Effects of Anisotropic Nanoconfinement on Rotational Dynamics of

Aug 27, 2012 - (12-14) In principle, the method can be applied to organic/inorganic systems. .... least-squares fitting with the Levenberg–Marquardt a...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JPCC

Effects of Anisotropic Nanoconfinement on Rotational Dynamics of Biomolecules: An Electron Spin Resonance Study Chia-Jung Tsai and Yun-Wei Chiang* Department of Chemistry and Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, 30013, Taiwan S Supporting Information *

ABSTRACT: The development of nanostructured materials for next-generation nanodevice technologies requires a better understanding of dynamics of the objects as confined in nanospace. Here, we characterize the rotational dynamics of a long (14-residue) proline-based peptide (approximately 4 nm in length) under anisotropic nanoconfinement using spinlabeling CW/pulsed ESR techniques as well as spectral simulations. We show by pulsed ESR experiments that the conformations of the peptide in several different nanochannels and a bulk solvent are retained. Parameters characterizing the dynamics of the peptide regarding temperature (200−300 K) and nanoconfinement are determined from nonlinear least-squares fits of theoretical calculations to the multifrequency experimental spectra. Remarkably, we find that this long helical peptide undergoes a large degree of rotational anisotropy and orientational ordering inside the nanochannels, but not in the bulk solvent. The rotational anisotropy of the helical peptide barely changes with the nanoconfinement effects and remains substantial, as the nanochannel diameter is varied from 6.1 to 7.1 and 7.6 nm. This finding is advantageous for addressing purposes of anisotropic nanoconfinement and for advancing our understanding of the rotational dynamics of nano-objects as confined deeply inside the nanostructures of materials.

■

INTRODUCTION Next-generation electronic, biomaterial, or spintronic devices will be based on nanoscale functional units, such as quantum dots, peptide-based building blocks, or molecular-scale inorganic devices.1−4 The important role of spatial confinement, as found in the channels and chambers of novel materials, on the shape and dynamics of nano-objects definitely needs to be explored.5,6 The motional dynamics of biomaterialbased devices inside the nanostructures is among the most interesting and important research topics.4,7,8 Elucidation of the nano-object dynamics in nanosized confinement will advance our knowledge regarding the design of the molecular-scale devices and also enhance our understanding of how to manipulate the bionanodevices using naturally formed nanostructures, such as the nanospace in pharmacological chaperones and the tobacco mosaic virus. Clearly, there is a great potential for using biological materials to develop entirely novel systems that display superior characteristics, but further research is necessary. Therefore, we must address the effects of confinement on the dynamics of biomolecules at a molecular detail. The leading challenge is the coupling of the molecularscale units to the macroscopic world, which is essential for control and detection purposes for the catalytic activity, as the nanodevice is deeply confined within the nanostructures. The existing tools such as fluorescent/optical methods and scanning tunneling microscopy, except computational dynamic simulations,6,9 are very limited in their ability to address the © 2012 American Chemical Society

challenge. This limitation restricts the study of nanodevice motion to the outside surface of materials.10 Electron spin resonance (ESR)11 spectroscopy with spinlabeling techniques has been established as a powerful tool for determining the protein structure and topology of protein complexes, and, more recently, for investigating the molecularscale force of a nanodevice in a bulk solvent and peptide backbone dynamics in nanoconfinement.12−14 In principle, the method can be applied to organic/inorganic systems. The method requires the introduction of a stable free-radical molecular reporter group, such as a nitroxide spin probe, into the system of interest. Nitroxide spin labels can be covalently attached to the system, and can, therefore, serve as probes of the local backbone dynamics of polymers, or protein/ biomolecules, providing information on the local orientation, structure, dynamics, and environment at a nanoscale level. There are several unique features of the ESR methods. The ESR spectra are most sensitive when the dynamics of the unpaired electron spin becomes intimately entangled with the molecular dynamics in the so-called “slow-motional regime” (e.g., 10 −6 −10 −11 s for the commonly used X-band frequency).15 The ESR spectra are known to change dramatically as the tumbling motion of the spin probe is in Received: May 17, 2012 Revised: August 25, 2012 Published: August 27, 2012 19798

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

Article

Figure 1. (a) The residue sequences of the three proline-based peptides used in the study. (b) The CD spectra of the PP-3R1 in water versus in nanochannels collected at 25 and 4 °C, showing a typical spectrum of a polyproline PPII helix with the maximum and minimum around 228 and 205 nm, respectively. (c) A model of a simple drawing to illustrate the PP-3R1 peptide carrying three nitroxide spin labels, as confined in a nanochannel of the hexagonal straight tunnel structure. An axial model (R⊥ = Rx = Ry, Rz = R∥) is employed to describe the rotational dynamics of the helical peptides.

the present study is to nvestigate how much extent the nanoconfinement anisotropy/effect could influence the rotational dynamics of nanoconfined molecules. The purpose of this work is to demonstrate the utility of the spin-labeling ESR techniques in probing locomotion of nanodevices and revealing local ordering and dynamics of the molecular-scale units as confined inside nanostructures to which other commonly employed tools are not easily accessible.

the slow-motional regime, thus providing great sensitivity to fluidity/dynamics nearby the spin probe. Therefore, the ESR spectra can be usefully exploited to acquire insights into molecular rotational motions in isotropic fluids, in liquid crystals, and in model/biological membranes, because of the inherent orientation dependence (anisotropy) of the magnetic interactions of the unpaired nitroxide spin with the applied magnetic field. In this work, we studied the rotational dynamics of a longhelical proline-based peptide, as encapsulated within the nanochannels of mesoporous materials, by CW/pulsed ESR techniques together with spectral simulations. We examine the structural conformation of the peptide by pulsed double electron−electron resonance (DEER) techniques16,17 and demonstrate that the conformation of the encapsulated peptides remains approximately unchanged as compared to the peptide conformation in a bulk solvent. Parameters describing the local ordering and dynamics of the confined peptides are determined from the theoretical fits of the stochastic Liouville equation (SLE)-based calculations to the experimental spectra.18 Two clearly resolved spectral components, distinct in both their ordering and dynamics, are resolved in the room-temperature studies and assigned to the dynamic modes associated with the backbone dynamics. The interest in

■

MATERIALS AND METHODS Polypeptides. All peptides of this study were customsynthesized by Kelowna International Scientific Inc. (New Taipei, Taiwan) with a purity greater than 95%. The PP-3R1 is a 14-mer-long polyproline peptide, with the 1st, 5th, and 14th residues substituted with cysteine for spin labeling. The PP-m2 and PP-m3 are also 14-mer-long polyproline model peptides, with seventh and ninth residues substituted with cysteine to be conjugated with spin labels, respectively. See Figure 1a for the peptide sequences. Nanochannels. The mesoporous silica MSU-H (abbreviated as MSU) and MCM41 materials were purchased from Aldrich. Mesoporous silica SBA15 materials were kindly provided by Professor C.-M. Yang. The materials were synthesized as previously described by Yang et al.,19 in which 19799

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

Article

CW/Pulsed-ESR Measurements. A Bruker ELEXSYS E580 CW/pulse spectrometer, equipped with a Bruker pulse ELDOR unit E580-400, a dielectric resonator (ER4118XMD5W), and a helium gas flow system (4118CF and 4112HV), was used. The X- and Q-band CW-ESR experiments were performed at the operating frequencies of 9.4 and 33.9 GHz, respectively. The swept magnetic range was 200 G for both frequencies. Samples in the bulk solution study were loaded in a microcapillary before being placed in 4 mm (O.D.) quartz ESR tubes. Samples in the nanochannel studies were directly loaded into quartz ESR tubes. Pulsed-DEER experiments were carried out using the four-pulse constant-time DEER sequence: π/ 2(ωA)−τ1−π(ωA)−(τ1 + t)−π(ωB)−(τ2 − t)−π(ωA)−τ2− echo. The detection pulses (ωA) were set to 32 and 16 ns for π and π/2 pulses, respectively, and the pump frequency (ωB) was set to approximately 70 MHz lower than the detection pulse frequency. The pulse amplitudes were chosen to optimize the refocused echo. The π/2-pulse was employed with the +x/−x phase cycle to eliminate receiver offsets. The durations of the pumping pulse was about 32 ns, and its frequency (ωB) was coupled into the microwave bridge by a commercially available setup from Bruker. All pulses were amplified via a pulsed traveling wave tube amplifier (E5801030). The field was adjusted such that the pump pulse is applied to the maximum of the nitroxide spectrum, where it selects the central mI = 0 transition of Azz together with the mI = ± 1 transitions. The echo was measured as a function of t, while τ2 was kept constant, depending on TM. Typical numbers of shots per points and scan number were set to 1024 and 30− 40, respectively. Accumulation time for each set of data was about >10 h. All pulsed ESR experiments of this study were performed at 50 K. The determination of the interspin distance distribution of the DEER data was performed in the timedomain analysis by Tikhonov regularization based on the Lcurve method. A common freezing approach was used. The sample tube was plunge-frozen in liquid nitrogen, and then transferred into the ESR probehead, where it was precooled to 50 K using a helium flow system. CW-ESR Spectral Simulations and Pulsed-DEER Analyses. In this study, we have performed theoretical analyses of the experimental CW-ESR spectra using a nonlinear leastsquares (NLLS) fitting program based upon the stochastic Liouville equation (SLE).18 The NLLS program is performed using a powerful and efficient computational methodology and by nonlinear least-squares fitting with the Levenberg− Marquardt algorithm.21 In the SLE theory, the spin degree of freedom of the system is treated in a quantum-mechanical fashion while the orientational dynamics of the spin probe is determined by a classical stochastic process. Such a rigorous SLE-based interpretation for CW-ESR spectral analysis has been demonstrated to be useful for analyzing the spectra of complex macromolecules, for example, proteins and membranes, to extract the dynamic parameters of spin probes from the experimental spectra. The microscopically ordered but macroscopically disordered (MOMD) model was included in the SLE-based line-shape analysis of the spectra.21 In this model, the nitroxide side chain is constrained by a local ordering potential while the globular core of the peptide is assumed immobilized on the ESR time scale and is statistically distributed with respect to the magnetic field. Thus, the simulated spectrum represents the integration of the spectra of all possible orientations over the distribution of the local director with respect to magnetic fields.

information about the material structures can be found. Briefly, tetraethoxysilane was added to the HCl solution of triblock copolymer Pluronic P-123 (EO20PO70EO20). The molar composition was 1 TEOS:0.54 HCl:100 H2O:0.017 P-123. The mixture was stirred at 35 °C for 24 h, aged at 90 °C (SBA15a) and 60 °C (SBA15b) for 24 h, and then filtered and dried. The copolymer molecules in the as-synthesized SBA15 samples were removed by treating the samples with concentrated sulfuric acid at 90 °C, followed by calcination at 350 °C. The structural properties of the mesoporous materials are summarized in the following order: SBA15a, SBA15b, MSU, MCM41. The pore diameters are 7.6, 6.1, 7.1, and 2.5 nm. The unit cell sizes are 11.6, 9.6, 11.6, and 4.7 nm. The sizes of the wall thickness are thus 4.0, 3.5, 4.5, and 2.2 nm. Experimental Procedures. In the spin-labeling experiment, peptides were labeled with a 10-fold excess of (1-oxy2,2,5,5-tetramethyl-3-pyrroline-3-methyl) methanethiosulfonate spin label (MTSL) (Alexis biochemicals, San Diego, CA) per cysteine residue for overnight in the dark at 4 °C. They were further purified by reverse-phase HPLC as previously described. MALDI-TOF experiments were conducted to confirm the identity of the peptides. The concentration of the spin-labeled peptides for the vitrified bulk solution studies was 0.5 mM in a mixture of 40% sucrose (cryoprotectant) and H2O. The solution volumes added into the ESR tube were 40 μL for the X-band studies. The encapsulation of the samples into the nanochannels was prepared as previously described.12 For the nanochannel studies, the peptide concentration and the added solution volume for the X-band (Q-band) studies were 0.5 mM (1 mM) in pure H2O and 20 μL (4 μL), respectively. The amount of the nanochannel material used was fixed to be 6 times of the added solution volume, as previously described.12,13 A higher concentration was required for the Q-band experiments to achieve a good SNR, as the solution volume was only one-fifth of the volume in the X-band experiments. We confirmed that the samples of the two different concentrations yielded the identical spectra in the Qband experiments. The circular dichroism (CD) experiments were performed at 25 and 4 °C to confirm the secondary structure of the peptides. Mesoporous materials (4 mg) containing the peptides were dispersed within 0.4 mL of pure glycerol in a 1 mm CD cell. The CD spectra of the studied peptides in the bulk solvent versus within the nanochannels were verified to be similar (Figure 1b), both of which possessed the maximum at 228 nm and minimum at 205 nm that were indicative of a PPII helix. This study followed the sample encapsulation procedure that was previously demonstrated as an effective procedure to have most of the peptides trapped in the nanochannels.13,20 Additionally, this study also performed the following experiment to ensure that the peptide variants are adequately encapsulated within the nanochannels. An excess buffer was added into the ESR tube containing the mesoporous materials, wherein the spin-labeled peptides were encapsulated as described. The tube was sent again for ESR measurements at room temperature. The collected spectra were found to remain identical to the spectra collected before the addition of the excess buffer, that is, typical slow-motional lineshapes. This experiment demonstrated that the spin-labeled peptides were not adsorbed/left on the outer surface of the materials and that the molecules are trapped significantly well within the nanochannels. No ESR signal was obtained for the supernatant liquid after centrifugation. 19800

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

Article

Table 1. Parameters Obtained from Nonlinear Least-Squares Fitting of Multifrequency CW-ESR Spectra (X and Q Bands) of the Spin-Labeled PP-m2 and PP-m3 Peptides at 300 and 200 K. Estimated Errors Are Given Belowf R⊥ (s−1)

T = 300 K sol. MSU

Im Mb Im Mb Im Mb R⊥ (s−1)

SBA15a SBA15b T = 200 K sol. MSU SBA15a SBA15b

2.35 1.27 3.82 1.27 6.39 1.06 4.39

× × × × × × ×

R∥ (s−1) 8

10 106 107 106 107 106 107

6.54 1.24 3.82 3.79 6.39 1.03 4.38 R∥ (s−1)

× × × × × × ×

S0

R∥/R⊥ 7

10 107 107 107 107 107 107

0.28 9.76 1.00 11.22 1.00 9.72 0.99 R∥/R⊥

0.03 0.56 0.15 0.54 0.16 0.48 0.14 S0

6.81 × 10 (0.13 ± 0.03) × 106

1.71 × 10 (8.15 ± 0.50) × 106

c

c

c

c

c

c

c

c

5

5

0.25 62.7

0.62 0.58 ± 0.05

Δ(0)a 0.5 2.7 2.1 3.0 1.7 2.4 1.4 Δ(0)d 7.5 1.9 1.4 1.4

(7.4) (1.2) (1.6) (1.8)

[Im]b 100% (100%) 48% (38%) 47% (32%) 45% (46%) Δ(2)e 0 (0) 4.6 (4.5) 2.6 (3.3) 3.9 (3.7)

a Gaussian inhomogeneous broadening (peak-to-peak line width, gauss). bThe population of the Im spectral component obtained in the spectral fits of the PP-m2 study. [Im] + [Mb] = 100%, if two spectral components are used. The values in parentheses represent the [Im] for the PP-m3 studies. c The obtained values are approximately the same as those of the MSU studies at 200 K. dGaussian inhomogeneous broadening (peak-to-peak line width, gauss) obtained for the PP-m2 studies. The values in parentheses represent the result for the PP-m3 studies. eOrientation-dependent Gaussian inhomogeneous broadening (peak-to-peak line width, gauss) obtained for the PP-m2 studies. The values in parentheses represent the result for the PP-m3 studies. fThe average errors estimated from the simulations of the present study were ca. 10% for R⊥ and R∥, 0.04 for S0, 10% for Δ, and 6% for the component population.

The average errors estimated from the simulations are given in Table 1. The parameter uncertainties were calculated from the covariance matrices returned from the SLE-based nonlinear least-squared fitting, as described in Budil et al.21 The spectra recorded at X- and Q-band frequencies were fit simultaneously using the NLLS program with a common set of the dynamic parameters. The determination of the interspin distance distribution of the DEER spectroscopy was performed in the time-domain analysis by Tikhonov regularization based on the L-curve method.24 The DEER technique was previously demonstrated to be useful for determining the interspin distances and distance distributions of a triple-labeled molecule.25

Presumably, the tumbling motion of the spin-labeled biomolecule, which is extremely slow in nanochannels or at low temperatures, is completely excluded out of the ESR time scale. The MOMD model is, therefore, an appropriate model to simulate the spectra of the spin-labeled peptides in the present study. The values for the magnetic A and g tensors were determined by fitting both the rigid-limit and the motionalnarrowing spectra of the two resonance frequencies (X- and Qband) using Matlab with the EasySpin toolbox.22 We started with the magnetic tensors of the spin-labeled polyproline peptides, derived previously, and allowed some reasonable variations of the magnetic tensors as well as line widths using the Nelder−Mead simplex minimization algorithm. The determined principal values for the A tensor are 6.39, 5.15, and 37.12 G. The determined principal values for the g tensor are 2.0077, 2.0055, and 2.0019. The rotational diffusion tensor (R) that describes the motion of the nitroxide spin probe is assumed to be axially symmetric (i.e., R⊥ = Rx = Ry, Rz = R∥) in the present study. The ordering potential of the anisotropic motion of the nitroxide is described by the order parameter S0 that is calculated from the ordering coefficients varied in the fitting. Two line-width parameters (Δ(0) and Δ(2)) were used to account for the orientation-independent and -dependent inhomogeneous Gaussian broadening, respectively. The total inhomogeneous broadening is defined by Δ = Δ(0) + Δ(2) sin2 ψ to account for the dependence of line width on the orientation angle (ψ) of the peptide helix director.21 In ordered systems, Δ(2) is necessary as the anisotropies in g or A tensors are pronounced in the spectral lineshape and cannot be treated as being isotropic. In nanochannels, water density varies distinctly along the transverse direction, but insignificantly along the longitudinal axis.23 Thus, the anisotropies are important in the spectra, making Δ(2) a necessary parameter for achieving a good theoretical fit. The requirement of Δ(2) is indicative of a preferential alignment. Multicomponent spectral fitting was allowed in the NLLS program. The populations of the spectral components were determined by the program of the nonlinear least-squares fits and were an output of the fitting.

■

RESULTS Three different nanochannel materials (SBA15a, SBA15b, and MSU) were used. The respective pore diameters are 7.6, 6.1, and 7.1 nm. Details of the textural properties are given in the Materials and Methods section. Structural Conformations of the Encapsulated Biomolecules. Figure 1a shows the residue sequences of the three proline-based peptides, PP-3R1, PP-m2, and PP-m3, of the present study; all of which are 14-residues long. The Cys sites indicate where the nitroxide spin probe is attached. Figure 1b shows the circular dichroism (CD) measurements as the PP3R1 peptides are in H2O versus in the nanochannels. They appear similar and display a typical spectrum for a polyproline PPII helix. (See the Materials and Methods section for the characteristic peaks of the CD spectra.) Figure 1c displays the structure of the materials and a cartoon model of the PP-3R1 carrying three spin labels within a nanochannel. The rotational diffusion rates of the spin-labeled peptide are described by an axial model composed of R⊥ and R∥, as illustrated. To study how much the structural conformation of the peptide is disrupted in the nanochannels, in this study, we performed DEER measurements for the PP-3R1 peptide with a concentration of 0.5 mM in three different conditions: (i) a vitrified bulk solvent composed of 40% sucrose and water, (ii) SBA15a containing pure water, and (iii) MSU containing pure 19801

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

Article

spatial density of the spin-labeled peptides is not notably changed with the studied conditions. The inset in Figure 2 shows the Fourier-transformed (FT) DEER signals, known as the Pake doublet, reporting the average of interspin distances in the ensemble, and the distance distributions P(r) obtained using the model-independent Tikhonov method.17 The average distances, as well as the peak positions in the P(r) distributions for the three studies, are very consistent. This consistency indicates that the structure of the PP-3R1 remains alike in the three different environments. The peak positions are approximately at 2.2, 3.0, and 3.9 nm, which are close to rigid-body model calculations (ca. 1.8, 2.8, and 4.2 nm) of a 14mer-long polyproline type II (PPII) structure with an assumption of the nitroxide side-chain length of 0.67 nm. (Note that PPII is a left-handed helix with all-trans peptide bonds and has backbone dihedral angles of (φ, ψ, ω) = (−75°, 145°, 180°).26,27 This model peptide has been extensively studied.28−30 Formed at the absence of intrachain hydrogen bonds, it has a relatively rigid structure and is quite extended with an axial translation of 0.31 nm per amino acid.29 Thus, the total persistence axial length is 4.03 nm for a rigid 14-prolinelong PPII structure.) The P(r) results together with the CD spectra provide sufficient evidence that the PP-3R1 remains approximately a PPII secondary structure in the conditions studied. See Figure S1 in the Supporting Information for additional evidence from the DEER measurements showing support for the determined structure of the peptides. Also, Figure S1 reports evidence that the confined peptides are homogeneously dispersed in the nanochannels. Furthermore, this study shows that the P(r) results are clearly better resolved (with peaks much narrower in width) in the nanochannels than in the bulk solution results, suggesting that (i) the P(r) resolution is improved in the nanochannel studies and (ii) the encapsulation of the PP-3R1 into the nanochannels leads to favorable free energies. This adds a valuable advantage to the approach combining ESR with nanochannels for biophysical study. In the FT plots (cf. Figure 2), the doublet splittings in the three studies are about the

water. (See the Materials and Methods section for details.) The experimental and analysis results of the DEER measurements are shown in Figure 2, with red, blue, and black lines

Figure 2. Time-domain DEER data for the PP-3R1 peptide in the conditions studied, including a vitrified bulk solvent (red) and two different nanochannels, SBA15a (blue) and MSU (black) at 50 K. The insets show the results of the FT plots, derived from the time-domain DEER data, and the distance distributions P(r), determined from the Tikhonov analyses. The determined structures remain alike and appear to be a PPII structure, in the three conditions studied.

representing the results of the time-domain data for the vitrified bulk solution, SBA15a, and MSU, respectively. The gray lines represent the exponential baselines that best fit the time-domain DEER data.17 The time-domain signals of the three experiments clearly display a similarity in the slope of the baseline as well as modulation depth. On the basis of the spincounting analysis principle,25 this observation suggests that the

Figure 3. X-band (a) and Q-band (b) CW-ESR spectra of the PP-m2 and PP-m3 peptides recorded as in the vitrified bulk solvent and three different nanochannels (MSU, SBA15a, and SBA15b) at temperatures of 200 and 300 K. The experimental and simulated spectra are plotted by solid and dashed lines, respectively. The multifrequency spectra were fit simultaneously using the NLLS program with a common set of the parameters. 19802

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

Article

Figure 4. (a) The X-band CW-ESR spectra of the various polypeptides as confined in the nanochannels (MCM41, SBA15b, and MSU) at 300 K. Two clearly resolved spectral components, distinct in both their ordering and rotational diffusion, are resolved in the spectra of the helix-shaped peptides (PC5, PP-m2, and n3-α) at room temperature and are assigned as Im and Mb components. The two simulated spectral components are shown in the inset. The experiments at high temperature (300 K) clearly show that the Mb notably appears in the spectra of the helix-type peptides, but almost disappears in the spectra of the hairpin-type peptide (n3-β). (b) The cartoon models of the studied peptide indicated in (a). The sequence of the PC5 is PPPPCPPPPPP. The sequence of the n3 is GNDYEDRYCRENMYRYPNQVYYRPVA. Cysteine residues on the peptides indicate the sites to which the spin probe is attached.

strains (cf. the Materials and Methods section). The orientation-dependent line-width broadening (Δ(2)) for the nanochannel studies is found to be approximately 2.6−4.6 G, which is distinctly greater (at least twice) as compared with the inhomogeneous (orientation-independent) line-width broadening (Δ(0)), which is about 1.2−1.9 G. The parameter Δ(2) accounts for the dependence of line width on the orientation angle of the helix director of the studied peptide relative to the magnetic field (cf. the Materials and Methods section). Thus, the requirement of Δ(2) for achieving a good quality of the theoretical fits in the nanochannels indicates an improved spectroscopic resolution in the orientation. Previously, the side-chain disordering of spin-labeled polypeptides was found to be substantially reduced in nanochannels as compared to the side-chain disordering studied in a bulk solution.20 Taken together, the improved orientational resolution demonstrated in the present study is likely due to the reduced disordering in the side chain and the long PPII helical structure of the studied peptides that tends to align favorably along the nanochannels featured with a hexagonal straight tunnel of the two-dimensional structure. Evidence supporting the latter reason can also be gained by a comparison of the present study and a previous study12 concerning a β-hairpin peptide (n3-β) confined in the same nanochannel at the same temperature, 200 K. In the previous study, the n3-β peptides were confined in the nanochannels, but it was not necessarily to have a preferential orientation relative to the axial director of the nanochannel. The n3-β peptides were homogeneously dispersed in the nanochannels. Therefore, the study indicated that a good quality of theoretical fits to the CW-ESR spectra of the n3-β at 200 K was obtained with R∥/R⊥ ∼ 0.15 and Δ(2) = 0, showing support to the aforementioned conjecture; that is, R∥/R⊥ and Δ(2) are sensitive to the preferential orientation of spin-labeled molecules in an anisotropic nanoconfinement. On the whole, the simulation results of the present study for 200 K indicate that the peptides in the nanochannels experience a notable

same. However, the half-height width of the FT plot is distinctly greater for the bulk solution than the nanochannel studies, suggesting a greater disordering in the side-chain or conformational mobility for PP-3R1 in the bulk solution. This observation based on the FT results is consistent with the aforementioned observations based on the P(r) results. Theoretical Simulations of the Multifrequency CWESR Spectra. To investigate the changes of the local environment on the spin-labeled sites concerning temperature and nanoconfinement effects, this study performed CW-ESR measurements of the singly spin-labeled peptides (PP-m2 and PP-m3) in the various conditions indicated in Figure 3, where the results for the X- and Q-band studies are displayed in (a) and (b), respectively. The 200 K experiments are to ensure that the unwanted (global) isotropic tumbling motions are frozen out in the ESR-sensitive time scale (micro- to nanoseconds) in both the bulk solution and the nanochannel experiments. As a result, only the local environment (anisotropy) is reported on the CW-ESR spectra. In Figure 3, the dashed and solid lines represent the theoretical fits and the experimental spectra, respectively. The multifrequency spectra were analyzed simultaneously using a common set of the dynamical parameters. Table 1 shows the summary of the parameters obtained from the theoretical analyses of the multifrequency spectra. Below, we first present the simulation results of 200 K. All of the spectra can be fit well using one spectral component, characterized by slow rotational diffusion rates (R⊥ and R∥) and high molecular ordering (S0 > 0.55). The obtained values for R⊥ and R∥ are very slow (105−106 s−1) and close to the slowmotional regime of the ESR time scale. The anisotropy ratio, R∥/R⊥, is found necessary to be as high as >62, which is substantial, for the nanochannel studies, whereas the ratio becomes only 0.25 for the bulk solution study at the same temperature, 200 K. Best fits were obtained by introducing an anisotropic (orientation-dependent) broadening that can model small effects such as unresolved hyperfine splitting, or g, A 19803

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

Article

The Significance of the Spectral Components. Figure 4a shows the CW-ESR spectra of four different polypeptides confined in various nanochannels (at 300 K), including MCM41 (2.5 nm), SBA15b (6.1 nm), and MSU (7.1 nm), where the values in parentheses indicate the pore diameter of the nanochannels. Three of the peptides were previously studied extensively as confined within nanochannels, and thus were used as a model in the present study.12,20,31 All of the spin-labeled sites in the peptides are solvent-exposed. The sequences and structures of the peptides are given in Figure 4b. In brief, PC5 is an 11-residue-long proline-based peptide carrying cysteine at the fifth position to be conjugated with the spin label. The peptides, n3-α and n3-β, represent a 26-residuelong polypeptide, derived from a domain of human prion protein, in two different secondary structures, α-helical (in a mixture of TFE/PB = 87/13 buffer) and β-hairpin (in PB buffer), respectively. The results show that the Mb component (cf. Figure 4a) is notably present in all of the spectra for the helical secondary structures, irrespective of the types of helical structure (PPII or α-helix), length (11-, 14-, or 26-residue long), solvents (H2O, PB, or TFE/PB mixture), and nanochannel pore sizes (2.5−7.1 nm), whereas the Mb is nearly absent in the spectra of the n3-β. If the Mb component is correlated with the increased side-chain internal motions with increasing temperature, it should be also present in the spectra of the n3-β, but fade away with decreasing pore diameter in the helix studies; however, it does not (cf. the spectra for the PC5 as confined inside SBA15b (6.1 nm) versus MCM41 (2.5 nm)). As shown in Figure 4a, Mb is, however, hardly observed in the spectra of n3-β in both the MSU and the SBA15b studies, but is clearly resolved in the spectrum of PC5 in the MCM41 study, that is, in a significantly smaller pore size as compared with MSU and SBA15b. These observations evidently suggest that the Mb is related to the temperature-dependent dynamics/ flexibility on the backbone of the helical structures rather than the side-chain rotamers of the spin label, nor the side-chain internal motions. Additionally, the Mb is not related to the global isotropic tumbling because the pore size of MCM41 (2.5 nm) is too small to allow the global tumbling of the peptides. This study indicates that a (relatively) mobile spectral component appears at high temperature and the component is related to the fast backbone dynamics of the helical structures. Nanoconfinement improves the capability of ESR spectroscopy to identify backbone dynamics on a helical nanodevice. Moreover, we give a brief comment on the relation of the present study to other studies. Previously, it was shown13 that the ESR spectra are sensitive to the backbone dynamics of the n3 polypeptides as confined in the nanochannels at 275−300 K, with the stipulation that the line-width contributions from the global rotational motion are not comparable with the contributions from the local backbone dynamics. In the present study, the use of the long helix-shaped peptide promotes an enhancement of the global rotational anisotropy in the nanochannels. The low temperature (200 K) also contributes to the ordering of the alignment, according to the Boltzmann law. As a result, the ESR spectra are verified in the present study to genuinely reflect the global rotational anisotropy of the rigid polyproline model peptides at low temperature, and both the global rotational dynamics and the backbone dynamics at high temperature. Dependence of Distance Distribution on Maximum Dipolar Evolution Time. Lastly, we comment on the

confinement. The confinement reduces the side-chain disordering effectively and facilitates the alignment of the helix-shaped peptides in the channels. As a result, the ESR spectra display an improved spectroscopic resolution and a large degree of rotational anisotropy, as quantitatively indicated in the obtained parameters. The simulation results for 300 K are described below. The study shows that one spectral component is sufficient to achieving a good fit to the spectra of the bulk solution study, whereas two spectral components are required to achieve a good quality of fits for the spectra of the nanochannel studies. The two spectral components are characterized by a (relatively) higher versus lower mobility and are denoted as mobile (Mb) and immobile (Im), respectively, as indicated in Table 1. The bulk solution study shows a substantial increase in R⊥ and R∥, but a large decrease in Δ(0) and S0, as compared with the study of 200 K. The anisotropy ratio remains approximately unchanged (0.25−0.28) with increasing temperature. Taken together, the changes of the bulk solution studies, in conjunction with temperature, are in agreement with the general expectation. Regarding the nanochannel studies, it shows that the Im component seems to be related to the spectral component observed at 200 K, because both (i.e., the Im at 300 K and the component of 200 K) are characterized by a high anisotropy ratio (>9 at 300 K and >62 at 200 K) and a substantial ordering S0. The major difference lies in the absence of Δ(2) in the studies of 300 K. This is a reasonable finding as the side-chain disordering increases with increasing temperature, thereby reducing the orientational resolution in the ESR spectra of the nanochannel studies and, consequently, resulting in the absence of Δ(2). The population of the Im is indicated by [Im] for both the studies of PP-m2 and PP-m3 peptides. The other spectral component Mb is found to be necessary for achieving a good quality of the fits at 300 K. The Mb component is featured with an isotropic rotational diffusion, that is, R⊥ = R∥, and a relatively low ordering (S0 < 0.15). The two simulated spectral components of the PP-m2 in the MSU nanochannels are shown in the inset of Figure 4a. They are distinctly different from each other. (See Table 1 for the simulation parameters.) The significance of the two spectral components will be discussed later (Figure 4). Collectively, the spectral simulations demonstrate that the anisotropic nanoconfinement restricts the isotropic tumbling of the peptides effectively and enhances the rotational anisotropy of the confined helix-shaped peptides. The rotational anisotropy (R∥/R⊥) barely changes with the nanochannel pore size. This ordering indicates a substantial nanoconfinement effect, restricting the encapsulated peptides to orient themselves in a preferential direction in the nanochannels. It suggests that the peptides are likely adapted to align along the longitudinal axis of the straight tunnels in the nanochannels studied, that is, in a thermodynamically favorable arrangement. Alternatively, this finding implies that some tolerance on the size of the confined molecules is allowed in a nanochannel as the nanoconfinement effects on rotational anisotropy persist. Besides, the confinement also reduces the side-chain disordering effectively. Given that the available configurational space of the side-chain rotamers becomes more impact as confined in nanochannels, the orientation resolution for the local ordering and dynamics is thus enhanced and better resolved in the ESR spectra. The ESR methods are clearly demonstrated as a powerful tool for probing the local environment of nanodevices as confined inside nanostructures. 19804

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

■

maximum evolution time shown in Figure 2 and its possible effect on the resultant P(r). The length of the evolution time was mainly restricted by the short TM value in the nanochannel studies. The 1.2 μs was the longest evolution that was attainable at a reasonable signal-to-noise ratio in our nanochannel experiments. Although the length is shorter than the optimum evolution time (1.5−2 μs), determined theoretically,17 for the determination of a P(r) ranging from 2 to 4 nm, it is considered to be sufficiently adequate for the P(r) of this study. The rationale is given below. Previously, a model study about the dependence of data quality on maximum evolution time was carefully studied.32 The studied P(r) has some similarity, in terms of distribution width and the maximum distance (∼4 nm), to the P(r) results in the present study. It was shown that the important P(r) distance information (e.g., the P(r) peak positions, distribution widths, and average distances) is changed slightly, and the changes are reasonably acceptable, as the optimum evolution time (1.75 μs) is decreased to 1 μs or increased to 2.5 μs. In terms of root-mean-square deviation (rmsd) values shown in the model study, the result for the evolution time of 1.2 μs is as good as the result for the optimum evolution time of 1.75 μs, and is superior to the results for 1 and 2.5 μs. Thus, it is legitimate to say that the evolution time of 1.2 μs in this study is sufficient enough for obtaining reasonable P(r) distances and distributions for the PP-3R1 peptide.

■

CONCLUSIONS

■

ASSOCIATED CONTENT

Article

AUTHOR INFORMATION

Corresponding Author

*Phone: +886-3-571-5131, ext. 33345. E-mail: ywchiang@mx. nthu.edu.tw. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by Taiwan NSC grant, NSC1002113-M-007-013-MY2. All of the CW/pulsed ESR measurements were carried out in the NSC Research Instrument Center of Taiwan located at NTHU.

■

REFERENCES

(1) Mirkovic, T.; Zacharia, N. S.; Scholes, G. D.; Ozin, G. A. Small 2010, 6, 159−167. (2) Gimenez-Lopez, M. D.; Moro, F.; La Torre, A.; Gomez-Garcia, C. J.; Brown, P. D.; van Slageren, J.; Khlobystov, A. N. Nat. Commun. 2011, 2, 407. (3) Gardeniers, H. J. G. E. Anal. Bioanal. Chem. 2009, 394, 385−397. (4) Gazit, E. Chem. Soc. Rev. 2007, 36, 1263−1269. (5) Zhou, H. X.; Rivas, G. N.; Minton, A. P. Annu. Rev. Biophys. 2008, 37, 375−397. (6) Zhang, S. Q.; Cheung, M. S. Nano Lett. 2007, 7, 3438−3442. (7) Luan, B. Q.; Martyna, G.; Stolovitzky, G. Biophys. J. 2011, 101, 2214−2222. (8) Lee, C. H.; Lin, T. S.; Mou, C. Y. Nano Today 2009, 4, 165−179. (9) Mittal, J.; Best, R. B. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 20233−20238. (10) De Feyter, S. Nat. Nanotechnol. 2011, 6, 610−611. (11) Borbat, P. P.; Costa-Filho, A. J.; Earle, K. A.; Moscicki, J. K.; Freed, J. H. Science 2001, 291, 266−269. (12) Huang, Y. W.; Lai, Y. C.; Tsai, C. J.; Chiang, Y. W. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 14145−14150. (13) Sung, T. C.; Chiang, Y. W. Phys. Chem. Chem. Phys. 2010, 12, 13117−13125. (14) Gulla, S. V.; Sharma, G.; Borbat, P.; Freed, J. H.; Ghimire, H.; Benedikt, M. R.; Holt, N. L.; Lorigan, G. A.; Rege, K.; Mavroidis, C.; Budil, D. E. J. Am. Chem. Soc. 2009, 131, 5374−5375. (15) Freed, J. H. Annu. Rev. Phys. Chem. 2000, 51, 655−689. (16) Pannier, M.; Veit, S.; Godt, A.; Jeschke, G.; Spiess, H. W. J. Magn. Reson. 2000, 142, 331−340. (17) Borbat, P. P.; Freed, J. H. Methods Enzymol. 2007, 423, 52−116. (18) Schneider, D. J.; Freed, J. H. In Spin Labeling: Theory and Application; Beerliner, L. J., Reuben, J., Eds.; Plenum: New York, 1989; Vol. 8, p 1−76. (19) Yang, C. M.; Zibrowius, B.; Schmidt, W.; Schuth, F. Chem. Mater. 2004, 16, 2918−2925. (20) Huang, Y. W.; Chiang, Y. W. Phys. Chem. Chem. Phys. 2011, 13, 17521−17531. (21) Budil, D. E.; Lee, S.; Saxena, S.; Freed, J. H. J. Magn. Reson., Ser. A 1996, 120, 155−189. (22) Stoll, S.; Schweiger, A. J. Magn. Reson. 2006, 178, 42−55. (23) Milischuk, A. A.; Ladanyi, B. M. J. Chem. Phys. 2011, 135, 174709. (24) Chiang, Y. W.; Borbat, P. P.; Freed, J. H. J. Magn. Reson. 2005, 172, 279−295. (25) Bode, B. E.; Margraf, D.; Plackmeyer, J.; Durner, G.; Prisner, T. F.; Schiemann, O. J. Am. Chem. Soc. 2007, 129, 6736−6745. (26) Stapley, B. J.; Creamer, T. P. Protein Sci. 1999, 8, 587−595. (27) Cowan, P. M.; McGavin, S. Nature 1955, 176, 501−503. (28) Schuler, B.; Lipman, E. A.; Steinbach, P. J.; Kumke, M.; Eaton, W. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2754−2759. (29) Yuan, H. F.; Xia, T.; Schuler, B.; Orrit, M. Phys. Chem. Chem. Phys. 2011, 13, 1762−1769. (30) Bonora, M.; Becker, J.; Saxena, S. J. Magn. Reson. 2004, 170, 278−283.

The use of multifrequency ESR spectra, which provide very good orientational resolution and sensitivity to the molecularscale local environment, has made possible an unambiguous determination of anisotropic nanoconfinement effects sensed by the nitroxide-labeled helix-shaped polypeptide as confined in nanochannels. Pulsed ESR study shows that the structural conformation of the peptide in the studied nanochannels remains approximately unchanged as compared with that in the bulk solvent. The studies of the CW-ESR experiments and theoretical simulations demonstrate that ESR spectroscopy has sufficient sensitivity to detect the local environment, providing quantitative characterizations for the rotational anisotropy and local ordering/dynamics of the peptides concerning temperature and nanochannel pore size. The virtue of the ESR approaches is demonstrated to usefully provide crucial information regarding the coupling of the molecular-scale units, as confined deeply inside nanostructures of materials, to the macroscopic world, offering an excellent solution to connecting the confined unit to the outside world. This approach shall greatly enhance the nanodevice technologies for control, design, and detection purposes.

S Supporting Information *

Additional DEER measurements of the doubly labeled PP-2R1 and the singly labeled PP-m2 peptides are reported in Figure S1. The results show strong evidence for the determined structure of the triply labeled PP-3R1 peptides and demonstrate that the peptides are dispersed homogeneously in the nanochannels. This material is available free of charge via the Internet at http://pubs.acs.org. 19805

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806

The Journal of Physical Chemistry C

Article

(31) Chiang, Y. W.; Zheng, T. Y.; Kao, C. J.; Horng, J. C. Biophys. J. 2009, 97, 930−936. (32) Jeschke, G. Struct. Bonding (Berlin, Ger.) 2012, 1−38.

19806

dx.doi.org/10.1021/jp306478c | J. Phys. Chem. C 2012, 116, 19798−19806