Solid-State NMR Investigation of Major and Minor ... - ACS Publications

Jan 3, 2008 - Gregory P. Holland,* Janelle E. Jenkins, Melinda S. Creager, ... Silks spun from the major (Ma) and minor (Mi) ampullate glands by the s...
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Biomacromolecules 2008, 9, 651–657

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Solid-State NMR Investigation of Major and Minor Ampullate Spider Silk in the Native and Hydrated States Gregory P. Holland,* Janelle E. Jenkins, Melinda S. Creager, Randolph V. Lewis, and Jeffery L. Yarger* Magnetic Resonance Research Center, Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, and Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071 Received August 26, 2007; Revised Manuscript Received October 21, 2007

Silks spun from the major (Ma) and minor (Mi) ampullate glands by the spider Nephila claVipes respond to water differently. Specifically, Ma silk supercontracts (shrinks 40–50% in length) while Mi silk does not contract at all when hydrated with water. In the present study, 1H f 13C cross polarization magic angle spinning (CP-MAS), 13 C MAS NMR collected with dipolar decoupling, and two-dimensional wide-line separation spectra are presented on Mi silk in its native and hydrated state and comparisons are made to Ma silk. This combination of NMR data demonstrates that water plasticizes Mi and Ma silk similarly, with an increase in chain dynamics observed in regions containing Gly, Glu, Ser, Tyr, Leu, and a fraction of Ala when the Mi silk is hydrated. Resonances that correspond to the poly(Ala) and poly(Gly Ala) motifs of Ma and Mi silk are predominately rigid indicating that water does not penetrate these β-sheet domains.

Introduction Through millions of years spiders have evolved to produce up to six different silks and one type of glue.1 Each silk is a remarkable biopolymer having unique mechanical properties that are optimized according to its specific use. The mechanical properties are a combination of strength and extensibility that surpass most man-made materials.2 The strength and elastic properties are correlated to specific amino acid sequence motifs.3,4 A better understanding of these motifs and their interaction with one another at the molecular level is needed in order to generate fibers for specific industrial applications. Multidimensional solid-state NMR experiments were conducted to help elucidate the molecular structure and dynamic features of these motifs for major (Ma) ampullate and minor (Mi) ampullate silk from the spider Nephila claVipes. Ma and Mi silks are good silks to compare because of the availability of the silks and their differing mechanical properties. Of specific relevance is the impact of water on these two types of silk. Specifically, Ma silk axially contracts up to 50% in length when it comes in contact with water while, hydrated Mi silk displays essentially no contraction.5,6 Ma and Mi silks have some similarities in their amino acid sequences and mechanical properties as well as distinct differences. Ma and Mi silk both are comprised of two proteins, MaSp1 and MaSp2 for major7 and MiSp1 and MiSp2 for minor (see Figure 1 for the consensus amino acid sequences).8 The poly(Ala)n domains present in Ma and Mi silks comprise the majority of the crystalline β-sheet structures.9,10 One distinct difference between these two silks is the length of these poly(Ala)n runs. Ma silk has runs with n up to 7 in MaSp1 and 10 in MaSp2 while, Mi has shorter poly(Ala)n regions with n ) 3–5.7,8,11 It is thought that the poly(Gly Ala)n region that is present in great abundance in Mi silk, is also incorporated in the crystalline β-sheet domains.8 These crystalline regions are * Corresponding authors. E-mail: [email protected], Jeff.yarger@ asu.edu.

Figure 1. The consensus amino acid sequence of Ma and Mi spider silk proteins. Runs of poly(Ala) that form a β-sheet structure are underlined.

aligned parallel to the fiber axis when the silks are dry as shown in X-ray diffraction5,12,13 and NMR14–16 and both Ma and Mi silk show a reorientation but preservation of the crystalline β-sheet domains when the silks are wetted.5,17 The structural model of spider silk most often proposed is poly(Ala) crystalline β-sheet domains embedded in an amorphous region, which is Gly rich in both silks. Rotational-echo double-resonance18,19 (REDOR) NMR measurements performed by Jelinski et al. support a type I β-turn for a fraction of the Gly-rich region of Nephila claVipes Ma silk.6,20 Recent solid-state NMR results on Ma silk from the spider Nephila edulis indicate that a substantial fraction of the Gly containing region has an approximate 31 helical secondary structure that has a preferred orientation along the fiber axis.15,21,22 This shows that the Glyrich region although amorphous by X-ray diffraction (XRD), has a substantial degree of local structural and orientational order. When exposed to water, the Ma silk contracts up to ∼50% ofitsoriginallengthaxially,aprocessknownassupercontraction,17,23 while Mi silk displays essentially no supercontraction.5,6 It has been shown that there are molecular and mechanical properties that change in the Ma silk when in contact water. Rubber-like mechanical properties are observed with a large decrease in elastic modulus and increase in extensibility24 when the silk is in contact with water while the stiffness decreases.23 Results from 13C cross-polarization magic angle spinning (CP-MAS) and 2H wide-line NMR showed that significant chain mobility occurs in a fraction of the Gly, Gln, Ser, Tyr, and Leu in the

10.1021/bm700950u CCC: $40.75  2008 American Chemical Society Published on Web 01/03/2008

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Ma silk while the poly(Ala)n crystallite regions remained rigid when the silk was exposed to water.25 Wide-line seperation26 (WISE) NMR was used to correlate the structural information with mobility by indirectly observing the 1H wide-line spectrum in a two-dimensional (2D) 13C detected experiment. These WISE results confirmed the presence of chain mobility in the Gly-rich region when the Ma silk was in contact with water while the poly(Ala) crystallites remained rigid and intact.27 In the present study, these NMR techniques are applied to Mi silk in its native and hydrated state, and comparisons are made to Ma silk in order to determine if the structural and/or dynamic properties differ between the two silks when hydrated with water. Structural and/or dynamic differences between the hydrated Ma and Mi silk could be related to supercontraction since the Ma silk displays this property and Mi silk does not.

Materials and Methods Materials. Nephila claVipes Ma and Mi silks were collected by mechanically silking adult female spiders at 2 cm/s to remove stored silk proteins. The silking process was monitored under a light microscope to ensure that the Ma and Mi silks were not mixed and kept separate. The spiders were fed U-13C6-glucose (Cambridge Isotope Laboratories, Inc.) in a 10% w/v aqueous solution and 1 cricket per week. The U-13C6-glucose feeding process was repeated at 48 h intervals. The 13C-labeled silks used in the NMR experiments were from the third silking and later. The 13C enrichment is approximately 8% as measured by comparing the intensity of the Ala and Gly resonances in the fully relaxed 13C MAS spectrum for a natural abundant Ma and Mi sample and the 13C-labeled samples. When silk samples were made wet, D2O was utilized to ensure that none of the observed 1H WISE NMR signals originated from the water but rather 1 H environments from the amino acid residues. Solid-State NMR. Solid-state NMR spectra were collected on a Varian VNMRS 400 MHz wide-bore spectrometer equipped with a 3.2 mm triple resonance MAS probe operating in double resonance mode (1H/13C). The silk samples were packed in zirconia rotors that were sealed with O-ring Kel-f inserts to maintain complete water saturation of silk samples exposed to D2O for hydration studies. The wet silk samples are referred to as hydrated or wetted silks. These silk samples are completely saturated in a pool of water within the rotor. The wetted silk samples were checked following each experiment to ensure that no water evaporation occurred due to heating by MAS and/ or high-power 1H decoupling. The thermal properties of spider silk indicate that the fiber will not be impacted by heating effects from MAS and/or 1H decoupling.28 1H f 13C CP-MAS spectra for natural abundant silk samples were collected with 10 kHz MAS with the CP condition matched on the -1 spinning sideband in the Hartmann–Hahn profile. Silk samples labeled with 13C were collected with 5 kHz MAS on the Hartmann–Hahn condition for both wet and hydrated silks. Typical experimental parameters for the 13C CP-MAS NMR spectra of 13C-labeled samples were a 4 µs 1H pulse, a 1 ms CP pulse with a 62.5 kHz rf field strength, 100 kHz two pulse phase modulated29 (TPPM) 1H decoupling during acquisition, 2048 data points, 1024 scan averages, a 50 kHz sweep width, and a 4 s recycle delay. The 13C CP-MAS spectra for natural abundant samples were collected with similar parameters with the exception that 12 K scan averages were obtained. Direct 13C MAS spectra collected with dipolar decoupling (DD-MAS) were obtained with short (1 s) and long recycle delays (100 s). The 13C DD-MAS spectra collected with a 1 s delay enhances resonances in the 13C spectrum that have shortened 13C T1 relaxation times due to local molecular mobility30 while the fully relaxed spectra were utilized to quantify various amino acid fractions of the silks. The 100 s recycle delay utilized to obtain quantitative data from 13C DDMAS spectra is known to be five times the longest T1 relaxation time in spider silks.9 Although these T1 data were collected at 200 MHz and the backbone 13C T1’s are likely to be linearly dependent on the

Figure 2. The natural abundance 13C CP-MAS NMR spectrum of (A) Ma and (B) Mi spider silk from the spider Nephila clavipes. Spectra were collected with 10 kHz MAS and a 1 ms CP contact time. Amino acid residue assignments are displayed in the figure. Spinning sidebands are labeled ssb.

field strength, a recycle delay of 100 s should still suffice for the quantification of all the 13C resonances with the exception of the carbonyl group. The carbonyl was not used for any quantification in this paper. The two-dimensional (2D) WISE spectra were collected as previously described26,27 with 5 kHz MAS and CP matched on the Hartmann–Hahn condition. The indirect 1H dimension had a sweep width of 200 kHz and was collected with 64 t1 points and 256 scans per point. The recycle delay was 2 s, and the hypercomplex (States) method was applied to obtain phase-sensitive 2D spectra.31 Processing parameters for 13C CP-MAS and DD-MAS spectra were zero-filling to 8192 points and 50 and 10 Hz of exponential line broadening, respectively. Processing of the 2D WISE spectra was accomplished with 100 Hz of exponential line broadening in the direct dimension and 1 kHz of exponential line broadening in the indirect dimension with zero-filling to 8192 and 1024 in the two dimensions, respectively. Peak fitting routines were conducted with the DMFIT software package.32

Results and Discussion Natural Abundance 13C CP-MAS NMR of Ma and Mi Silks. The natural abundance 13C CP-MAS NMR spectrum of dry Ma and Mi silk from the spider Nephila claVipes is displayed in Figure 2. The two spectra are similar and contain resonances that can be assigned to specific amino acids based on previous results.9,10 Most of the peaks are heterogeneously broadened due to a distribution in chemical shift that results from various structural conformations. This is particularly evident in the Ala Cβ resonance of both silks where a low ppm shoulder is clearly present indicating two conformations for Ala. The carbonyl resonance also displays a heterogeneously broadened resonance with a full width at half-maximum (fwhm) that is greater than 5 ppm for both silks. There are some subtle differences in the natural abundance 13 C CP-MAS spectra of the two silks. Specifically, the fwhm of the Ala CR is smaller (∼6%) and the Gly CR is larger (∼9%) for Ma silk compared to Mi silk. The fwhm in these samples is a measure of the heterogeneity at the specific amino acid site. This result can be rationalized based on the amino acid sequences of the silk proteins (see Figure 1). In both Ma and Mi silk, the Ala is primarily organized in β-sheet crystalline domains.5,9,10,13,14,33-38 However, the difference for Mi silk is

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Figure 3. The 13C CP-MAS NMR spectra of 13C-enriched (A, C) Ma and (B, D) Mi silk spider silk from the spider Nephila clavipes in the dry and hydrated states. The spectra of the hydrated silks (dotted line) display a decrease in intensity compared to the dry silks. Spectra were collected with 5 kHz MAS and (A, B) 1 ms and (C, D) 100 µs CP contact time. Amino acid residue assignments are displayed in the figure. Spinning sidebands are labeled ssb. Background signals from the O-ring and Kel-F inserts are assigned an asterisk.

that it will mainly be in the poly(Gly Ala) form compared to poly(Ala) in Ma silk. The poly(Ala) domains in Ma are presumably more ordered than the poly(Gly Ala) in Mi, and this reflected in a broader fwhm for the latter. The opposite will be the case for Gly. In Ma silk, Gly is found predominantly in GGX repeats that are amorphous by XRD,13,38 while in Mi silk a significant fraction of Gly is located in poly(Gly Ala) that is incorporated in the β-sheets. Thus, the latter displays a smaller fwhm because the poly(Gly Ala) repeats that dominate the amino acid sequence of MiSp1 and MiSp2 form β-sheet domains that are crystalline and, therefore, more ordered than the GGX repeats prevalent in Ma silk. Another noticeable difference between the 13C CP-MAS spectra of the two silks is that Ma silk displays a more intense signal for Gln Cβ,γ. This is in rough agreement with amino acid compositions reported for Nephila edulis where the Gln content was three times higher in Ma compared to Mi silk.39 However, it disagrees with previous amino acid compositions reported for Nephila claVipes where the Gln content was reported to be two times higher for Mi compared to Ma silk.10 There appears to be significant variability in the amino acid compositions of spider silks reported in the literature. 13 C CP-MAS NMR of Labeled Ma and Mi Silks and the Impact of Hydration. Silk samples were 13C labeled to accelerate experiment time and make two-dimensional experiments feasible. The 13C CP-MAS spectra for the labeled Ma and Mi silk samples in the dry and hydrated states are shown in Figure 3. The 13C enrichment was estimated to be ∼8% for Ala and Gly by comparing the intensities of a fully relaxed 13C DD-MAS spectrum of the labeled silk to a natural abundant silk sample of similar weight. The more complex amino acids get labeled to a lesser extent. For example, Tyr displays no increase in 13C enrichment. This is particularly evident when

comparing the 13C CP-MAS NMR spectrum of the natural abundant silks (Figure 2A,B) with those that have been fed the 13 C-enriched glucose source (Figure 3A,B). An inability to label Tyr has been discussed before in the context of liquid state NMR on 13C-enriched Ma ampullate silk gland fibroin40 and is due to the fact that Tyr is produced de novo from phenyalanine.41 The 13C CP-MAS NMR spectra of the hydrated silks display significant differences compared to the dry silks. Particularly, a decrease in intensity is observed for the Gly CR, Leu CR/Gln CR/Ser CR/Tyr CR, Tyr Cγ,δ,,ζ, and the CdO resonances, while the CR and Cβ Ala resonances display similar intensities in the dry and wetted states. This indicates that water penetrates the Gly-rich region but not the poly(Ala) and poly(Gly Ala) β-sheet domains in Ma and Mi silk. The presence of water causes an increase in chain mobility and consequential loss in CP signal for those amino acids that are hydrated by water. It should be noted that there is a small decrease in the intensity for the low (upfield) and high (downfield) ppm shoulder of the Ala Cβ resonance in both silks that results due to the presence of water near these sites. These subtle features are more evident when observing the Ala Cβ resonance at short (100 µs) CP contact times (see Figure 3C,D). The low ppm shoulder can be ascribed to a helical Ala fraction (discussed below) while, the high ppm shoulder can be assigned to Leu Cδ (this is in agreement with solid-state NMR HETCOR results that will be published in a subsequent paper). When the intensity of the Gly CR resonance is monitored as a function of CP contact time in the wetted state, the overall intensity was 30% and 20% lower at the maximum compared to the dry state for Ma and Mi silk, respectively. This provides strong evidence for the presence of near isotropic mobility with correlation times less than 10-5 s for the fraction of Gly CR CP signal that is lost. The presence of near isotropic motions is

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Figure 4. The 13C DD-MAS NMR spectra of 13C-enriched (A) hydrated Ma spider silk, (B) dry Ma spider silk, (C) hydrated Mi spider silk, and (D) dry Mi spider silk from the spider Nephila clavipes. Spectra were collected with 10 kHz MAS and 1 s recycle delay. Amino acid residue assignments are displayed in the figure. Spinning sidebands are labeled ssb. Background signals from the O-ring and Kel-F inserts are assigned asterisks.

confirmed by 13C DD-MAS spectra discussed below for hydrated silks. The measured 30% decrease in intensity for this mobile Gly fraction for Ma silk agrees well with the value of 35% extracted by Jelinski et al. from 13C MAS Hahn spin-echo spectra collected on wet Ma silk with short 1 s delay times.25 The larger decrease in signal intensity provides evidence that the impact of hydration on the Gly-rich region of the silk is larger in Ma compared to Mi silk. This could potentially be due to the larger fraction of Gly in Mi silk that is present in poly(Gly Ala) repeats that maintain β-sheet structure when the silk is in contact with water. 13 C DD-MAS NMR of Labeled Ma and Mi Silks and the Impact of Hydration. The 13C DD-MAS spectra obtained with a 1 s recycle delay are presented in Figure 4 for dry and hydrated Ma and Mi silk. In this experiment, residues that exhibit shortened T1 relaxation times will have enhanced signal intensity. In addition, these resonances are typically sharp due to lengthening of the T2 relaxation times. Such behavior is observed for the Gly CR, Gln Cβ,γ, LeuR/GlnR, Ser CR,β, the low ppm component of Ala Cβ, and components of CdO resonance when comparing 13C DD-MAS spectra collected on the dry silk to spectra collected on the wetted silk samples. The enhanced signals observed here are due to the mobile components lost in the 13C CP-MAS spectra of the wet silks discussed

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above (see Figure 3). This agrees with the presence of near isotropic mobility for a fraction of these amino acid residues and in agreement with previous results on mobility in hydrated Ma spider silks where a decrease in 13C T1 was observed for similar residues.25,42 The larger fraction of sharp components in the 13C DD-MAS spectra of Mi silk compared to Ma silk indicates that the plasticizing effect of water on Mi silk appears to be greater. It should be noted that some resonances could be absent or less intense in the 13C DD-MAS spectrum of Ma silk due to motional fluctuations on the MAS (10 kHz) or 1H decoupling (100 kHz) time scales.30 These incoherent interference effects43,44 are currently being explored in our laboratory but will take a number of experiments at different MAS and 1H decoupling frequencies to sort out and will be the subject of a future publication. Nonetheless, it is particularly interesting to note that water indeed plasticizes Mi silk and this plasticizing effect is often the focal point when proposing a mechanism for supercontraction in Ma silk,25,27,45 yet Mi silk shows a similar plasticizing effect without supercontraction. This strongly indicates that although the penetration of water into the Gly-rich region of the silk could help explain the supercontraction process, this observation alone cannot account for supercontraction in Ma silk. The 13C DD-MAS spectra of hydrated Ma and Mi silk display significantly improved resolution compared to the dry silks. The chemical shifts remain constant when the silks are wetted; however, a significant sharpening of the resonances is observed allowing for accurate chemical shift measurements to be made compared to the dry silks. These chemical shifts are reported in Table 1. The 13C chemical shifts of various amino acids are known to depend on the secondary structure in the solid state46-51 and have been extensively utilized in the characterization of secondary structure in spider silks.5,9,10,27,52 Comparisons can be made between reference chemical shifts of polypeptides having known secondary structures with those observed for Ma and Mi spider silks. For Ala, the Cβ chemical shift of the primary resonance positioned at 20.8–20.9 ppm and the CR resonance at 49.0–49.3 ppm can clearly be assigned to Ala in the β-sheet structure in both Ma and Mi silks, in agreement with previous interpretations.9,10 Unfortunately, the chemical shifts observed for the other amino acids are ambiguous, and assignment to a distinct secondary structure is impossible to determine. For example, the broad 13C resonance observed for Gly spans the range of both a β-sheet and 31helical structure indicating a static mixture of the two conformations. It is also worth noting that the 13C chemical shifts for most of the amino acids present in Ma and Mi silk fall outside the range for an R-helical conformation. The Ala Cβ resonance displays a sharp component in the 13C DD-MAS spectrum of the wet Ma and Mi silk that has a chemical shift of 17.4–17.5 ppm. This chemical shift is nearly identical to that observed for Ala in a 31-helical structure (see Table 1). This component is present in the dry silks, although it is significantly broader and convoluted with the main Ala component at 20.8–20.9 (see Figures 2 and 3).40 Doublequantum/single-quantum correlation (DOQSY) NMR experiments indicate that a significant fraction of the Gly-rich region is present in a 31-helical structure;15 thus the Ala fraction that displays a sharp resonance in the 13C DD-MAS spectrum of hydrated Ma and Mi silk can be tentatively assigned to those Ala present in this region of the silk. A peak fitting routine was performed on the fully relaxed 13 C DD-MAS spectrum of wet Ma and Mi silk to extract the

Solid-State NMR of Major and Minor Spider Silk Table 1.

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C Chemical Shifts of Major Silk, Minor Silk, and Reference Chemical Shiftsa

residue

major silk

minor silk

R-helix47,54

β-sheet47,54

random coil55

Ala Cβ Ala CR Gln Cβ Gln Cγ Gly CR Leu CR Gln CR Ser CR Ser Cβ Tyr Cγb Tyr Cδb Tyr Cb Tyr Cζb CdOc

17.5, 20.9 49.3 27.7 31.8 43.2 53.7 53.7 55.9 61.9 129.0 129.0 118.8 157.0 172.4

17.4, 20.8 49.0 28.0 31.9 43.1 53.8 53.8 56.3 62.1 129.1 129.1 116.4 156.6 172.6

14.8–15.5 52.8–52.3

19.9–20.3 48.2–49.3

47.2–49.2 55.7–55.8

43.2–44.3 50.5–51.2

60.3–62.3 62.0–63.8

55.5–58.0 63.5–67.3

176.2–176.8

171.6–172.2

19.1 52.5 29.4 33.7 45.1 55.1 55.7 58.3 63.8 130.6 133.3 118.2 157.3 172.1

a

31-helix40,46,50,51 17.4 48.7 41.7–43.2

175.5

Chemical shift from the DD-MAS spectra of hydrated silk (Figure 4). Chemical shift from the natural abundant CP data (Figure 2). c Chemical shift measured at the center of the resonance, reference chemical shifts are for Ala.

ratio of the two Ala fractions (see Supporting Information). The integrated ratio of the Ala Cβ component at 20.8–20.9 ppm to the component at 17.4–17.5 ppm is 85 and 80% for Ma and Mi silk, respectively. The fraction of Ala in poly(Ala), poly(Gly Ala), and those Ala found in the remaining Gly-rich fraction of the silk was calculated from the known amino acid sequences of Ma11 and Mi8 silk proteins. The breakdown was found to be 70% poly(Ala), 16% poly(Gly Ala), and 14% for the Ala found in the remaining Gly-rich fraction of the amino acid sequence for the MaSp1 and MaSp2 silk proteins. The fraction of Ala in nonpoly(Ala) and nonpoly(Gly Ala) regions of the amino acid sequence from the Ma silk proteins is 14%, which is very close to the value of 15% extracted from the deconvolution of the 13C DD-MAS spectrum of hydrated Ma silk for the resonance at 17.5 ppm. This is consistent with the assignment made above to a 31 helical structure since preliminary evidence exists that this Gly-rich region of the silk is in a 31 helical structure15 and the integrated ratios appear to agree with those Ala in non-βsheet-forming motifs. For Mi silk, the situation is more complicated since, the ratio of MiSp1 to MiSp2 is not known.8 The breakdown for poly(Ala), poly(Gly Ala), and the Ala found in the remaining Gly-rich fraction is 35%, 43%, 22% and 32%, and 48%, and 20% for MiSp1 and MiSp2, respectively. Even without knowing the ratio of the two proteins, it is clear that the fraction of Ala in the remaining Gly-rich region that is not present in poly(Ala) or poly(Gly Ala) is 20–22%. This agrees well with the integrated fraction for the resonance at 17.4 ppm from the deconvoluted 13 C DD-MAS spectrum of hydrated Mi silk where a value of 20% was extracted. The assignment of the Ala Cβ resonance near 17 ppm to Ala present in a 31-helical conformation disagrees with our previous assignment of this resonance in hydrated Ma silk to the poly(Gly Ala) motif.27 The combination of quantitative results presented here appear to be a more accurate interpretation of this resonance particularly in light of the fact that the fraction for Mi silk is 20%, which is significantly smaller than the fraction of Ala located in the poly(Gly Ala) motif that is 43–48%. Thus, it is believed at this point that the poly(Ala) and poly(Gly Ala) motifs present in Ma and Mi silk maintain a rigid β-sheet structure, while only those Ala present in the remaining Gly-rich fraction of silk become mobile when the silks are hydrated. 1 H/13C WISE NMR of Labeled Mi Silk and the Impact of Hydration. WISE NMR spectra were collected on Mi silk to correlate structural information from the 13C chemical shift with mobility by indirectly observing the 1H wide-line spectrum. The WISE NMR spectrum of dry Mi silk is presented in Figure

b

Figure 5. The 1H/13C WISE NMR spectrum of dry Mi silk from the spider Nephila clavipes. The (A) carbonyl region, (B) low ppm region, and (C) 1H slices taken at specified 13C chemical shifts are shown. Spectra were collected with 5 kHz MAS and a 1 ms CP contact time.

5. The projection in the 1H dimension is a single broad resonance having a fwhm of 40 kHz. Slices taken at the Ala Cβ, Gly CR, Ala CR and CdO resonance show that each of these amino acid resonances is broad with no sharp components (see Figure 5C). This is consistent with a completely rigid organic system on NMR time scales26 and is similar to previous WISE NMR results present on dry Ma silk.27 The WISE NMR spectrum of hydrated Mi silk is shown in Figure 6. This WISE spectrum differs significantly from the WISE spectrum of the dry Mi silk. Specifically the 1H components of these spectra are greatly narrowed due to the presence of local molecular mobility compared to the WISE spectrum of the dry silk. These WISE spectra were collected

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these larger amino acids are not incorporated in the β-sheets that remain rigid when the Mi silk is wetted. This is consistent with previous interpretations from XRD data that indicated the larger amino acids were not incorporated in the β-sheet structure in silk fibroins.53

Conclusions 13

Figure 6. The 1H/13C WISE NMR spectrum of hydrated Mi silk from the spider Nephila clavipes. The (A) carbonyl region, (B) low ppm region, and (C) 1H slices taken at specified 13C chemical shifts are shown. Spectra were collected with 5 kHz MAS and a 1 ms CP contact time.

with a CP contact time of 1 ms. At contact times this long there is a question of whether 1H spin diffusion causes some similarity in the 1H line widths and line shapes of the different amino acid residues. In order to determine whether 1H spin diffusion was influencing the observed line widths, a WISE spectrum of hydrated silk was collected with a 200 µs contact time (data not shown). This spectrum was nearly identical to the spectrum displayed in Figure 6 indicating that 1H spin diffusion during CP was not greatly impacting the 1H line widths and line shapes observed with a CP contact time of 1 ms. Slices taken in the 1H dimension at the 13C chemical shift of Ala Cβ (20.9 ppm) and CR (49.2 ppm) are similar to the dry silk and remain primarily broad with only a small fraction of a narrow component. This indicates that these Ala residues that have chemical shifts representative of the β-sheet conformation (see Table 1) remain rigid in the wetted Mi silk. The Ala Cβ resonance at 17.6 ppm that was ascribed to Ala Cβ residues present in the Gly-rich region in a 31-helical structure display primarily a narrow line width in the 1H dimension (see Figure 6D). The 13C resonances corresponding to Gln, Leu, and Ser display 1H wide-line spectra that are narrow with fwhm between 3 and 5 kHz indicating a significant degree of chain mobility at these residues due to the presence of water. The 1H slice taken at the 13C chemical shift of Gly CR shows primarily a narrow component with a fwhm of 5 kHz; however, there is a broad component present. The latter is assigned to Gly present in the poly(Gly Ala) runs that form a β-sheet structure. Note this in contrast with the 1H wide-line spectrum of Gln, Leu, and Ser where only a narrow component is observed. This indicates that

The combination of C CP-MAS, DD-MAS, and WISE NMR spectroscopies have shown that water plasticizes Ma and Mi silk similarly with some subtle differences. There are three dynamic motional regimes that can be distinguished when water interacts with Ma and Mi silk. One motional regime shows nearly isotropic mobility where CP signal is lost and significantly sharpened peaks with shortened T1 relaxation times are observed in the DD-MAS spectra. Gly, Glu, Ser, Leu, and a small component of Ala show this behavior. The latter has been assigned to Ala that is incorporated in the 31-helical Gly-rich region of the silk. For Gly, the loss in CP signal was found to be larger for Ma silk compared to Mi silk. This was attributed to the larger poly(Gly Ala) content in Mi silk that are believed to maintain a β-sheet structure when hydrated. The two final motional regimes are those that can be distinguished in the WISE spectrum. These are residues that CP but, either display a sharp (3–5 kHz) or broad (40 kHz) 1H wide-line spectrum. For Ma and Mi silk the results are similar, where Ala is the only amino acid that remains almost entirely rigid while Gly, Gln, Ser, and Leu display significant fractions with intermediate mobility characterized by 1H wide-line spectra with fwhm ) 3-5 kHz. These results highlight that water plasticizes supercontracting Ma and nonsupercontracting Mi silk similarly; therefore, this observation alone cannot account for supercontraction in Ma silk. The plasticizing effect of water may be a general property of spider silks and can be exploited to enhance the resolution of 13C solid-state NMR spectra. Acknowledgment. This research was supported by grants from the National Science Foundation (CHE-0612553 and CMMI-0304494) and the National Institutes of Health (NIBIB5R01EB000490-05). We thank Dr. Brian Cherry for help with NMR instrumentation, student training, and scientific discussions. Supporting Information Available. NMR spectra of hydrated (A) Ma and (B) Mi spider silk. This material is available free of charge via the Internet at http://pubs.acs.org.

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