HRMAS 1H NMR Conformational Study of the Resin

Feb 15, 2010 - Ow This paper contains enhanced objects available on the Internet at http://pubs.acs.org/JPCA. The conversion of soluble proteins to in...
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J. Phys. Chem. A 2010, 114, 3457–3465

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HRMAS 1H NMR Conformational Study of the Resin-Bound Amyloid-Forming Peptide GNNQQNY from the Yeast Prion Sup35 Samuel B. Andrey, Michael L. Chan, and William P. Power* Department of Chemistry, UniVersity of Waterloo, Waterloo, Ontario, Canada N2L 3G1 ReceiVed: October 15, 2009; ReVised Manuscript ReceiVed: January 19, 2010 嘷 w This paper contains enhanced objects available on the Internet at http://pubs.acs.org/JPCA.

The conversion of soluble proteins to insoluble amyloid fibrils is associated with numerous human diseases. The peptide GNNQQNY is a short segment of the yeast prion protein Sup35 that previously has been found to form amyloid fibrils in a similar manner to the protein itself. The approach taken in this work was to attach this peptide sequence to an insoluble polymer matrix through solid phase peptide synthesis and give it the internal freedom to fold into its local conformation in an organic solvent. Observation of its monomeric structure, free from the effects of aggregation, entropic solvent effects, and neighboring molecules, was possible by two-dimensional high-resolution magic angle spinning 1H NMR spectroscopy. Analysis of the throughbond correlations and through-space interactions observed in the spectra, combined with global energy minimization via computational studies, led to the observation that the peptide chain adopts a compact β-like turn at the central hydrophilic residues. The technique of peptide attachment to a polymer resin and observation by NMR may allow for future study of single peptide fragments prone to aggregation. Introduction The inability of a protein to adopt or remain in its native conformation and the subsequent formation of insoluble protein aggregates, known as amyloid fibrils, are associated with 40 known human diseases, including Alzheimer’s disease, type II diabetes, Parkinson’s disease, and transmissible spongiform encephalopathies.1 Despite the fact that each of the aggregating proteins associated with amyloidosis diseases have unrelated primary sequences and distinct native structures, it has been established by X-ray diffraction that a structural “cross-β spine” is common among amyloid fibrils, in which β-sheets align perpendicular to the fibril axis.2 A more detailed structural characterization has remained elusive until recently due to the difficulty in preparing samples via crystallization or dissolution for analysis by the conventional methods of structural determination, namely X-ray crystallography and solution-state NMR.1 While the recent use of solid-state NMR has resulted in significant insights into amyloid fibril conformations, the use of shorter peptide fragments from known amyloidogenic proteins has allowed for the development of model systems for research into the details of amyloid fibril structure and aggregation mechanics.3,4 One of the most studied5-21 amyloid model systems is derived from the yeast protein Sup35, which is natively a translation termination factor, but has been shown to undergo fibril formation and consequently gain prionlike transmission.22-24 The prion-forming domain of the 685-residue protein has been traced to the amino terminal 123 amino acids,25 and a seven-residue segment within this domain (residues 7-13) is independently capable of fibril formation in Vitro.3 This aggregating segment, GNNQQNY, displays the amyloidic properties of the entire Sup35 protein including cooperative kinetics of aggregation, binding of the dye Congo red and thioflavin T, and the cross-β * To whom correspondence should be addressed. E-mail: wppower@ uwaterloo.ca.

spine diffraction pattern.3,8 X-ray diffraction of GNNQQNY microcrystals reveals a double β-sheet atomic structure in which each sheet is formed from parallel peptide chains held together by 11 stacks of backbone-backbone and side chain-side chain hydrogen bonds between identical residues on neighboring chains and the favorable π-π interactions between stacked tyrosine side chains.8,11 The tightly packed glutamine and asparagine side chains of the two sheets form a dry “steric zipper” in which van der Waals interactions, not hydrogen bonds, keep an unusually tight 8.5 Å interface together.8 The opposing 15 Å wet interface, which is considerably larger, is hydrated by water molecules, and has only one contact between neighboring sheets by means of the tyrosine residues.8 Solidstate 13C and 15N MAS NMR studies of this peptide under a variety of crystallization conditions have characterized the degree of conformational variability and polymorphism available to this short peptide sequence in crystalline and fibril forms.14 The finding that the conformational stability of fibril aggregation can rely solely on steric interactions between a short peptide fragment rather than high sequence specificity and hydrogen bonding is consistent with the structural polymorphism that has been seen by this peptide and with the common cross-β spine structure exhibited by unrelated aggregating proteins.5,8,12 While much progress has been made in understanding amyloid fibril structure and stability in its aggregated crystalline formation, less is understood about the structure of the monomeric and oligomeric species that initially form to propagate fibril formation; even less is known about the breakdown in cellular machinery that triggers the formation of these aggregated states or the pathogenicity that is derived from their formation.1 There is growing evidence to suggest that it is early oligomeric peptides that are the cytotoxic species, rather than the fibrils themselves.26-30 This information, combined with the belief that the standard free energy of oligomeric formation is not strongly negative,8 leads to the assumption that translation regulation or chaperone proteins that isolate single proteins may be of critical

10.1021/jp909899w  2010 American Chemical Society Published on Web 02/15/2010

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Figure 1. Skeletal structure of the Fmoc-protected GNNQQNY peptide sequence. By linking the chain to a polymer matrix resin, the local monomeric conformation of the peptide can be observed by HRMAS NMR spectroscopy without the interference of neighboring peptide chains or aggregation present in solution state.

importance in the prevention of amyloid aggregation. The use of molecular dynamics simulations of GNNQQNY aggregation has led to possible insights into the formation of dimers and oligomers in the early steps of fibril formation;13,15,16,18-20 however, the ability to isolate and conduct conformational studies on single noninteracting GNNQQNY monomers free in solution remains a fundamental challenge. This study utilizes Merrifield’s solid phase peptide synthesis technique (SPPS) to covalently bind the carboxyl end of the GNNQQNY peptide fragment to an insoluble resin (displayed in Figure 1), followed by conformational analysis of the resinbound peptide by 1H NMR spectroscopy. Gel-phase samples such as these have restricted internal mobility, compared to those studied by solution-state NMR, disrupting the averaging of nuclear spin direct dipolar coupling.31 Additionally, a variation in the magnetic field results from a difference in the magnetic susceptibility of the resin and the solvent. These two occurrences result in significant line broadening; however, both can be overcome by the use of high-resolution magic angle spinning (HRMAS) NMR spectroscopy.32-34 Rapid spinning of the sample at an angle of 54.7° with respect to the magnetic field averages the (3 cos2 θ - 1) orientation dependence of dipolar coupling and susceptibility differences to zero, allowing for a reduction in line broadening and for the conformation to be deduced through a series of two-dimensional 1H-1H throughbond (COSY and TOCSY) and through-space (ROESY) experiments.35 The technique of binding the peptide to a resin confers the advantage of being able to study the structural conformation of the single monomeric peptide fragment, demonstrated previously by Dhalluin et al.36 and Rousselot-Pailley et al.37 for other small peptides. Here, we focus on the peptide GNNQQNY. Anchoring the terminal tyrosine residue to the resin and removing the terminal charges also more closely replicates the environment in which this peptide fragment exists within Sup35, namely six residues away from the amino terminus of the 685 amino acid protein. Furthermore, by studying this peptide in an organic solvent, the formation of secondary structures is driven by enthalpy, rather than entropic solvent forces, with hydrogen bonding, van der Waals forces, dipole-dipole, and quadrupolar interactions as the primary contributors to structure.38 The result is a three-dimensional model of the resinbound peptide without interference from aggregation, solvent effects, or neighboring molecules.

Experimental Methods Sample Preparation. Solid phase peptide synthesis was used to generate the resin-bound GNNQQNY sequence. The amino acids used contained a 9-fluoromethoxycarbonyl (Fmoc) group for protection and quantification of coupling success. Coupling of the first amino acid Fmoc-Tyr(t-Bu) to Wang resin (0.6-1.0 mmol/g functionality, 200-400 mesh, 1% divinylbenzene crosslinker) was performed in dichloromethane (DCM) with the 2,6dichlorobenzoyl chloride (DCBC) method using 3.3 mol equiv of both DCBC and pyridine (22 h, rt); the tert-butyl group on tyrosine acted as a protecting group to prevent the terminal hydroxyl group from undergoing side reactions during the synthesis. Uncoupled sites on the resin were capped using 5.0 mol equiv of both acetic anhydride and N,N-diisopropylethylamine (DIPEA) (15 min, rt) to prevent incorrect chain synthesis in successive coupling reactions. Fmoc deprotection was achieved by addition of piperidine (20%) in N,N-dimethylformamide (DMF) (20 min, rt). The six successive coupling reactions were carried out using the 2-(1H-Benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling reagent with an N-hydroxybenzotriazole (HOBt) coupling additive (minimum of 15 h, rt). The solvent used was dependent upon the amino acid being coupled; Fmoc-Asn-OH, Fmoc-GlnOH, and Fmoc-Gly-OH were coupled to the resin-bound peptide in DMF, DMSO, and DCM, respectively. The residues N2, N3, Q4, and Q5 each required a second coupling reaction to achieve sufficient substitution between 85% and 95%. Measurement of substitution level at each coupling step was performed by deprotection of a 2 mg sample with piperidine (20%) in DMF (20 min, rt) and subsequent quantification by UV absorbance of the dibenzofulvene-piperidine adduct (λ ) 301 nm, ε ) 7800 M-1 cm-1); this method was also used to confirm successful deprotection of the sample at each step by a zero absorbance detection. The final substitution level of the resinbound heptapeptide, after thorough washing and drying, was measured to be 0.285 mmol/g. Due to the nature in which the amino acids are coupled sequentially from the carboxyl terminus to the amino terminus, 56% of the peptide chains on the resin contain the full-length sequence of seven residues. The remaining peptide chains are approximately 2%, 4%, 11%, 10%, 13%, and 4% of the six, five, four, three, two, and one residue chains, respectively. Electrospray ionization mass spectroscopy was used to verify the mass of the peptide sequence after cleavage from a 2 mg sample of the resin using trifluoroacetic acid (TFA)

1H

NMR Study of Peptide GNNQQNY

in DCM (1:1, 30 min, rt). The Fmoc protecting group was removed from an isolated portion of the resin to test for its effects; removal was accomplished by shaking with 20% piperidine in DMF and subsequent cleavage check by UV spectroscopy. Samples were prepared for NMR by filling half the volume of a Doty Scientific 4 mm XC4 thin wall Kel-F sealing cell with the resin-bound peptide (6-8 mg). The cell was filled with DMF-d7 (D, 99.5%, 0.05% v/v TMS) to swell the resin, and the cell was subsequently sealed with an XC4 Teflon plug cap. NMR Equipment. All NMR spectra were obtained on a Bruker Avance 500 MHz NMR spectrometer (11.75 T superconducting magnet, 1H Larmor frequency of 500.13 MHz) equipped with a Doty Scientific triple-tuned (1H/X/2H-lock) DSIXC4 HRMAS probe at 295 K; all spectra were collected under field-frequency locked conditions and referenced internally to the residual 1H signal of tetramethylsilane (TMS). A Doty Scientific XC4 4-mm silicon nitride thin wall rotor was loaded with the sample cell and capped with Torlon short turbine caps. The rotor was spun at the magic angle between 4.3 and 4.6 kHz. Bruker XWIN-NMR 3.5 pl6 software was used to process all spectra. 1D 1H NMR Spectroscopy. The 1D spectrum was collected with the DIPSI-2 pulse program available in XWIN-NMR using the technique of polymer signal attenuation39 with an isotropic mixing time of 30 ms. The spectrum was collected with 1024 transients, each with an acquisition time of 1.24 s and a spectral width of 6613 Hz. A recycle delay of 2.00 s was used. The 90° 1 H pulse lengths of 10.12 and 35.00 µs were applied at high and low power level, respectively. The resulting 16k complex data points were zero-filled to 32k data points for Fourier transform and apodized using the exponential windowing function with a Lorentzian broadening constant of 0.30 Hz. 2D 1H NMR Spectroscopy. In the COSY experiment, a set of 2048 × 256 data points, acquired in the t1 and t2 dimensions, respectively, with 64 scans per increment, were accumulated with a spectral width of 5165 Hz in both dimensions. A 90° 1H pulse length of 10.12 µs and acquisition time of 0.20 s were followed by a 2.00 s relaxation delay. The TOCSY experiment, which used the above DIPSI-2 sequence for mixing, collected a set of 4096 × 512 data points with 32 scans per increment and a spectral width of 6127 Hz in both dimensions. A mixing time of 35 ms and acquisition time of 0.33 s were utilized. After Fourier transformation, both COSY and TOCSY data sets underwent apodization using the sine windowing function with a Lorentzian broadening constant of 0.30 and 1.00 Hz in the t1 and t2 dimensions, respectively. The ROESY experiments resulted in sets of 2048 × 156 data points with 200 scans per increment and a spectral width of 6127 Hz in both dimensions. The 90° 1H pulse length of 10.12 µs was followed by a delay for power switching of 20 µs. A 2.00 s relaxation delay was used. Three ROESY experiments were run with the following pulse lengths for spinlock: 65 ms, 55 ms, and 45 ms. The resulting Fourier-transformed data set was apodized using the squared sine windowing function with a Lorentzian broadening constant of 0.30 and 2.00 Hz in the t1 and t2 dimensions, respectively. Results and Discussion Two samples of the GNNQQNY peptide were studied to determine the resin-bound conformations: one with the Fmoc protecting group from the synthetic procedure still attached to the N-terminus, and another with it removed. For clarity, the method used to determine conformation and the results for the

J. Phys. Chem. A, Vol. 114, No. 10, 2010 3459 TABLE 1: 1H NMR Chemical Shift Assignments of the Resin-Bound Fmoc-Protected GNNQQNY Peptide (δ in ppm Relative to TMS) NH Fmoc G1 N2 N3 Q4 Q5 N6 Y7 resin linker

RH

βH 3.98

7.65 8.42 8.59 8.30 8.31 8.90 8.14

3.97, 4.81 4.71 4.65, 4.33 4.77, 4.61,

γ H NH2 7.83

other 7.38 (δ), 7.32 ()

3.94a 4.52b 4.92b 4.81b

2.84 2.83 2.30 2.34 1.97 2.34 3.12, 2.71a 3.08

6.89 6.70 7.75 7.46 7.14 (phenyl), 1.27 (t-butyl)

a

5.10, 4.57

a

b

Inequivalent protons resulting in second chemical shift. Chemical shift in alternate conformation.

Fmoc-protected sample are outlined in detail initially, while the results of the Fmoc-free sample are similarly determined and presented in summary afterward. Sequence Assignment. Proton sequence assignment of the Fmoc-protected GNNQQNY peptide was successfully achieved through spectral analysis of the series of 1H-1H 2D experiments; these assignments are summarized in Table 1 and are consistent with the peaks of the 1D spectrum, which is included in the Supporting Information as Figure S1. Using the COSY and TOCSY spectra (displayed in Figure 2), seven spin systems containing an amide proton coupled to an R proton were identified, corresponding to the seven amino acids in the peptide sequence; six of these spin systems had R protons coupled to β protons, and two of these spin systems also had these R protons coupled to γ protons. The amide, R, and β protons of these spin systems were initially assigned in the COSY and TOCSY spectra on the basis of the predicted random coil chemical shift values for amino acids in solution.40 While this strategy effectively assigned the glycine (G1) and tyrosine (Y7) residues, the middle five amino acids which contained three asparagine residues (N2, N3, N6) and two glutamine residues (Q4, Q5) were spectrally congested and therefore required more thorough analysis. Differentiating the Q residues from the N residues was feasible because the glutamine’s amide, R, and β protons tend to be 0.3-0.5 ppm lower than the typical values of asparagine protons, in addition to the fact that the β and γ proton groups were resolved along the same R shift for both Q residues in the TOCSY spectrum. The remaining three spin systems were assigned to the three N residues. It was predicted that N2 and N3 would have more similarity in chemical shift than each would have with N6, given their spatial proximity, and that N2 would have a lower amide proton chemical shift than N3 given N2’s position toward the end of the peptide chain; these predictions allowed for a tentative differentiation of the N residues. This total sequence-specific assignment was subsequently verified by ROESY’s throughspace interactions. The assignment of G1 was consistent with this residue being the only spin system without a corresponding β proton in the TOCSY spectrum. Its amide-R coupling was present in all three 2D experiments, and while the chemical shift of the G1 R proton was at the expected chemical shift, the amide proton was shifted to a considerably lower frequency (7.65 ppm) compared to the expected value (8.33 ppm); however, this was unsurprising given its position at the N-terminus of the peptide chain next to the large Fmoc protecting group. G1’s R peak split into two overlapping signals at 3.97 and 3.94 ppm, suggesting that the two R protons of glycine were inequivalent.

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Figure 2. 500 MHz 1H TOCSY spectrum of the Fmoc-protected peptide. The key regions of interest, namely the amine/R proton and R/β proton interactions, are expanded, and the key through-bond correlations are labeled. This spectrum, along with the COSY spectrum, were used to identify and spectrally assign the seven spin systems corresponding to the peptide’s amino acids, along with protons from the Fmoc protecting group and resin linker.

The spin system assigned to N2 had its amide-R interaction evident in all three 2D experiments, in addition to the R-β interaction which was present in both the COSY and TOCSY spectra. The β protons appeared to be equivalent and were at the expected chemical shift. Similarly, N3 had its amide-R and R-β interactions present in both the COSY and TOCSY spectra and contained equivalent β protons. As expected, neither N2 nor N3 had the protons in their chain’s terminal NH2 group coupled sufficiently to be present in the COSY and TOCSY spectra. However, values (6.89 and 6.70 ppm) close to the expected value (6.85 ppm) of one of asparagine’s inequivalent NH2 protons appeared in through-space ROESY interactions and were assigned to N2 and N3’s NH2 groups, respectively. The amide-R and R-β interactions of Q4 were both present in COSY and TOCSY at expected shifts. The R-γ interaction in TOCSY was present; however, the β and γ protons did not resolve well and the γ proton appeared to couple to the R proton at a slightly lower frequency (4.60-4.52 ppm) than the R proton that coupled to the amide and β protons (4.65 ppm). This finding indicated a possible conformational flexibility in the Q4 chain. Q5 had its amide-R interaction present in all three 2D spectra, in addition to its R-β coupling in both COSY and TOCSY. The Q5 β and γ protons resolved more clearly than in Q4 and coupled to the same R proton. The Q5 γ protons also interacted with the chain’s NH2 protons in the ROESY spectrum, allowing assignment of the NH2 group to 7.46 ppm, which was consistent with one of glutamine’s expected NH2 shifts. Similar to N2 and N3, both Q4 and Q5 appeared to have their two β protons equivalent or unresolvable, as did their γ protons. N6 had its amide-R interaction present in all three spectra, and its R proton coupled to two inequivalent β protons at 3.12 and 2.71 ppm. The NH2 group of N6 was not evident in any of

the spectra and remains the only proton group unassigned. Past Q5, the presence of incomplete two-residue chains containing only N6 and Y7, as discussed in the Experimental Section, began to appear in the spectra, resulting in alternative chemical shifts for the R protons in N6 and Y7. These shorter chains became evident in COSY and TOCSY where one of the N6 β protons and an unknown proton in the amide region coupled to a peak at 4.92 ppm in TOCSY, which was thought could be the R proton in N6 in this truncated sequence. The amide-R and R-β interactions of Y7 appeared in both COSY and TOCSY at shifts typical of tyrosine; as in most of the other amino acids, the β protons appear to be equivalent. In addition, both the R and β protons displayed an interaction in ROESY with a peak at 7.14 pm, which was subsequently assigned to tyrosine’s phenyl ring protons. Similar to N6, Y7 appears to have a second R shift at 4.81 ppm due to the shortened chain conformation, given that peak coupled to the Y7 amide proton in both COSY and TOCSY. A strong peak at 1.27 ppm was assigned to the nine protons of the Y7 t-butyl protecting group. This group produced a cross peak in ROESY at 7.75 ppm; this shift indicated a glutamine NH2 proton and was therefore assigned to the remaining Q4 NH2 group. In addition to the seven core spin systems corresponding to the peptide’s seven amino acids, peaks corresponding to both the Fmoc protecting group and the linker chain attaching the peptide sequence to the resin were evident. The β and γ protons of Fmoc showed a strong interaction in both COSY and TOCSY. Finally, the two inequivalent protons at the R position of the resin linker coupled in COSY and TOCSY, producing a cross peak at 4.57 and 5.10 ppm. Structural Determination. Twelve major through-space interactions (NOEs) evident in the ROESY spectra (displayed

1H

NMR Study of Peptide GNNQQNY

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Figure 3. 500 MHz 1H ROESY (65 ms spin lock) spectrum of the Fmoc-protected peptide. The through-space interactions are labeled and the key inter-residue interactions are numbered to correspond to Table 2 and Figure 4. The spectrum verified the sequence assignment determined by the correlation experiments and allowed for further spectral assignment. Additionally, 12 major through-space interactions were identified, which allowed for determination of the three-dimensional conformation.

TABLE 2: Inter-Residue NOE Assignments and Distance of Interaction in Fmoc-Protected Optimized Structure from 65 ms Spin Lock ROESY Experiment 1 2 3 4 5 6 7 8 9 10 11 12

F1

F2

distance of interaction

Y7-CH3 Y7-CH3 Y7-CH3 Q5-R Q5-β N2-R N2-R G1-R Q5-R Q5-R Q5-β Y7-β

Q4-NH2 Q4-NH N2-NH2 N3-NH N3-NH2 N3-NH Y7-NH N2-NH Q4-NH2 Y7-NH Q4-NH N6-NH

4.0 Å 4.9 Å 5.4 Å 6.1 Å 3.3 Å 2.4 Å 5.4 Å 2.8 Å 6.9 Å 6.5 Å 4.5 Å 5.8 Å

in Figure 3) were unobserved in the COSY and TOCSY correlation experiments; these interactions are summarized in Table 2 and are displayed in Figure 4. Five of these NOEs (numbered 6, 8, 9, 11, and 12) were due to 1H-1H interactions among adjacent amino acids. The NOE between G1 and N2 (interaction 8), between N2 and N3 (interaction 6), and additionally between N6 and Y7 (interaction 12) helped confirm that the sequence assignment of the asparagine residues was in the correct order. While the NOE between the Q5 β proton and the Q4 amide proton (interaction 11) remained through all three ROESY experiments as spinlock times were decreased, the remaining four were not visibly present in the 55 and 45 ms experiments, indicating that these interactions may not be key polarization transfers. The cross peak at 6.70 ppm and 1.61 ppm may represent an interaction between the terminal NH2 group of N3 and a methylene proton within the resin linker; this interpretation is supported by modeling which indicated the proximity between these two groups (Vide infra). However, the assignment of 1.61 ppm to the resin linker is not verifiable by

Figure 4. Key through-space interactions of the Fmoc-protected peptide are pictorially displayed and numbered to correspond to Table 2 and Figure 3. Carbon, hydrogen, oxygen, and nitrogen atoms are displayed in gray, white, red, and blue, respectively. The resin polymer matrix is denoted in orange, and the R carbons are depicted in green. The strong interactions of Y7 with N2 and Q4 indicated the presence of a turn in the peptide chain, and interactions between N3 and Q5, in addition to Q5 and Y7, revealed important information about the shape of the peptide chain in its monomeric conformation.

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any of the other spectra and, as such, this cross peak has not been designated as a major through-space interaction. The remaining seven NOEs (numbered 1-5, 7, and 10) allowed for determination of the resin-bound peptide’s threedimensional structure. The t-butyl protecting group of Y7 interacted strongly with both the amide proton of Q4 (interaction 2) and the NH2 group of N2 (interaction 3), in addition to producing a relatively weak interaction with the NH2 group of Q4 (interaction 1). Furthermore, the amide proton of Y7 interacted with the R proton of N2 (interaction 7). This indicated strongly that the Y7 residue was in close proximity to both N2 and Q4. The fact that the peptide chain remained bound to the resin polymer and that substitution on the resin was relatively low eliminated the possibility of two or more peptide chains interacting and producing these cross peaks. Therefore, the position of Y7 on the end of the chain, three residues away from Q4 and a full five residues from N2, led to the conclusion that the peptide chain must be bending back upon itself in its isolated resin-bound conformation. This was further verified by the presence of several other cross peaks that indicated a bent conformation with the N2 and N3 residues coming back into proximity with the C-terminus of the chain. The Q5 R proton interacted with the amide proton of N3 (interaction 4), while the Q5 β proton produced a strong NOE with the NH2 group of N3 (interaction 5). Also, the amide proton of Y7 interacted with the Q5 R proton (interaction 10), although this interaction was weak and was the only long-range interaction to not appear throughout all three ROESY experiments. The lack of longrange interactions involving G1 and Fmoc pointed to the likelihood that the end of the chain furthest from the resin was not in close proximity to the remaining residues. These parameters led to an initial structural proposal with two antiparallel β chains connected by a β-turn involving Q4 and Q5. A β-turn is characterized by the close approach of two R carbons separated by two residues, which seemed consistent with the observed interactions. This initial structure was optimized using the Hartree-Fock method in Gaussian 0341 on the SHARCNET network42 to obtain a global minimum energy. This structure is displayed in Figure 5 and a 3D rotatable image is available online in a web-enabled format. The resulting bond lengths of the key interamino acid interactions are summarized in Table 2. The resulting structure has Q4 and Q5 contributing to a turn in the peptide chain, N2 and N6 coming into close proximity on either end of the turn, and the phenyl group of Y7 creating a kink in the chain and lying parallel to the Q4 side chain. The terminal end of the chain containing G1 and Fmoc extends outward toward the resin linker and is uninvolved with the nonadjacent residues, which is consistent with the lack of long-range interactions. The structure agrees well with the observed long-range interactions and proposes these interactions have distances between 2 and 7 Å, which is within the realistic distance for observation of NOEs by HRMAS NMR spectroscopy. A closer examination of the intensities of the ROESY interactions 1 and 9, which both involve the terminal NH2 group of Q4, reveals that the conformation flexibility of Q4 suggested in the TOCSY spectrum may exist. Interaction 1 between Q4 and Y7 is indicated by the optimized structure to be much closer together (4.0 Å) than interaction 9 between Q4 and Q5 (6.9 Å). However, the intensity of interaction 9 is considerably stronger than interaction 1 in the ROESY spectrum, indicating that the side chain of Q4 may in reality exist closer to Q5 than Y7 or that the side chain fluctuates in conformation somewhat given its position on the axis of the peptide turn. Although the interaction between the NH2 group of N3 and the resin linker

Andrey et al.

Figure 5. Three-dimensional structure of the Fmoc-protected GNNQQNY peptide monomer, optimized to a global minimum energy. Carbon, hydrogen, oxygen, and nitrogen atoms are displayed in gray, white, red, and blue, respectively, while the resin polymer matrix is denoted in orange. (a) To better illustrate the peptide backbone, the residue side chains are not shown in full and are denoted in purple. (b) The R carbons are depicted in green. A turn occurs amidst the hydrophilic minimum from Q4 to Q5 bringing the N and C terminal ends of the peptide chain close together. The Y7 residue has its phenyl side chain directed outward parallel to Q4, while G1 remains a considerable distance from the other residues. The shape is similar to that of a β-turn and is a distinctly different conformer from the linear shape required to form dimers and propagate aggregation and fibril formation. A 3D rotatable image of this structure is available online in a web-enabled format.

methylene group, noted previously, was not used as a constraint in this model, the optimized structure resulted in a close proximity of these two groups, explaining its presence in the ROESY spectra (Figure 3). Effect of Protecting Groups. A portion of the resin-bound peptide sample had the Fmoc protecting group removed, and the previous HRMAS NMR experiments were repeated with the sample terminated by a free NH2 group on G1. The same methodology for sequence assignment and structural determination as outlined above was utilized. This was done to test for the effects of the aromatic protecting group on the conformation adopted by the peptide chain. The overall result is a moderate change in the peptide conformation that straightens the kink in the chain at the turn and alters the position of the Y7 residue, while retaining the two-residue turn among the Q4 and Q5 residues. This change is summarized schematically in Figure 6. The change in structure is best displayed by comparing the ROESY spectra of the peptide with and without Fmoc, as displayed in Figure 7. A number of the chemical shift assignments have changed, particularly within the central NNQQN residues, indicating that a change in the structure has resulted in modifications to the local environment experienced by the nuclei. These chemical shift assignments are summarized in Table S1 in the Supporting Information. Interactions 1 and 2,

1H

NMR Study of Peptide GNNQQNY

Figure 6. Schematic of the peptide conformations of the two samples with and without the Fmoc protecting group. (a) Fmoc-protected sample has a kink in its chain conformation with the Y7 residue lying parallel to the side chain of Q4. (b) Fmoc-removed sample has a more classic β-turn with two antiparallel chains, and the Y7 residue interacting with N2 and N6.

both of which involve the t-butyl group of Y7 interacting with Q4, are no longer evident, while a new interaction between the t-butyl group and N6 is observable (interaction A). Additionally, interaction 3 between this group and the NH2 group of N2 is stronger in this new spectrum. These observations together indicate that the tyrosine side chain has changed position and now lies further from Q4 and closer to N6 and the side chain of N2. Interaction B indicates magnetization transfer between the R carbon of Q5 and the amide proton of Q4, while interaction 4 between N3 and Q5 appears much weaker as a shoulder on this new interaction. Interaction D also shows an NOE exists between the R proton of Q4 and the amide proton of Q5. Interaction 11 between the β proton of Q5 and the amide proton of Q4 remains intact. Three other interactions involving Q5, namely interactions 5, 9, and 10 between N3, the side chain of Q4, and Y7, respectively, are lost in the new spectrum. The observations of these new and retained interactions, and the loss of the other Q5 interactions, support the notion that the turn of Q4 and Q5 on the peptide backbone is now more isolated and further from the remaining residues, in particular Y7 and N3. Interaction 7 between N2 and Y7 is retained, and there is also a new interaction observed between the R proton of N6

J. Phys. Chem. A, Vol. 114, No. 10, 2010 3463 and the amide proton of N3 (interaction C). Both of these residue pairs are in register on the antiparallel chains of a β-turn, giving strong support for the proposed structure. Interaction 6 and the newly observed interaction E are between sequential amino acids on the backbone. The previously observed interaction between the R and amide protons of N2 is likely still present at a slightly higher frequency from interaction D. No through-space interactions involving G1 are observable, including the lost interaction 8; this is likely due to a high degree of motional flexibility by the free G1 NH2 group without Fmoc present. The weakly observed interaction 12 between N6 and Y7 is also no longer present, which is possibly explained by a change in environment for both residues and the increased magnetization transfer of the β proton of Y7 to the Y7 phenyl protons. These observed parameters are in good agreement with the global minimum energy optimized structure created for this peptide, depicted in Figure 8. This structure is consistent with the probability that the removed Fmoc group interacted favorably with the similarly hydrophobic and aromatic linker region. Regardless, the interactions observed point to the retention of the previously proposed β-turn. The primary limitation of this model is the distance proposed between the t-butyl group of Y7 and the amide proton of N6 to create the NOE observed in interaction A. It is likely that the position of the tyrosine side chain is in fact closer to the N6 residue than it is to G1, though interaction 3 continues to support the proximity of the Y7 and N2 side chains. It is not clear if the t-butyl protecting group on Y7 or the resin linker are significant factors in the peptide conformation, and it is hoped that further work currently being undertaken by our group on this peptide and others will clarify what roles these elements play in the contribution to structure. Structural Significance. The determined conformations are consistent with the tendency for peptide chain turns to occur where hydrophobicity is at a local minimum and for hydrophobic and aromatic residues to aggregate.43,44 The five hydrophilic

Figure 7. 500 MHz 1H ROESY (65 ms spin lock) spectra of the resin-bound peptide with the Fmoc protecting group removed. The ROESY spectrum of the Fmoc-protected sample from Figure 3 is depicted in gray. The through-space interactions are labeled and the key inter-residue interactions are numbered to correspond to Table 2. Interactions A, B, C, D, and E indicate the newly observed through-space interactions.

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Andrey et al. solvent effects, or aggregation. HRMAS 1H NMR spectroscopy on the resin-bound peptide allowed for the determination of its monomeric three-dimensional conformation: a compact β-like turn among the central glutamine residues bringing the N and C terminal ends of the chain closer together. This structure is consistent with a conformation previously determined by molecular mechanics and has been found to have a low potential energy. The effects of the Fmoc protecting group were tested for and the turn was conserved, though moderate changes to the peptide conformation were determined. The observation of a turn in the predominant conformation of the peptide chain suggests that a change to a more linear shape may be necessary to propagate fibril formation. This finding may be informative in the pursuit to understand the mechanistic and structural details behind the early steps of aggregation as a means to prevent amyloid fibril formation. Finally, while a practical limit exists for the maximum peptide length that can be correctly attached to a polymer resin, this research is an illustration of the ability of HRMAS NMR spectroscopy to study the detailed monomeric conformation of small peptides. Acknowledgment. The authors gratefully acknowledge financial support by the Natural Science and Engineering Research Council of Canada (NSERC) through Discovery and Equipment Grants to W.P.P. and a 2008 Undergraduate Student Research Award to S.B.A. We also thank Mr. Kamal Mroue and Mrs. Janet Venne for their assistance.

Figure 8. Three-dimensional structure of the GNNQQNY peptide monomer with Fmoc removed, optimized to a global minimum energy. Carbon, hydrogen, oxygen, and nitrogen atoms are displayed in gray, white, red, and blue, respectively, while the resin polymer matrix is denoted in orange. (a) To better illustrate the peptide backbone, the residue side chains are not shown in full and are denoted in purple. (b) The R carbons are depicted in green. The structure proposed straightens the kink in the chain at the turn, compared to the Fmoc-protected structure, and alters the position of the Y7 residue, while retaining the two-residue turn among the Q4 and Q5 residues. A 3D rotatable image of panel A and panel B of this structure is available.

residues NNQQN participate in a β-like turn and the more hydrophobic residues, namely G1 and Y7, are brought into closer proximity. Additionally, the structures are similar to one of the monomer models previously determined using replica exchange molecular dynamics by Wales et al.13 Their region T structure also models a two-residue turn involving Q4 and Q5 and brings the side chain of Y7 near N2 and Q5. Additionally, Q4 and Y7 are more or less parallel, and G1 is in a position to remain relatively uninvolved with the nonadjacent residues. This compact structure was found to have low potential energy, but relatively high harmonic free energy due to its stiffness.13 The same study suggested that free energy barriers between conformations for this peptide sequence are low at around 2 kcal mol-1, indicating that fluctuation between conformations may occur rapidly in solution. Furthermore, it has been suggested that a more linear conformation than is observed here is necessary for the formation of the β-sheet dimers and to propagate fibril formation.13,19 Conclusions By attaching the amyloid-forming peptide sequence GNNQQNY to a polymer matrix through solid phase peptide synthesis and placing it in an organic solvent, the monomer was given the internal freedom to fold into its local conformation, free from the effects of neighboring peptides, entropic

Supporting Information Available: Assigned 1D 1H HRMAS NMR spectrum of the resin-bound sample, the chemical shift assignments of the peptide with Fmoc removed, and the complete citation to ref 41. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Chiti, F.; Dobson, C. M. Annu. ReV. Biochem. 2006, 75, 333–366. (2) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. F. J. Mol. Biol. 1997, 273, 729–739. (3) Balbirnie, M.; Grothe, R.; Eisenberg, D. S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2375–2380. (4) Makin, O. S.; Atkins, E.; Sikorski, P.; Johansson, J.; Serpell, L. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 315–320. (5) Diaz-Avalos, R.; Long, C.; Fontano, E.; Balbirnie, M.; Grothe, R.; Eisenberg, D.; Caspar, D. L. D. J. Mol. Biol. 2003, 330, 1165–1175. (6) Gsponer, J.; Haberthu¨r, U.; Caflisch, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5154–5159. (7) Lipfert, J.; Franklin, J.; Wu, F.; Doniach, S. J. Mol. Biol. 2005, 349, 648–658. (8) Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. Ø.; Riekel, C.; Grothe, R.; Eisenberg, D. Nature 2005, 435, 773–778. (9) Esposito, L.; Pedone, C.; Vitagliano, L. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11533–11538. (10) van der Wel, P. C. A.; Hu, K.; Lewandowski, J.; Griffin, R. G. J. Am. Chem. Soc. 2006, 128, 10840–10846. (11) Zheng, J.; Ma, B.; Tsai, C.; Nussinov, R. Biophys. J. 2006, 91, 824–833. (12) Sawaya, M. R.; Sambashivan, S.; Nelson, R.; Ivanova, M. I.; Sievers, S. A.; Apostol, M. I.; Thompson, M. J.; Balbirnie, M.; Wiltzius, J. J. W.; McFarlane, H. T.; Madsen, A. Ø.; Riekel, C.; Eisenberg, D. Nature 2007, 447, 453–457. (13) Strodel, B.; Whittleston, C. S.; Wales, D. J. J. Am. Chem. Soc. 2007, 129, 16005–16014. (14) van der Wel, P. C. A.; Hu, K.; Lewandowski, J.; Griffin, R. G. J. Am. Chem. Soc. 2007, 129, 5117–5130. (15) Zhang, Z.; Chen, H.; Bai, H.; Lai, L. Biophys. J. 2007, 93, 1484– 1492. (16) De Simone, A.; Esposito, L.; Pedone, C.; Vitagliano, L. Biophys. J. 2008, 95, 1965–1973. (17) Guo, Z.; Eisenberg, D. Protein Sci. 2008, 17, 1617–1623. (18) Meli, M.; Morra, G.; Colombo, G. Biophys. J. 2008, 94, 4414– 4426. (19) Vitagliano, L.; Esposito, L.; Pedone, C.; De Simone, A. Biochem. Biophys. Res. Commun. 2008, 377, 1036–1041.

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