Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
Solid-State NMR Spectroscopy and Isotopic Labeling Target Abundant Dipeptide Sequences in Elastin’s Hydrophobic Domains Kosuke Ohgo, Chester L. Dabalos, and Kristin K. Kumashiro* Department of Chemistry, University of Hawaii, 2545 McCarthy Mall, Honolulu, Hawaii 96822, United States S Supporting Information *
ABSTRACT: Isotopic labeling strategies are coupled with solid-state NMR spectroscopy to characterize elastin’s abundant glycines (Gly) and prolines (Pro) and the sequences that include these residues. Elastin is prepared with isotopic labels at Gly, Pro, and Valthree of its four most abundant amino acids. Solid-state 15N−1H nuclear magnetic resonance (NMR) spectra show four resolved glycine populations, whereas three features are observed for the prolines. Selection of the Val-Pro and GlyGly pairs found exclusively and abundantly in the hydrophobic domains confirms these assignments and also provides more information on the conformational ensembles of elastin. The 1HN and 15N temperature coefficients indicate that discrete secondary structures are present, even among significant populations with rapid conformational fluctuations typical of a random coil. 13C−15N couplings and the 13C chemical shift anisotropy (CSA) are also utilized to confirm assignments and structure, respectively. Implications for the evolving conformational ensemble model for elastin are also discussed.
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coils.9,10 This “conformational ensemble”10 or “structure distribution”11−13 is supported by infrared spectroscopy and circular dichroism data on model peptides, or mimetics, of elastin.10,14−16 Among the more likely secondary structures in the hydrophobic domains of elastin is the β-turn.17 “Sliding βturns” were proposed as a model for elasticity.9,18 The β-turn occurs in the structures of Pro-containing elastin-like sequences, such as VPGVG.19,20 However, direct observation of this structure in native elastin has not yet been reported. Solid-state nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for the study of the structure and dynamics of elastin.21−24 The inherent mobility in hydrated elastin precludes the application of dipolar-based methods like crosspolarization.21,25 Alternatively, J-coupling-based experiments are compatible with the rapid fluctuations in this protein. Recently, we reported26 the characterization of hydrated 15Nenriched recombinant elastin using the refocused insensitive nuclei enhanced by polarization transfer (rINEPT) technique.27,28 Detailed structural and dynamic information about the prolines in elastin from NMR spectroscopy requires isotopic enrichment. Cultures of neonatal rat smooth muscle cells (NRSMC) provide enriched elastin samples in NMR-scale quantities.29 Notably, this cell line retains its native crosslinking mechanism in vitro. The labeling of the Gly residues utilized a straightforward approach that substituted the unenriched amino acid with its isotopically enriched counter-
INTRODUCTION Elastin imparts lifelong elasticity and resiliency to vertebrate tissue.1 It is composed of alternating hydrophobic and crosslinking domains and has a relatively unique composition. The hydrophobic domains, with its characteristic tetra-, penta-, and hexapeptide repeats of the predominant amino acids, Gly, Pro, Ala, and Val, are typically targeted for studies that aim to elucidate the basis of elasticity in terms of these structural elements and how they change during stretch-and-recoil cycles. Glycine (Gly, G) is the most abundant amino acid in elastin, accounting for ∼40% of its residues.2,3 Glycines are found almost exclusively in the hydrophobic domains, typically in repeating sequences. Glycines are also likely to be next to another glycine in the primary structure of the protein. Notably, others have hypothesized that glycine is believed to play an important but yet undefined role in the elasticity of elastomeric proteins like elastin.4 Prolines (Pro, P) comprise about 10% of the amino acids in elastin. Its presence is believed to inhibit the formation of longrange order that is found in extensive secondary structures, such as long α-helices or other conformations that require stabilization via hydrogen-bonding networks. The majority of the prolines in elastin are found throughout the Gly-rich hydrophobic domains, and roughly one-fourth of them are preceded by Val (ValPro). The structural properties of proline favor a β-turn, especially when followed by Gly.5 Notably, the Val-Pro-Gly triad is abundant in rat tropoelastin and is conserved across vertebrate species.3 Several structural models have been proposed to explain the elasticity of elastin.6−9 Favored among these models is an equilibrium of defined secondary structures and random © XXXX American Chemical Society
Received: December 8, 2017 Revised: February 7, 2018
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DOI: 10.1021/acs.macromol.7b02616 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules part at equimolar quantities (in the growth media).29 The analogous approach was successfully utilized for the enrichment of the valines in elastin. However, this strategy yielded low incorporation levels for labeling of elastin’s proline residues, so a different approach is presented herein. In this study, a 1H−15N rINEPT with heteronuclear correlation (rINEPT-HETCOR) experiment is applied to a hydrated sample of [15N-Gly]-enriched, naturally cross-linked elastin. As with a recombinant elastin,26 multiple populations of Gly are observed, and temperature coefficients for each of the resolved 1HN chemical shifts are reported. Furthermore, an unambiguous assignment of glycine-neighboring-glycine (GlyGly) in [U-13C, 15N-Gly]NRSMC elastin is achieved by a pulse sequence based on a 13C−15N J-based transfer. In addition to analyzing the Gly-rich domains, 15N variabletemperature direct-polarization with magic-angle-spinning (DPMAS) experiment was conducted on [15N-Pro]elastin. Again, multiple populations of the prolines are resolved, and tentative assignments are made alongside variable-temperature experiments that distinguish the peaks and their constituent populations’ likely involvement in hydrogen-bonded structures. The [13CCO-Val, 15N-Pro]elastin sample was used to confirm the assignment of the 15N-Pro in ValPro and to determine the principal values for the 13CCO-Val tensor. The spectroscopic data from three of the major four amino acid residues in elastin support the existence of β-turns and random coil in its hydrophobic domains. These findings support Tamburro’s conformational ensemble model for elasticity.
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deionized (or ultra pure) water. A denatured elastin sample was prepared by soaking lyophilized protein in 8 M urea overnight at 4 °C. The isotopic incorporation for the Gly residues was measured by 13 C solution NMR spectroscopy,29,31 while the labeling for Pro and Val was assessed by derivatization of the hydrolysate with a modified Marfey’s reagent, Nα-(2,4-dinitro-5-fluorophenyl)-L-leucinamide, followed by mass spectrometry.32 For the Pro and Val quantification, the mixture of amino acid derivatives was analyzed by LC-MSD TOF (Agilent Technologies, Santa Clara, CA). The [MH]+ and [MH + 1]+ peaks were integrated using Mass Hunter (Agilent Technologies, Santa Clara, CA) to determine the abundance (Ab) of the isotopically enriched and unlabeled amino acids. The enrichment as a percentage of the given amino acid was calculated using the equation
% isotopic enrichment = 100 × (R e − R c)/(1 + R e − R c) where Re = Ab[MH+1]+/Ab[MH]+ of enriched sample and Rc = Ab[MH+1]+/ Ab[MH]+ of unenriched sample.33 Synthesis of poly(Gly[15N-Pro]) begins with the preparation of the protected tetrapeptide BocGPGPOBzl (Bzl = benzyl).34 (Attempts to synthesize the polymer from a dipeptide resulted in cyclization and negligible linear polymer.) A 1:2 (w/w) mixture of 15N-Pro and unenriched (natural-abundance) proline (TCI, OR) was used as starting materials to prepare BocGly[15N-Pro]Gly[15N-Pro]OBzl (15N NMR (50 MHz, 15N-Gly) δ: 127.2 and 128.8 ppm). Deprotection took place over two steps; first, hydrogenolysis of the C-terminal group followed by coupling with 4-nitrophenol afforded the nitrophenyl ester. The IR spectrum showed an intense band at 1767 cm−1. In the second step, Boc was removed by reaction with trifluoroacetic acid. Upon removal of the excess acid and solvent, polymerization was initiated with the addition of triethylamine, the reaction mixture was stirred for 2 days. Then, addition of acetone to the reaction mixture isolated the polymer as an oil. The oil was redissolved in methanol and reprecipitated with diethyl ether. The residual solvent was removed under N2 gas to give a light yellow powder. The IR spectrum of the product showed intense bands at 3093 cm−1 (NH) and 1649 cm−1 (CO). The dry polymer was hydrated by placing in a chamber saturated with water vapor overnight, yielding a viscous, yellow liquid, which was then transferred to the MAS rotor. Solid-State NMR Spectroscopy. Data were acquired on a Varian Inova NMR spectrometer (or Agilent DD2 NMR spectrometer) (Agilent Technologies, Santa Clara, CA) equipped with a wide-bore (89 mm) superconducting magnet (Oxford Instruments, Oxford, UK) with a 1H resonance frequency of 399.964 MHz. The probe used for these experiments was a 4 mm triple-resonance (HXY) T3 magicangle-spinning (MAS) probe (Chemagnetics/Varian NMR, Ft. Collins, CO). The sample temperatures were calibrated using lead nitrate, Pb(NO3)2,35 under MAS with an 8000 Hz spinning rate. 15N chemical shifts were referenced to the NH3 scale [δ(15N) = 0 ppm], using 15Nglycine [δ(15N) = 32.0 ppm at 37 °C] as an external standard. 1H chemical shifts were externally referenced to sodium 2,2-dimethyl-2silapentane-5-sulfonate (DSS) in D2O [δ(1H) = 0.0 ppm at 32 °C]. 13 C chemical shifts were referenced to the tetramethylsilane scale, using hexamethylbenzene as an external standard [δ(13C) = 17.0 ppm at 25 °C]. An 8.0 kHz MAS rate was used for all experiments, except the CSA measurement. For direct polarization (DP), or single-pulse excitation, of the 15N, a 9.5 μs 15N 90° pulse was used with a 20 s recycle delay for [15NGly]elastin and 30 s for [15N-Pro]elastin, unless otherwise stated. Baselines of the DP spectra were corrected using the algorithm described by Golotvin and Williams.36 For cross-polarization (CP), a 3.3 μs 1H 90° pulse was followed by a 2 ms contact time with a 5 s recycle delay. The CP field strengths were set to γHB1H/2π = 34 kHz and γNB1N/2π = 26 kHz, fulfilling the γHB1H/2π = γ NB1N/2π + 1*νr Hartman−Hahn condition, where νr is a spinning rate of rotor. The MAS spinning speed is νr = 8 kHz, as noted above. The 1H−15N rINEPT27,28 spectra were obtained using 90° 1H and 15N pulse lengths of 4.0 and 9.0 μs, respectively. Delays for the first spin−echo (τ−π−τ)
EXPERIMENTAL SECTION
Preparation of Enriched Samples and Assay for Isotopic Incorporation. The preparation and purification of the enriched samples from the NRSMC line were reported previously.29 Briefly, the smooth muscle cells were isolated from the aorta of 2−3 day old rat pups and incubated in a growth media that includes Dulbecco’s modified Eagle’s medium (DMEM) and minimum essential medium (MEM) nonessential amino acids. All growth media components for the normal (unlabeled, control) cultures were purchased from ThermoFisher (Hanover Park, IL), unless otherwise specified. Upon reaching confluency, the cells initiate the highest rate of elastin synthesis, and the labels are introduced, as described below. After 6−8 weeks of growth, the elastin-rich matrix was harvested. Elastin was purified by the cyanogen bromide method. Labeling of the amino acids required modification of the growth media. For the labeling of the Gly and Val residues, a custom DMEM formulation without these amino acids was used. The culture media was supplemented with isotopically enriched Gly (15N-Gly or [U-13C,15N]Gly) to a concentration of 0.45 mM. For the extensive 15 N labeling, the concentration of α-15N-Gln in the cell culture media was 4 mM. For the enrichment of the Pro residues, a MEM nonessential amino acids solution (100X stock) was prepared with 100 mM 15N-Pro. The [13CCO-Val, 15N-Pro]elastin was prepared by using 0.7 mM [1-13C]Val and 1.0 mM 15N-Pro in the growth media All enriched amino acids were purchased from Cambridge Isotope laboratories (Andover, MA). The isotope purity for the singly enriched amino acids [1-13C]Val and 15N-Pro are 99% and 98%, respectively; the enrichment of the uniformly labeled [U-13C, 15N]Gly is 98%. Each sample was packed into a 4 mm rotor that was sealed to preserve water content with a Kel-F spacer (Revolution NMR, Ft. Collins, CO) that was fitted with fluorosilicone micro-O-rings (Apple Rubber Products Inc., Lancaster, NY).30 The dry weight of the samples is ∼20 mg. The approximate wet weight of a typical sample for ssNMR is 60 mg. The water content of hydrated elastin is ∼70% (w/w). The enriched elastin samples used in this study were hydrated with B
DOI: 10.1021/acs.macromol.7b02616 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules and the second (τ′−π−τ′) of rINEPT were optimized to τ = τ′ = 2.2 ms. A 1.5 s recycle delay was used. Two-pulse phase modulation (TPPM) 1H decoupling37 was applied during acquisition for DP, CP, and rINEPT experiments, using an applied field strength of γHB1H/2π ∼ 60 kHz. The 15N-edited 1H spectra were obtained with the pulse sequence shown in Figure S1. The 1H magnetization is transferred to its directly bonded 15N via rINEPT. An 15N 90° pulse then restores the transferred magnetization to the longitudinal axis. The water signal is suppressed by the MISSISSIPPI scheme (γHB1H/2π ∼ 20 kHz).38 Finally, the 15N magnetization is transferred to its directly bonded 1H via rINEPT followed by 1H detection with WALTZ-16 15N decoupling (γNB1N/2π ∼ 4 kHz).39 The 90° pulse lengths for 1H and 15N were 4.2 and 8.5 μs, respectively. Each period τ was set to 2.2 ms. A 3 s recycle delay was used. For the 1H−15N 2D rINEPT-HETCOR experiment, the rINEPT parameters were the same as described for the 1D experiments. In addition, the spectral width in the indirect dimension was 1200 Hz, with a maximum t1 evolution time of 20.0 ms over 25 increments. 128 scans were acquired per t1 point, such that each 2D experiment required 3.0 h of measuring time. “Semi-constant-time” 1H chemical shift evolution in the indirect dimension was used.26,40 Data were acquired at 5, 13, 21, 29, and 37 °C. The {13CO}-15N-1H HETCOR spectrum for [U-13C, 15NGly]elastin was obtained with using the pulse sequence shown in Figure 1. Initially, the 13C transverse magnetization is prepared by DP
between directly bonded 15N−13CO. The relaxation parameters were experimentally obtained as T′C2 = 12.8 ms and T′N2 = 33.4 ms, with the τ−π−τ sequence under 1H TPPM decoupling (γHB1H/2π ∼ 42 kHz). For the T′C2 measurement, DP excitation with steady-state NOE and a 13CO selective π pulse were applied; 1H−15N rINEPT with a nonselective π pulse was used for the T′N2 measurement. The 13 C−15N rINEPT delays were calculated as τ1 = 6.0 ms and τ2 = 10.5 ms from the above equation to maximize the transfer efficiency (9.5%). The 15N−1H rINEPT delays of τ3 = 2.5 ms and τ4 = 2.25 ms were experimentally optimized. A 1.4 s recycle delay was used. The spectral width in the indirect dimension was 800 Hz, with a maximum t1 evolution time of 18.8 ms over 16 increments. 256 scans were acquired per t1 point. The spectrum was acquired at 42 °C to increase the transfer efficiencies from 13C-to-15N and 15N-to-1H. The {13CO}-15N spectrum for [13CCO-Val, 15N-Pro]elastin was obtained with using the pulse sequence shown in Figure S2. The initial 13 C transverse magnetization was prepared by DP excitation with steady-state NOE enhancement.41 τ1 and τ2 delays were set to 7 and 10.5 ms, respectively. TPPM decoupling was applied during rINEPT and acquisition periods with γHB1H/2π ∼ 37 kHz. The 90° pulse lengths for 13C and 15N were 4.5 and 9.0 μs, respectively. The recycle delay was set to 4.2 s. Chemical shift anisotropy (CSA) of 13CCO-Val in Val-Pro was determined from a slow spinning MAS (νr = 2.0 kHz) experiment with 1 H−15N−13C double CP (DCP) (Figure S3)46 of frozen [13CCO-Val, 15 N-Pro]elastin. CP contact times were set to 1 and 4 ms for 1H−15N CP and 15N−13C CP, respectively. The CP field strengths were set to γHB1H/2π = 30 kHz and γNB1N/2π = 28 kHz for first CP and γNB1N/2π = 28 kHz and γCB1C/2π = 26 kHz for second CP. The bandwidth on matching is relatively broad, such that even with the apparent 2 kHz differences, Hartmann−Hahn matching is achieved. Ramp-CP was applied to 1H channel for the first CP and to 13C channel for the second CP with 95−105% linear ramp.47 CW decoupling was applied during the second CP with γHB1H/2π ∼ 77 kHz. TPPM decoupling was applied during acquisition with γHB1H/2π ∼ 55 kHz. The recycle delay was 5 s. The simulation of the spinning sidebands was based on the Herzfeld−Berger analysis.48 The extracted principal values of the CSA tensor were used to compute the asymmetry parameter, η. η = (δ22 − δ11)/(δ33 − δiso) The three principal values were assigned according to the convention |δ33 − δiso| ≥ |δ11 − δiso| ≥ |δ22 − δiso|.
Figure 1. Pulse sequence for {13CO}-15N-1H HETCOR experiment. The water suppression was achieved by MISSISSIPPI.38 Filled narrow and unfilled wide rectangles represent π/2 and π pulses, respectively. HS indicates homospoil gradients.
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RESULTS AND DISCUSSION Enrichment of Elastin at Its Abundant Glycines, Valines, and Prolines. Previously, we described a method by which elastin is labeled at its abundant glycines, using an enrichment strategy that utilizes the NRSMC line.29 Briefly, upon reaching confluency, the growth media is changed to a formulation that includes the desired amino acid, such as 13C-, 15 N-, and/or 2H-enriched Gly. The isotopically enriched amino acid is included in media at the same concentrations as the unenriched amino acid in the control (unenriched) media. This approach yields elastin with enrichment at 35−40% of its glycines, and the incorporation of valines, an essential amino acid, is even higher (>90%). In contrast, the nonessential prolines are not extensively labeled by this matched approach. Incorporation of isotopically labeled prolines was 0),79 effectively creating a steric barrier to rotation or motion, thus “locking” the delocalization into place. In contrast, an Ala that precedes Pro does not experience this type of
Figure 11. Sideband patterns for 13CCO-Val in elastin at 2 kHz spinning speed. (a) Experimental (DCP) line shape for 13CCO-Val (4096 scans, −20 °C); (b) best-fit values from Herzfeld−Berger analysis.48 The spinning sideband intensities were calculated using a Gaussian line shape with fwhm = 300 Hz. The 13C−15N dipolar coupling is negligible in the sideband pattern.
Table 2. Principal Values and Asymmetry Parameter, η, of the 13CCO-Val Chemical Shift Tensor
[13CCO-Val,15N-Pro] elastin [13CCO-Val6] in (VPGVG)3a [13CCO-Val29] in [U−13C,15N] GB1b 13 [ CCO-Val1] in ValGlyGlyc a
local structure
δ11
δ22
δ33
δiso
η
234
194
87
172.0
0.47
243
185
88
171.7
0.69
253
191
93
179.0
0.72
type II βturn α-helix
245
170
93
169.2
0.98
β-sheet
Reference 11. bReference 77. cReference 74.
Hence, the 13CCO-Val is a likely hydrogen-bond acceptor. In addition, the asymmetry parameter η = 0.47 of the [13CC=OVal]Pro is closest to that determined for the Type II β-turn
Figure 12. Distribution of VP, GG, VPG, VPGG, and GXGGX in rat tropoelastin. Hydrophobic and cross-linking domains are shown in magenta and cyan, respectively. Hydrophobic domains are labeled at the top of the schematic. The signaling and C-terminal domains are not shown. Populations of these sequences are shown by dark blue rectangles. The number of occurrences of each sequence in each domain of tropoelastin is reflected as the height of each rectangle (between 0 and 5). The primary sequence is obtained from ElastoDB.3 I
DOI: 10.1021/acs.macromol.7b02616 Macromolecules XXXX, XXX, XXX−XXX
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delocalization and undergoes more rapid, large-amplitude motions. This proposed model is consistent with that of Urry, who reported the relative proximity of valine and proline side chains in the VPGG and APGG elastin mimetics.80 Likely hydrogen-bond donors are the amide protons of glycines that are preceded by another glycine (GlyGly). The GlyGly pair is found throughout the protein, but it is more abundant in the hydrophobic domains (i.e., as compared to cross-linking domains). In sharp contrast to the Pro or ValProcontaining regions, the lack of residues on two neighboring glycines characterizes a relatively small (compact) and mobile segment, such the proton(s) on these sites may have greater access to acceptor sites, such as the CO of ValPro. A tentative β-turn assignment for ValPro and GlyGly in elastin is consistent with previous structural studies on its mimetics19 and single-domain peptides.10 Furthermore, the βturn is highly favored in sequences in which Pro is preceded by Val and followed by Gly.5,81 For example, the tetrapeptide VPGG82 and its polymer, (VPGG)n,83 adopt β-turns (Figure 13). About 90% of the ValPro pairs in rat tropoelastin occur as
AUTHOR INFORMATION
Corresponding Author
*(K.K.K.) Phone 1-808-956-5733; Fax 1-808-956-5908; e-mail
[email protected]. ORCID
Kristin K. Kumashiro: 0000-0002-5208-1119 Funding
This material is based upon work supported by the National Science Foundation under Grants MCB-1022526 and CHE1532310. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Dr. Walter P. Niemczura for helpful discussions and technical assistance. REFERENCES
(1) Rosenbloom, J.; Abrams, W. R.; Mecham, R. Extracellular matrix. 4: The elastic fiber. FASEB J. 1993, 7, 1208−1218. (2) Pierce, R. A.; Deak, S. B.; Stolle, C. A.; Boyd, C. D. Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning. Biochemistry 1990, 29, 9677−9683. (3) He, D.; Chung, M.; Chan, E.; Alleyne, T.; Ha, K. C. H.; Miao, M.; Stahl, R. J.; Keeley, F. W.; Parkinson, J. Comparative genomics of elastin: Sequence analysis of a highly repetitive protein. Matrix Biol. 2007, 26, 524−540. (4) Rauscher, S.; Baud, S.; Miao, M.; Keeley, F. W.; Pomes, R. Proline and glycine control protein self-organization into elastomeric or amyloid fibrils. Structure 2006, 14, 1667−1676. (5) Hutchinson, E. G.; Thornton, J. M. A revised set of potentials for β-turn formation in proteins. Protein Sci. 1994, 3, 2207−2216. (6) Hoeve, C. A. J.; Flory, P. J. Elastic properties of elastin. Biopolymers 1974, 13, 677−686. (7) Gray, W. R.; Sandberg, L. B.; Foster, J. A. Molecular model for elastin structure and function. Nature 1973, 246, 461−466. (8) Venkatachalam, C. M.; Urry, D. W. Development of a linear helical conformation from its cyclic correlate. β-Spiral model of the elastin poly(pentapeptide) (VPGVG)n. Macromolecules 1981, 14, 1225−1229. (9) Debelle, L.; Tamburro, A. M. Elastin: molecular description and function. Int. J. Biochem. Cell Biol. 1999, 31, 261−272. (10) Tamburro, A. M.; Bochicchio, B.; Pepe, A. Dissection of Human Tropoelastin: Exon-By-Exon Chemical Synthesis and Related Conformational Studies. Biochemistry 2003, 42, 13347−13362. (11) Yao, X. L.; Hong, M. Structure Distribution in an ElastinMimetic Peptide (VPGVG)3 Investigated by Solid-State NMR. J. Am. Chem. Soc. 2004, 126, 4199−4210. (12) Ohgo, K.; Ashida, J.; Kumashiro, K. K.; Asakura, T. Structural Determination of an Elastin-Mimetic Model Peptide, (Val-Pro-GlyVal-Gly)6, Studied by 13C CP/MAS NMR Chemical Shifts, TwoDimensional off Magic Angle Spinning Spin-Diffusion NMR, Rotational Echo Double Resonance, and Statistical Distribution of Torsion Angles from Protein Data Bank. Macromolecules 2005, 38, 6038−6047. (13) Kumashiro, K. K.; Ohgo, K.; Niemczura, W. P.; Onizuka, A. K.; Asakura, T. Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB. Biopolymers 2008, 89, 668−679. (14) Vrhovski, B.; Jensen, S.; Weiss, A. S. Coacervation characteristics of recombinant human tropoelastin. Eur. J. Biochem. 1997, 250, 92−98. (15) Reichheld, S. E.; Muiznieks, L. D.; Stahl, R.; Simonetti, K.; Sharpe, S.; Keeley, F. W. Conformational Transitions of the Crosslinking Domains of Elastin during Self-assembly. J. Biol. Chem. 2014, 289, 10057−10068. (16) Debelle, L.; Alix, A. J. P.; Jacob, M.; Huvenne, J.; Berjot, M.; Sombret, B.; Legrand, P. Bovine Elastin and κ-Elastin Secondary
Figure 13. Delocalization of the electron pair of proline’s nitrogen in VPGG. At lower temperatures, the equilibrium is shifted to the right.
VPG,3 such as the commonly known repeating VPGVG motif. Previous NMR and CD studies on VPGVG peptides11,12,65,84,85 indicate significant Type II β-turn character in model systems. Finally, GlyGly in G1X2G3G4X5 repeats are abundant in the hydrophobic domains.3 CD and NMR measurements provide evidence of dynamic β-turns as the dominant feature in these mimetics.18 The G1X2G3G4X5 motifs are found in a rapid equilibrium of two conformations that differ in the position of the hydrogen-bond, i.e., Gly1 (CO) and Gly4 (NH) versus X2 (CO) and X5 (NH), or the “sliding β-turn”.9,18 Finally, these conclusions also are consistent with structural studies on other elastomeric proteins. A recurring theme among these mechanical proteins is the occurrence of multiple populations of motifs that have low energy barriers for interconversion. For example, distributions of β-turns, coil, and extended structures are found in mimetics of titin86 and lamprin.87 This observation suggests that the mechanism for elasticity is not purely entropic, as predicted by the random coil theory. As more residue-specific information on this complex biopolymer is acquired, a more refined model for elasticity will emerge.
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Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b02616. Pulse sequences used in this study, information on NMR spectra of the samples (chemical shift, line widths, peak area, or volume), and additional NMR spectra (PDF) J
DOI: 10.1021/acs.macromol.7b02616 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules Structure Determination by Optical Spectroscopies. J. Biol. Chem. 1995, 270, 26099−26103. (17) Reichheld, S. E.; Muiznieks, L. D.; Keeley, F. W.; Sharpe, S. Direct observation of structure and dynamics during phase separation of an elastomeric protein. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E4408−E4415. (18) Tamburro, A. M.; Guantieri, V.; Pandolfo, L.; Scopa, A. Synthetic Fragments and Analogues of Elastin. II. Conformational Studies. Biopolymers 1990, 29, 855−870. (19) Urry, D. W.; Mitchell, L. W.; Ohnishi, T. Carbon-13 magnetic resonance assignments of the repeat peptides of elastin and solvent delineation of carbonyls. Biochemistry 1974, 13, 4083−4090. (20) Chang, D. K.; Venkatachalam, C. M.; Prasad, K. U.; Urry, D. W. Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin. J. Biomol. Struct. Dyn. 1989, 6, 851−858. (21) Perry, A.; Stypa, M. P.; Tenn, B. K.; Kumashiro, K. K. Solid-state 13 C NMR reveals effects of temperature and hydration on elastin. Biophys. J. 2002, 82, 1086−1095. (22) Pometun, M. S.; Chekmenev, E. Y.; Wittebort, R. J. Quantitative Observation of Backbone Disorder in Native Elastin. J. Biol. Chem. 2004, 279, 7982−7987. (23) Ohgo, K.; Niemczura, W. P.; Muroi, T.; Onizuka, A. K.; Kumashiro, K. K. Wideline Separation (WISE) NMR of Native Elastin. Macromolecules 2009, 42, 8899−8906. (24) Ohgo, K.; Niemczura, W. P.; Kumashiro, K. K. Probing the Natural-Abundance 13C Populations of Insoluble Elastin Using 13C-1H Heteronuclear Correlation (HETCOR) NMR Spectroscopy. Macromolecules 2009, 42, 7024−7030. (25) Kumashiro, K. K.; Ohgo, K.; Elliott, D. W.; Kagawa, T. F.; Niemczura, W. P. Backbone motion in elastin’s hydrophobic domains as detected by 2H NMR spectroscopy. Biopolymers 2012, 97, 882−888. (26) Ohgo, K.; Niemczura, W. P.; Seacat, B. C.; Wise, S. G.; Weiss, A. S.; Kumashiro, K. K. Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy. J. Biol. Chem. 2012, 287, 18201−18209. (27) Morris, G. A. Sensitivity Enhancement in 15N NMR: Polarization Transfer Using the INEPT Pulse Sequence. J. Am. Chem. Soc. 1980, 102, 428−429. (28) Burum, D. P.; Ernst, R. R. Net Polarization Transfer via a JOrdered State for Signal Enhancement of Low-Sensitivity Nuclei. J. Magn. Reson. 1980, 39, 163−168. (29) Perry, A.; Stypa, M. P.; Foster, J. A.; Kumashiro, K. K. Observation of the glycines in elastin using 13C and 15N solid-state NMR spectroscopy and isotopic labeling. J. Am. Chem. Soc. 2002, 124, 6832−6833. (30) Martin, R. W.; Paulson, E. K.; Zilm, K. W. Design of a triple resonance magic angle sample spinning probe for high field solid state nuclear magnetic resonance. Rev. Sci. Instrum. 2003, 74, 3045−3061. (31) Kummerlen, J.; vanBeek, J. D.; Vollrath, F.; Meier, B. H. Local structure in spider dragline silk investigated by two-dimensional spindiffusion nuclear magnetic resonance. Macromolecules 1996, 29, 2920− 2928. (32) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide: Combination of Marfey’s method with mass spectrometry and its practical application. Anal. Chem. 1997, 69, 5146−5151. (33) Wolfe, R. R. Tracers in Metabolic Research: Radioisotope and Stable Isotope/Mass Spectometry Methods; A.R. Liss, Inc.: New York, 1984; p 232. (34) Wolfe, S.; Bowers, R. J.; Shin, H. S.; Sohn, C. K.; Weaver, D. F.; Yang, K. Y. Phenceptin: a biomimetic model of the phenytoin receptor. Can. J. Chem. 1988, 66, 2751−2762. (35) Bielecki, A.; Burum, D. P. Temperature-Dependence of Pb-207 MAS Spectra of Solid Lead Nitrate. An Accurate, Sensitive Thermometer for Variable-Temperature MAS. J. Magn. Reson., Ser. A 1995, 116, 215−220.
(36) Golotvin, S.; Williams, A. Improved Baseline Recognition and Modeling of FT NMR Spectra. J. Magn. Reson. 2000, 146, 122−125. (37) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin, R. G. Heteronuclear Decoupling in Rotating Solids. J. Chem. Phys. 1995, 103, 6951−6958. (38) Zhou, D. H.; Rienstra, C. M. High-performance solvent suppression for proton detected solid-state NMR. J. Magn. Reson. 2008, 192, 167−172. (39) Shaka, A. J.; Keeler, J.; Frenkiel, T.; Freeman, R. An Improved Sequence for Broad-Band Decoupling - WALTZ-16. J. Magn. Reson. 1983, 52, 335−338. (40) Helmus, J. J.; Surewicz, K.; Surewicz, W. K.; Jaroniec, C. P. Conformational Flexibility of Y145Stop Human Prion Protein Amyloid Fibrils Probed by Solid-State Nuclear Magnetic Resonance Spectroscopy. J. Am. Chem. Soc. 2010, 132, 2393−2403. (41) Paul, E. G.; Grant, D. M. Carbon-13 Magnetic Resonance. I. Improved Carbon-13 Magnetic Resonance Spectra Obtained by Proton Decoupling and Rapid Sample Spinning. J. Am. Chem. Soc. 1964, 86, 2977−2983. (42) Kupce, E.; Boyd, J.; Campbell, I. D. Short Selective Pulses for Biochemical Applications. J. Magn. Reson., Ser. B 1995, 106, 300−303. (43) Li, Y.; Wylie, B. J.; Rienstra, C. M. Selective refocusing pulses in magic-angle spinning NMR: Characterization and applications to multi-dimensional protein spectroscopy. J. Magn. Reson. 2006, 179, 206−216. (44) Chen, L. L.; Kaiser, J. M.; Lai, J. F.; Polenova, T.; Yang, J.; Rienstra, C. M.; Mueller, L. J. J-based 2D homonuclear and heteronuclear correlation in solid-state proteins. Magn. Reson. Chem. 2007, 45, S84−S92. (45) Lesage, A.; Bardet, M.; Emsley, L. Through-bond carbon-carbon connectivities in disordered solids by NMR. J. Am. Chem. Soc. 1999, 121, 10987−10993. (46) Schaefer, J.; Skokut, T. A.; Stejskal, E. O.; McKay, R. A.; Varner, J. E. Asparagine amide metabolism in developing cotyledons of soybean. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 5978−5982. (47) Metz, G.; Wu, X. L.; Smith, S. O. Ramped-Amplitude CrossPolarization in Magic-Angle-Spinning NMR. J. Magn. Reson., Ser. A 1994, 110, 219−227. (48) Herzfeld, J.; Berger, A. E. Sideband Intensities in NMR Spectra of Samples Spinning at the Magic Angle. J. Chem. Phys. 1980, 73, 6021−6030. (49) Vogel, H. J.; Davis, B. D. Glutamic γ-Semialdehyde and Δ1Pyrroline-5-carboxylic Acid, Intermediates in the Biosynthesis of Proline. J. Am. Chem. Soc. 1952, 74, 109−112. (50) Valle, D.; Downing, S. J.; Phang, J. M. Proline inhibition of pyrroline-5-carboxylate reductase: Differences in enzymes obtained from animal and tissue culture sources. Biochem. Biophys. Res. Commun. 1973, 54, 1418−1424. (51) Wishart, D. S.; Sykes, B. D.; Richards, F. M. The Chemical-Shift Index - a Fast and Simple Method for the Assignment of Protein Secondary Structure through NMR Spectroscopy. Biochemistry 1992, 31, 1647−1651. (52) Cornilescu, G.; Delaglio, F.; Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 1999, 13, 289−302. (53) Smith, C. K.; Withka, J. M.; Regan, L. A Thermodynamic Scale for the β-sheet forming tendencies of the amino-acids. Biochemistry 1994, 33, 5510−5517. (54) Pace, C. N.; Scholtz, J. M. A helix propensity scale based on experimental studies of peptides and proteins. Biophys. J. 1998, 75, 422−427. (55) Chou, P. Y.; Fasman, G. D. Empirical Predictions of Protein Conformation. Annu. Rev. Biochem. 1978, 47, 251−276. (56) Zhang, H. Y.; Neal, S.; Wishart, D. S. RefDB: A database of uniformly referenced protein chemical shifts. J. Biomol. NMR 2003, 25, 173−195. (57) Tomlinson, J. H.; Williamson, M. P. Amide temperature coefficients in the protein G B1 domain. J. Biomol. NMR 2012, 52, 57−64. K
DOI: 10.1021/acs.macromol.7b02616 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules (58) Doyle, C. M.; Rumfeldt, J. A.; Broom, H. R.; Sekhar, A.; Kay, L. E.; Meiering, E. M. Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts. Biochemistry 2016, 55, 1346−1361. (59) Cierpicki, T.; Otlewski, J. Amide proton temperature coefficients as hydrogen bond indicators in proteins. J. Biomol. NMR 2001, 21, 249−261. (60) Cierpicki, T.; Zhukov, I.; Byrd, R. A.; Otlewski, J. Hydrogen bonds in human ubiquitin reflected in temperature coefficients of amide protons. J. Magn. Reson. 2002, 157, 178−180. (61) Naito, A.; Tuzi, S.; Saito, H. A High-Resolution 15N Solid-State NMR Study of Collagen and Related Polypeptides - the Effect of Hydration on Formation of Interchain Hydrogen-Bonds as the Primary Source of Stability of the Collagen-Type Triple-Helix. Eur. J. Biochem. 1994, 224, 729−734. (62) Kricheldorf, H. R. 15N NMR Spectroscopy 30. Structure-Shift Relationships of Oligopeptides and Copolypeptides, Including Gramicidin-S. Org. Magn. Reson. 1981, 15, 162−177. (63) Grzesiek, S.; Stahl, S. J.; Wingfield, P. T.; Bax, A. The CD4 Determinant for Downregulation by HIV-1 Nef Directly Binds to Nef. Mapping of the Nef Binding Surface by NMR. Biochemistry 1996, 35, 10256−10261. (64) Hong, M.; Isailovic, D.; McMillan, R. A.; Conticello, V. P. Structure of an Elastin-Mimetic Polypeptide by Solid-State NMR Chemical Shift Analysis. Biopolymers 2003, 70, 158−168. (65) McMillan, R. A.; Conticello, V. P. Synthesis and Characterization of Elastin-Mimetic Protein Gels Derived from a Well-Defined Polypeptide Precursor. Macromolecules 2000, 33, 4809−4821. (66) Kawamura, I.; Ohmine, M.; Tanabe, J.; Tuzi, S.; Saito, H.; Naito, A. Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 3090−3097. (67) Li, B.; Alonso, D. O. V.; Daggett, V. The Molecular Basis for the Inverse Temperature Transition of Elastin. J. Mol. Biol. 2001, 305, 581−592. (68) Schwarzinger, S.; Kroon, G. J. A.; Foss, T. R.; Chung, J.; Wright, P. E.; Dyson, H. J. Sequence-dependent correction of random coil NMR chemical shifts. J. Am. Chem. Soc. 2001, 123, 2970−2978. (69) Wang, Y. J.; Jardetzky, O. Investigation of the neighboring residue effects on protein chemical shifts. J. Am. Chem. Soc. 2002, 124, 14075−14084. (70) Merutka, G.; Dyson, H. J.; Wright, P. E. Random Coil 1H Chemical-Shifts Obtained as a Function of Temperature and Trifluoroethanol Concentration for the Peptide Series GGXGG. J. Biomol. NMR 1995, 5, 14−24. (71) Schwarzinger, S.; Kroon, G. J.; Foss, T. R.; Wright, P. E.; Dyson, H. J. Random coil chemical shifts in acidic 8 M urea: implementation of random coil shift data in NMRView. J. Biomol. NMR 2000, 18, 43− 48. (72) Yao, J.; Chung, J.; Eliezer, D.; Wright, P. E.; Dyson, H. J. NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding. Biochemistry 2001, 40, 3561−3571. (73) Gu, Z. T.; Zambrano, R.; McDermott, A. Hydrogen-Bonding of Carboxyl Groups in Solid-State Amino-Acids and Peptides Comparison of Carbon Chemical Shielding, Infrared Frequencies, and Structures. J. Am. Chem. Soc. 1994, 116, 6368−6372. (74) Kameda, T.; Takeda, N.; Kuroki, S.; Kurosu, H.; Ando, S.; Ando, I.; Shoji, A.; Ozaki, T. Hydrogen-bonded structure and 13C NMR chemical shift tensor of amino acid residue carbonyl carbons of peptides and polypeptides in the crystalline state. Part I. J. Mol. Struct. 1996, 384, 17−23. (75) Wei, Y. F.; Lee, D. K.; Ramamoorthy, A. Solid-state 13C NMR chemical shift anisotropy tensors of polypeptides. J. Am. Chem. Soc. 2001, 123, 6118−6126. (76) Wylie, B. J.; Sperling, L. J.; Frericks, H. L.; Shah, G. J.; Franks, W. T.; Rienstra, C. M. Chemical-shift anisotropy measurements of
amide and carbonyl resonances in a microcrystalline protein with slow magic-angle spinning NMR spectroscopy. J. Am. Chem. Soc. 2007, 129, 5318−5319. (77) Hung, I.; Ge, Y. W.; Liu, X. L.; Liu, M. L.; Li, C. G.; Gan, Z. H. Measuring 13C/15N chemical shift anisotropy in 13C, 15N uniformly enriched proteins using CSA amplification. Solid State Nucl. Magn. Reson. 2015, 72, 96−103. (78) Ahmed, Z.; Myshakina, N. S.; Asher, S. A. Dependence of the AmII ’ p Proline Raman Band on Peptide Conformation. J. Phys. Chem. B 2009, 113, 11252−11259. (79) Ho, B. K.; Brasseur, R. The Ramachandran plots of glycine and pre-proline. BMC Struct. Biol. 2005, 5, 14. (80) Urry, D. W.; Khaled, M. A.; Rapaka, R. S.; Okamoto, K. Nuclear Overhauser enhancement evidence for inverse temperature dependence of hydrophobic side chain proximity in the polytetrapeptide of tropoelastin. Biochem. Biophys. Res. Commun. 1977, 79, 700−706. (81) Macarthur, M. W.; Thornton, J. M. Influence of Proline Residues on Protein Conformation. J. Mol. Biol. 1991, 218, 397−412. (82) Broch, H.; Moulabbi, M.; Vasilescu, D.; Tamburro, A. M. Quantum molecular modeling of the elastinic tetrapeptide Val-ProGly-Gly. J. Biomol. Struct. Dyn. 1998, 15, 1073−1091. (83) Khaled, M. A.; Prasad, K. U.; Venkatachalam, C. M.; Urry, D. W. Nuclear Magnetic Resonance and Conformational Energy Characterization of Repeat Peptides of Elastin - the Cyclohexadecapeptide, cyclo-(L-VAL1-L-PRO2-GLY3-GLY4)4. J. Am. Chem. Soc. 1985, 107, 7139−7145. (84) Urry, D. W. Characterization of Solubule Peptides of Elastin By Physical Techniques. Methods Enzymol. 1982, 82, 673−716. (85) Reiersen, H.; Clarke, A. R.; Rees, A. R. Short elastin-like peptides exhibit the same temperature-induced structural transitions as elastin polymers: implications for protein engineering. J. Mol. Biol. 1998, 283, 255−264. (86) Ma, K.; Wang, K. Malleable conformation of the elastic PEVK segment of titin: non-co-operative interconversion of polyproline II helix, β-turn and unordered structures. Biochem. J. 2003, 374, 687− 695. (87) Bochicchio, B.; Pepe, A.; Tamburro, A. M. On (GGLGY) synthetic repeating sequences of lamprin and analogous sequences. Matrix Biol. 2001, 20, 243−250.
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DOI: 10.1021/acs.macromol.7b02616 Macromolecules XXXX, XXX, XXX−XXX
