in various stretches of contiguous residues in the segment Ser 14 e Tyr 50 and the extended b-strand like preferences in the residues Val 81 e Ser 88 (as obtained from 3J(HN
Ha) couplings) are mostly native-type preferences since these regions correspond to the a1, a2 helices and the b5 strand respectively. These native like residual structural propensities in denatured DLC8 may actually facilitate folding by minimizing the search in the conformational space. However, there is no indication of stable secondary structure in the denatured ensemble as predicted from the absence of any long range NOEs. These topological preferences must be accompanied by motional restrictions. From the dynamics data it appears that in the DMSO-denatured state, the protein chain is not randomly fluctuating, but exhibits concerted slow motions due to conformational exchange in certain segments of the protein. It is known that exchange in the denatured state arises due to the rugged energy landscape at the initial folding coordinates and is indicative of stable population of conformational sub-states with long lifetimes, thereby having mobility on the milli to microsecond timescale, which is often taken to indicate potential folding initiation sites. The denatured chain of DLC8 experiences slow motions in certain contiguous stretches in the segment Ser 14 e His 55 as depicted by high J(0) values arising from conformational exchange. This is further supported by the presence of high values of Rex in certain residues in this segment. The above mentioned stretch of residues coincides with the a1 and a2 helices and the loop between a2 and b2 and edge of the b2 strand in the native folded form of the protein. The residues exhibiting high J(0) values encompass highly

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polar residues in the segment Lys 43 e Lys 49 with 2 sets of 2 adjacent Lysine residues. The calculated J(0) values at 800 MHz and Kyte Dolittle Hydrophobicity index [87] calculated with the ExPASy tool ProtScale [http://www.us.expasy.org/tools/protscal.html] for DMSO-d6 denatured DLC8 are depicted in Fig. 7A to indicate that regions with increased polarity correspond to the segment exhibiting slow motions. In Fig. 7B, the segments corresponding to high J(0) are marked on the folded monomer (PDB ID: 1RHW) in blue and the polar residues (negative Hydrophobicity Index) are indicated on the other monomer in green to describe their positions on the native structure of the monomer. The monomer has been chosen for demonstration since the hydrophobicity indices as calculated from the primary sequence mostly reflect on the monomeric property. The observation of high J(0) in the polar segment Lys 43 e Lys 49 may be explained by the fact that there is considerable repulsive interaction between the similar charges of the Lysine side chains. The vacuum electrostatic surface potential for the segment Lys 43 e Asn 51 on the folded dimeric protein (PDB ID 1F3C) is shown in Fig. 7C to indicate the high concentration of positive charge in the segment Lys 43 e Lys 49 at physiological pH. The residues showing conformational exchange coincide with the polar segment of the protein indicating that charge interactions may play some role in transient structure formation and deformation resulting in exchange between partially structured and open conformations in the ensemble, making this segment a potential folding initiation site. It may also be pointed out here that DMSO is reported to stabilize helices in peptides [88]. Our observation of high conformational exchange in the segment corresponding to the native helices is consistent with this report. It is worthwhile to mention here that native state H/D exchange [42] revealed these two helices and the beta strand 5 to be the folding nucleation sites. Though in the DMSO-denatured state we do not find any contribution from slow timescale motions in the beta

Fig. 7. Folding initiation sites. (A) Plots of calculated J(0) values at 800 MHz (filled circles) and Hydrophobocity (Kyte Dolittle Hydrophobicity index) for DMSO-d6 denatured DLC8. The Hydrophobocity values were calculated with the ExPASy tool ProtScale [http://www.us.expasy.org/tools/protscal.html]. Regions with increased polarity correspond to the segment exhibiting slow motions due to conformational exchange. The helices and the beta strands in the native DLC8 are indicated by cylinders and arrows, respectively, on top of the graphs. (B) The segments corresponding to high J(0) are marked on the folded monomer (PDB ID: 1RHW) in blue and the polar residues (negative Hydrophobicity Index) are indicated on the other monomer in green to describe their positions in the native structure of the monomer. (C) The vacuum electrostatic surface potential for the segment Lys 43 e Asn 51 of the folded dimeric protein (PDB ID: 1F3C) to show the high concentration of positive charge in the mentioned segment (http://noch.sourceforge.net/). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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strand 5, we do find that the other two structural elements exhibit fair degree of conformational exchange. However the presence of native b-strand like preference in the segment Val 81 e Ser 88 indicates a probability for this segment to be also involved in the early folding events. From the above detailed analysis, the likely sequence of events in the folding may be summarized as follows: the two alpha helices are the earliest structural elements formed. The helix formation is followed by the formation of b-strand 2 and the highly hydrophobic b5. Then the other structural elements form to give the final folded form of the protein. However the detailed visualization of the folding trajectory is only possible with a complete investigation of the different equilibrium states created by denaturant dilution. 5. Conclusion We have successfully investigated here, using a variety of NMR probes, the nature of the DMSO-denatured state of DLC8. The detailed analysis of structural preferences in the denatured state indicates that the protein exhibits native helical preferences in the segment of the polypeptide chain that corresponds to the two helices in the native state. Several residues (Val 81 e Ser 88) populate the space corresponding to the b-strand and others mostly exhibit random coil like behavior. The presence of native like structural propensities may direct the protein toward the native folded conformation by diminishing the search of the conformational space. The polypeptide chain has significant motional restrictions in the segment Ser 14 e His 55 arising from slow conformational exchange in the micro to milli-second timescale. It is important to point out here that the DLC8 in DMSO exists as monomer which is consolidated by the absence of slow timescale motion arising from conformational exchange in the segments corresponding to the structural elements (b3 strand and the loop between b3 and b4) present at the interface of the native dimer. As the structural propensities and the motional characteristics in the denatured state influence the folding pathways of proteins to a great extent, the present observations suggest that, folding of DLC8 monomer from the DMSO-denatured state may get initiated around the a1-loopea2-region along the polypeptide chain. Charge interactions between the adjacent positively charged side chains of Lysine may play a crucial role in structure formation. This is probably followed by the formation of b2 and b5 strands. However, the detailed elucidation of the folding trajectory demands the complete characterization of the different intermediates along the folding trajectory. Acknowledgments We thank the Government of India for providing financial support to the National Facility for High Field NMR at TIFR and Dr. Anindya Ghosh-Roy for the DLC8 clone. Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biochi.2011.10.013. References [1] K.A. Dill, D. Shortle, Denatured states of protein, Annu. Rev. Biochem. 60 (1991) 795e825. [2] H.J. Dyson, P.E. Wright, Equilibrium NMR studies of unfolded and partially folded proteins, Nat. Struct. Biol. 5 (1998) 499e503. [3] G.D. Rose, P.J. Fleming, J.R. Banavar, A. Maritan, A backbone-based theory of protein folding, Proc. Natl. Acad. Sci. U.S.A 103 (2006) 16623e16633.

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