Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native

Article pubs.acs.org/JPCB

Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations Heleen Meuzelaar,† Kristen A. Marino,† Adriana Huerta-Viga,† Matthijs R. Panman,† Linde E. J. Smeenk,† Albert J. Kettelarij,† Jan H. van Maarseveen,† Peter Timmerman,†,‡ Peter G. Bolhuis,† and Sander Woutersen*,† †

Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands Pepscan Therapeutics B.V., Zuidersluisweg 2, 8243 RC Lelystad, The Netherlands

‡

S Supporting Information *

ABSTRACT: Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage’s exact folding mechanism is still a matter of debate. Here we investigate Trp-cage’s relaxation dynamics in the amide I′ spectral region (1530− 1700 cm−1) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label (13C18O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding−unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I′ region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time- and frequency-resolved data. We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined α-helix precedes the rapid formation of the hydrophobic cage structure, implying a native-like folding intermediate, that mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone. We find that the main free-energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the α-helix, the 310-helix, and the Asp9− Arg16 salt bridge. Our results suggest that at low temperature (T ≪ Tm) a folding path via formation of α-helical contacts followed by hydrophobic clustering becomes more important.

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INTRODUCTION Understanding the mechanism by which proteins fold into their specific three-dimensional structure is a central issue in molecular biology and remains a largely unsolved problem.1,2 Moderately large proteins (>70 residues) are commonly believed to follow a hierarchal folding mechanism that proceeds through the occurrence of one or more intermediate states, represented by free-energy minima along the reaction coordinate.2−5 It has been postulated that the formation of globule-like folding intermediates facilitates the fast folding process as they restrict the conformational search of the protein, thereby directing it toward a well-defined folding pathway.6 However, since the discovery of several two-state folding proteins,7−12 of which the free-energy minima along the folding pathway are only occupied by the unfolded and folded state, the role of folding intermediates is under considerable discussion.1,6,13−19 Particularly, the existence of folding © XXXX American Chemical Society

intermediates involved in the folding process of small-sized, structurally simple proteins is under debate. Probably one of the best known examples of small-sized folding problems is the 20-residue “miniprotein” Trp-cage (NLYIQ WLKDG GPSSG RPPPS) designed by Neidigh et al.20 In spite of its short sequence and compact structure, Trpcage folds rapidly and spontaneously into a globular structure with well-defined secondary and tertiary structure elements. It folds on the microsecond time scale21 to a native state with an N-terminal α-helix, followed by a short 310-helix, a C-terminal polyproline II region, and a hydrophobic core in which the aromatic side chains of Tyr3 and Trp6 pack against residues Gly11, Pro12, Pro18, and Pro19 (Figure 1).20 Enhanced fold Received: May 13, 2013 Revised: August 23, 2013

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dx.doi.org/10.1021/jp404714c | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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tigations.37,39,40,43 Subsequently, it was proposed that, beside a predominant pathway involving the early formation of hydrophobic contacts with less helical content, a parallel, coexisting route exists in which α-helical contacts are formed prior to hydrophobic clustering.40,43,50 Here, we report a systematic investigation of Trp-cage’s (TC9b) folding mechanism using time-resolved infrared (IR) spectroscopy following a laser-induced temperature jump (Tjump). Time and structural resolution is obtained in the amide I′ spectral region (1530−1700 cm−1) which is very sensitive to conformational changes.51 Four key frequencies providing structural specificity are studied in particular, viz., the carbonyl stretch of 13C18O labeled Gly11 (1554 cm−1), the side-chain carboxylic group of Asp9 residues (1575 cm−1), the imide groups of the polyproline (II) helix (1624 cm−1), and the high frequency amide I′ mode (1664 cm−1) related mainly to the amount of α-helical content. Our T-jump IR-probe study at a final T-jump temperature close to Trp-cage’s melting temperature reveals relaxation kinetics that can be well described by a biexponential function, and involves fast fluctuations in polyproline II helical structure. The observed relaxation time constants derived from a global biexponential fit are 100 ± 10 and 770 ± 40 ns at 322 K. Accordingly, our findings do not support that Trp-cage’s folding pathway can be represented by a simple two-state transition. In contrast, SVD and global fitting analysis provide evidence that Trp-cage follows a hierarchical folding mechanism and proceeds through the formation of an effective intermediate state, the nature of which can be characterized from a combined analysis of the time- and frequency-resolved T-jump data, and molecular dynamics (MD) simulations.

stabilization of Trp-cage’s native state is provided through the formation of a salt bridge between residues Asp9 and Arg16.

Figure 1. Stick (a) and ribbon (b) representation of Trp-cage’s (TC9b) folded structure (PDB entry: 1L2Y) showing the α-helix (blue), the 310-helix (green), the polyproline II region (red), the hydrophobic side chains, and the salt-bridging side chains of Asp9 and Arg16. An isotopic label (13C18O) was incorporated into the amide carbonyl group of residue Gly11 (yellow). Pictures rendered with Chimera.52

Although Trp-cage’s folding mechanism has been extensively investigated both experimentally20−35 and theoretically,36−48 controversies regarding the existence of intermediate states along its folding pathway remain. Circular dichroism (CD) spectroscopy, differential scanning calorimetry, and nuclear magnetic resonance (NMR) spectroscopy indicated that Trpcage’s folding mechanism can be represented by a simple twostate model.20,26,49 The absence of folding intermediates was also suggested by the results of two different temperature-jump studies that showed single-exponential relaxation kinetics monitored using tryptophan fluorescence spectroscopy21 and amide I′ infrared spectroscopy.30 On the other hand, fluorescence correlation spectroscopy revealed hierarchical folding through the early formation of a compact globule-like intermediate state.23 This finding was supported by UV-resonance Raman spectroscopy measurements that show a compact folding intermediate with a partially unfolded α-helix, in which the polyproline and/or 310-helix is in closer proximity to Trp6, indicating reduced distances between Gly11, Pro12, and Trp6.22 Simulated vibrational circular dichroism and two-dimensional infrared spectroscopy indicated a similar folding pathway.48 Additionally, temperature-jump relaxation data probed at three different frequencies in the amide I′ region revealed bimodal relaxation kinetics at temperatures below 293 K, demonstrating the existence of a low-temperature folding intermediate.31 The authors proposed that local unfolding of the 310-helix occurs at lower temperatures ( 0.25 nm and rmsdαhx > 0.2 nm. A local free-energy minimum representing an intermediate state is located at rmsdαhx ≈ 0.02 nm and at rmsdPro ≈ 0.20 nm, with the main free-energy barrier positioned between the folding intermediate and the unfolded state ensemble. Rapid fluctuations (indicated by the red arrow) can occur between the minima in the folded free-energy basin (approximately at rmsdαhx < 0.08 nm and at rmsdPro < 0.40 nm), while the main free-energy barrier between the intermediate and the unfolded state ensemble corresponds to a relatively slow component of the folding process. This indicates that a possible folding pathway proceeds through a G



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310-helix (Gly11, 1554 cm−1), and the Asp9−Arg16 salt bridge (COO−, 1575 cm−1) participate can be described by a single slow phase (Figure 6). The REMD results (Figure 8) indicate the presence of a native-like intermediate with a fully formed αhelix but that differs mainly in the geometrical orientation of the (intact but detached) polyproline II helix with respect to the rest of the peptide. The main free-energy barrier is positioned between the folding intermediate and unfolded state ensemble. Possibly, the folding intermediate contains a welldefined α-helix but lacks a completely formed hydrophobic cage structure due to the larger average distance of the polyproline II helix relative to the aromatic residues Tyr3 and Trp6. These findings are consistent with the simulation results published recently by Lai et al.,48 who reported that the αhelical structure is formed prior to the formation of tertiary contacts (the coupling between the N-terminal α-helix and the C-terminus). On the other hand, our T-jump results show that at low temperature (T ≪ Tm) the rapid fluctuations involve both the polyproline II and the α- and 310-helical frequency region (Figure 7c). The REMD results suggest that this is due to an additional local free-energy minimum (Figure 8c), in which the α-helix and the orientation of the polyproline II helix are less well-defined, and in which residue Pro12 is detached from the hydrophobic cage structure, thereby disrupting the 310-helix (see structure Pro12d, Figure 8e). Indeed, the formation of such a low-temperature folding intermediate has been reported previously.31,72

that the majority of the paths follow the route via the experimentally observed native-like intermediate. k-Medoid clustering analysis shows that the intermediate state at rmsdαhx = 0.02 nm and rmsdPro = 0.20 nm found in Figure 8d is a native-like structure, in which the C-terminal polyproline II helix is not completely packed against the Nterminal α-helix, thereby partially disrupting the native hydrophobic core cluster (Figure 8f). The different orientations of the Pro rings are clearly visible when comparing both views of the folded and intermediate conformations. This is supported by other free-energy landscapes calculated for Trpcage (Figure S8, Supporting Information), which show that the polyproline II helix stays folded (small values of rmsdPro fit) regardless of the overall structure of the protein. Therefore, the fast kinetic phase observed in our T-jump relaxation data at frequencies where polyproline II helix absorption change occurs likely involves rapid environmental rearrangements of the (conformationally intact) polyproline II helix. These fluctuations of the intact polyproline II helical structure will increase its hydration, a process which is known to result in an IR intensity loss of the proline peak,56 in agreement with the observed decay-associated spectrum (Figure 7c). In Figure 8c and d, we compare Trp-cage’s free-energy landscapes calculated at 320 and 281 K, respectively. We note that it is often difficult to extract reliable free-energy barriers from projection onto arbitrary order parameters.47 Nevertheless, it is clear that there is a qualitative difference between the free-energy landscapes at the two temperatures. The relative free-energy barrier between the folded and unfolded state ensemble is 2kBT higher in energy at 281 K compared to 320 K. Both energy landscapes exhibit a local free-energy minimum positioned at rmsdαhx = 0.02 nm and at rmsdPro = 0.20 nm, representing a native-like intermediate conformation. The relative free-energy difference between this folding intermediate and the native state at low temperature (281 K) is similar to that close to the melting temperature (320 K). The accessible conformational space in the folded energy basin at low temperature ranges from approximately rmsdαhx = 0 to 0.10 nm and rmsdPro = 0 to 0.30 nm. An additional, relatively shallow, local free-energy minimum positioned at rmsdαhx = 0.09 and at rmsdPro = 0.20 is found at low temperature (Figure 8a and c), in which both the helical structure elements and the orientation of the polyproline II helix are less well-defined. The corresponding conformation (structure Pro12d, see Figure 8e) lacks well-defined 310-helical structure elements and shows that apart from the polyproline II helix (Pro17, 18, and 19) residue Pro12 is also detached from the hydrophobic core.40,47 Indeed, our T-jump relaxation data suggest that re-equilibration at low temperature (T ≪ Tm) involves fast fluctuations in not only the polyproline II region but also the remainder of the backbone (α- and/or 310-helical region, see Figure 7c). This is in agreement with the T-jump results reported by Culik et al.,31 who found evidence for the occurrence of a low-temperature folding intermediate lacking a fully formed 310-helix. Taken together, a combined analysis of our T-jump and REMD results provides evidence that Trp-cage’s folding pathway proceeds through the formation of a native-like intermediate conformation. The T-jump relaxation dynamics monitored at different structure-sensitive modes close to the transition temperature can be well-described by a biexponential function, and involves fast fluctuations of the polyproline II region (1624 cm−1) (Figure 5). The relaxation kinetics observed at frequencies where the α-helix (1664 cm−1), the

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CONCLUSION Our T-jump IR-probe study shows bimodal relaxation dynamics, providing kinetic evidence that Trp-cage’s folding process cannot be represented by a simple two-state transition and that the folding proceeds through the formation of an intermediate state. The nature of the folding intermediate was characterized on the basis of the time- and frequency-resolved T-jump relaxation data combined with the results of REMD simulations. We find that the re-equilibration dynamics at temperatures close to the transition midpoint involve rapid fluctuations in the polyproline II content, whereas the conformational rearrangement involving the α-helix, the formation of the salt bridge between Asp9 and Arg16, and the secondary structure elements involving residue Gly11 (310helix) are represented by a single slow kinetic phase. Our REMD data suggest that formation of α-helical structure precedes the formation of the tertiary structure: the correct folding of the polyproline II helix to complete the hydrophobic core. The folding intermediate contains a well-defined α-helix but lacks a completely formed hydrophobic cage structure due to a larger average distance of the polyproline II helix relative to the aromatic residues Tyr3 and Trp6. However, our T-jump results show that, at low temperature (T≪ Tm), rapid fluctuations occur due to rearrangements not only of the polyproline II region but also of the other parts of the miniprotein. The REMD results indicate that this is due to an additional local free-energy minimum, in which the α-helix and the hydrophobic cage structure are less well-defined. This suggest the existence of an additional, low-temperature folding intermediate, in agreement with previous observations.31,72 In the present experiments, we have used conventional (onedimensional) time-resolved vibrational spectroscopy to investigate the folding dynamics. A recent theoretical study48 has shown that using time-resolved two-dimensional vibrational spectroscopy can provide more detailed information about the H



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after the T-jump by mid-infrared (mid-IR) probe pulses, using the optical setup described previously.74 In brief, an optical parametric amplifier (OPA) based on BBO is pumped by the 800 nm output of a commercial Ti:sapphire laser (SpectraPhysics Hurricane, 1 mJ, 100 fs fwhm). Subsequent differencefrequency generation of signal and idler in AgGaS2 results in mid-IR pulses with a duration of ∼150 fs at 1620 cm−1, a bandwidth of 200 cm−1 (fwhm), and an energy of 1 μJ. Probe and reference pulses are obtained by means of a 50/50 beamsplitter, and are focused through the sample using a 100 mm off-axis parabolic mirror. Transient absorption changes are measured using frequency-dispersed detection of the probe and reference pulses using a liquid-nitrogen-cooled 2 × 32 HgCdTe (MCT) array detector (Infrared Associates). The reference pulse is focused through the sample in an area that is not influenced by the T-jump pulse to correct for small pulse-topulse fluctuations in the intensity of the IR-probe pulses. Spatial overlap between the probe and T-jump pulse is found by optimizing the transmission through a 250 m pinhole (focal diameters of 250 and 500 m for probe and T-jump pulse, respectively, as determined from the transmission through the pinhole) and is optimized by maximizing the transmission of the probe pulse through D2O. The polarization of the T-jump and mid-IR pulse are perpendicular with respect to each other. Synchronization between the two laser outputs is based on the electronic configuration described elsewhere:74 a fast photodiode detects the residual output from the Ti:sapphire oscillator (80 MHz). The signal from the photodiode is amplified and divided to produce a 1 kHz signal. This signal triggers a pulse generator, which provides the timing for (1) the YLF pump laser and Pockels-cell driver of the regenerative amplifier, (2) the electronic gated amplifier used to detect the signals of the MCT array, and (3) a computer-controlled electronic delay generator (Berkeley Nucleonics Corporation Model 575-4C). The latter provides triggering for the Nd:YAG laser. The delay time τ = 0 between T-jump pulse and mid-IR probe pulse is set at the point where the transmission increase in D2O is half of its maximum. To obtain transient IR spectra, we determine absorption changes ΔA = A − A0 = log(T/T0), where T and T0 are the transmission in the presence and absence of the T-jump pulse, respectively, as described previously.74 Each delay value is the average of approximately 1000 T-jumps. Care is taken to ensure that the sample has relaxed back to its initial temperature before the subsequent T-jump. The initial temperature of the sample was controlled using a thermostatted cell-holder with a circulating heat bath and was calibrated with an IR camera (FLIR ThermaCAM E2). Reference samples containing D2O were measured under identical conditions to calibrate the size of the T-jump and to correct for contributions of the solvent to the transient signals of Trp-cage. The frequency-dispersed detection allows measurements in a spectral window of approximately 100 cm−1, and two consecutive measurements with different mid-IR center frequencies are required to detect the transient absorption changes in the amide I′ spectral region between 1530 and 1700 cm−1 and to construct the transient spectra, as shown in Figures 3 and 4. From comparison of data sets with overlapping frequency ranges, we find that no scaling of the transient signals measured in different spectral windows is necessary. Molecular Dynamics Simulations. Although Trp-cage is a very small protein, the accessible time scale of standard MD simulations (microseconds) is too short to allow for many

Trp-cage folding pathway. We are currently working on experimentally implementing such experiments.

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EXPERIMENTAL SECTION Peptide Synthesis and Purification. A mutant of the Trp-cage miniprotein (TC9b) consisting of the sequence NAYAQ WLKDG GPSSG RPPPS is used in all experiments.30 The Trp-cage miniprotein was purchased from GL Biochem (Shanghai). Peptide purity (≥95%) was assessed by reversedphase HPLC, and the identity of the purified product was confirmed by mass spectrometry. An isotopic label (13C18O) was incorporated into the amide carbonyl group of the peptide backbone of residue Gly11. For introduction of the 13C18O isotopic label, 18O/16O exchange was carried out starting from commercially acquired 13C(1)-labeled glycine (99% 13C, SigmaAldrich) according to a previously reported method73 (progress of the 18O/16O exchange was monitored via 13C NMR of the C(1) signal, see Figure SI9, Supporting Information). After Fmoc protection, the thus obtained 13C18O isotopic labeled glycine was used to prepare the labeled Trp-cage NAYAQ WLKDG G*PSSG RPPPS using standard Fmoc solid phase peptide chemistry on a Protein Technologies Prelude automated synthesizer. After purification by preparative reversed-phase HPLC, the peptide was lyophilized against a 35% DCl/D2O solution to remove residual trifuoroacetic acid (TFA) and to achieve H/D exchange. Trp-cage stock solutions of 15−20 mM were prepared by directly dissolving dry peptide in D2O. The pH* of the peptide solutions was adjusted to neutral pH level (pH* = 6.9) by addition of NaOD solution (pH* = uncorrected pH meter reading in D2O). Equilibrium Temperature-Dependent Measurements. UV circular dichroism (CD) spectra were measured using an Olis DSM-1000 CD spectrophotometer equipped with a Julabo (CF31) temperature controller. The peptide concentration used for the CD measurements was 40 M in a 20 mM phosphate buffer (pH = 6.8) and all measurements were performed using 2 mm quartz cuvettes. The temperature dependency of the ellipticity was monitored between 257.5 and 190 nm with incremental steps of 0.75 nm and averaging over 5 s of data acquisition time (0.15 nm/min scanning speed). Singular value decomposition (SVD) and global fitting and of the temperature-dependent data set to an apparent two-state model was performed using the Olis GlobalWorks software package. Temperature-dependent equilibrium Fourier-transform infrared (FTIR) spectra were collected using a Bruker Vertex 70 spectrometer. The peptides were kept at 15−20 mM concentration between 2 mm thick CaF2 windows separated by a 50 m Teflon spacer. Temperature control was achieved by a circulating water bath. Typically, 75 scans were signal averaged at a resolution of 2 cm−1 to generate one spectrum. Time-Resolved T-Jump IR Experiments. Laser-induced T-jumps of 5 K were obtained by nanosecond excitation of the OD-stretch overtone of D2O. The T-jump pulse is generated using a beta barium borate (BBO)-based optical parametric oscillator (OPO) pumped by the second harmonic (λ = 532 nm) of a 20 Hz, Q-switched Nd:YAG laser (Quanta-Ray INDI, Spectra-Physics). The idler pulse (λ = 1.98 m, 12.5 mJ, 5 ns fwhm) is focused (f = 75 mm, CaF2) to 500 m diameter at the sample, leading to an almost spatially uniform T-jump within the pulse width of the laser. The frequency-dependent transient absorption changes induced in the sample are detected at various time delays I



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the 16O/18O exchange. This material is available free of charge via the Internet at http://pubs.acs.org.

transitions between the folded and unfolded states at room temperature. Replica exchange molecular dynamics (REMD)75 overcomes this problem by allowing transitions at higher temperatures. In REMD, a number of parallel MD simulations (replicas) are run, each with a different temperature. After a short time, exchanges are attempted between the different replicas, and exchanges are accepted according to Pacc = min[1,eΔβΔE]. E is the potential energy and β = 1/kBT, where kB is the Boltzmann constant and T is the temperature. REMD simulations of Trp-cage TC5b (NLYIQ WLKDG GPSSG RPPPS) were performed over the temperature range 270.0− 556.0 K using 64 replicas.47 Exchanges were attempted every 2 ps, and 50 000 cycles were performed. Two independent simulations were started from a folded and unfolded Trp-cage structure for an aggregate simulation time of 200 ns for each temperature. In the simulation box, Trp-cage TC5b was solvated with 2796 TIP3P waters and one Cl-atom to neutralize the charge. The MD simulations were performed with the GROMACS MD engine76 using the AMBER99SB force field,77 and a perl wrapper script was used to perform the exchanges. See ref 47 for further details. The simulation trajectories of ref 47 were analyzed further by calculating several order parameters, λ, which were then used to calculate two-dimensional free-energy landscapes from probability histograms, F(λ1, λ2) = kBT ln P(λ1, λ2). In order to improve statistics, data from the rejected exchange attempts was included in P(λ1, λ2) through virtual move parallel tempering.78 The order parameters used for this work were chosen to characterize the formation of the α-helix and the polyproline II helix. They include the root-mean-square deviation of the Cα atoms (rmsdCα) with respect to the NMR structure (PDB entry 1L2Y) and the α-helical residues (rmsdαhx) with respect to an ideal α-helix. In addition to these order parameters, which were also used in ref 47, we computed the rmsd of the polyproline II helix in two ways. First, the rmsdPro is calculated by fitting the entire protein backbone to the equilibrated NMR structure, so a large value indicates that the polyproline II helix is far from its position in the equilibrated structure and not that the polyproline II helix itself is unfolded. In order to quantify how much the helix is unfolded, the rmsd (rmsdPro fit) is also calculated by first fitting only the three Pro residues of the polyproline II helix to the equilibrated, folded Trp-cage structure. Low values of rmsdPro fit indicate the helix is formed, while higher values indicate that it is unfolded. To find representative structures of the minima in the free energy landscape, k-medoid clustering was performed on the trajectories at 281 and 320 K using the same procedure as in ref 47.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +31 (0)20 5257091. Fax: +31 (0)20 5256456. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We kindly acknowledge Prof. Dr. R. B. Dyer (Emory University) for his advice regarding the T-jump setup and Rolf Pfister (University of Zurich) for his advice about the 18Oisotope labeling. S.W. would like to acknowledge European Research Council for funding through grant 210999. This work is financially supported by the “Nederlandse Organisatie voor Wetenschappelijk Onderzoek”.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures showing unfolding in thermal equilibrium monitored by UV-CD and FTIR spectroscopy, T-jump control measurements, SVD analysis of the T-jump data obtained close to the transition midpoint, bi-exponential global fit of the Tjump data obtained close to the transition midpoint, relative amplitude of the instantaneous response amplitude, SVD analysis of the T-jump data obtained at low temperature, biexponential global fit of the T-jump data obtained at low temperature, calculated free-energy landscapes indicative of a folding intermediate containing an intact but detached polyproline II helix, and NMR spectra at different stages of J



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