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Detection of Labile Conformations of Elastin’s Prolines by Solid-State NMR and FTIR Techniques Chester L. Dabalos, Kosuke Ohgo, and Kristin K. Kumashiro Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00414 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Biochemistry

Detection of Labile Conformations of Elastin’s Prolines by Solid-State NMR and FTIR Techniques

Chester L. Dabalos, Kosuke Ohgo and Kristin K. Kumashiro*

Contribution from the Department of Chemistry, University of Hawaii, 2545 McCarthy Mall, Honolulu, Hawaii, 96822 (USA)

*Address correspondence to: Kristin K. Kumashiro, University of Hawaii, Department of Chemistry, 2545 McCarthy Mall, Honolulu, Hawaii 96822 (USA); Phone: 1-808-956-5733; Fax: 1-808-956-5908; Email: [email protected]

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Abstract Samples of native elastin are prepared with high levels of enrichment at its prolines, which are believed to play a major role in the elasticity of elastin. Major and minor populations of trans and cis isomers at the XaaPro imide bonds are detected in two-dimensional (2D) 13C NMR experiments. One- and two-dimensional 13C NMR and isotope-edited FTIR experiments are also used to identify the prolines’ folded and unfolded states, Type II β-turn and random coil, respectively, at physiological temperatures. This study provides new details on elastin’s conformational ensemble. In addition, the cis-trans isomerization of its abundant prolines provides an additional mechanism of fiber elongation in tissue.


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Biochemistry

Introduction The extensibility of blood vessels and other tissues is attributed to the biopolymer elastin. Most of the domains of elastin’s monomer, tropoelastin, fall into two general categories, i.e., crosslinking and hydrophobic.1 Putatively, the former confers durability2 while the latter is believed to be the origin of elasticity.3 Elastin’s hydrophobic domains are abundant in glycines (Gly, G), alanines (Ala, A), valines (Val, V), and prolines (Pro, P), so characterization of these residues may be the key to defining the relationships between structure and function in this protein. Our recent study on the backbone atoms of VP and GG dipeptide sequences, found abundantly in the hydrophobic domains of elastin, identified the presence of both Type II -turn and random coil.4 Turns and (random) coils do not have long-range order, thus imparting macroscale flexibility to this macromolecule. The molecular mechanism of elastin’s elasticity is not fully established, and several contradicting models exist. The earliest model suggested that elastin is devoid of local structure and behaves like rubber; i.e., elongation and restoration (to the original shape) are characterized by loss and gain of entropy, respectively.5 In contrast, the repeating β-turn was proposed for the elastin mimetic poly(VPGVG).6 Solution nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) studies of peptides with the sequences of single hydrophobic or crosslinking domains support an equilibrium of distinct secondary structures and the (unordered) random coil state.7 More recently, NMR studies on mimetics incorporating both domain types8 and native elastin4,9 provided additional evidence to support Tamburro’s model, which features a distribution of structures (or conformational ensemble).7


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Proline is abundant in elastomeric proteins, such as titin,10 major ampullate spindroin (MaSp2) silk,11 and the storage proteins of dough.12 Proline content differentiates ampullate silks with and without rubber-like properties,13-14 and it comprises about a tenth of elastin’s amino acids.15 Notably, elastin mimetics with greater proline content have physical properties that are representative of higher elasticity, whereas the proline-poor peptides are stiffer in nature.16 In contrast to all other amino acids, proline’s sidechain is covalently bonded to the backbone nitrogen. The absence of a NH proton in proline’s backbone disallows the long-range order that is found in helices and sheets and, consequently, reduces the extent of packing; i.e., proline’s uniqueness in this regard makes it key to its prevalence in elastomeric proteins.17 Hence, prolines favor labile “structures”, such as turns, loops, PPII, and coil.18 These environments involve fewer intramolecular hydrogen-bonds. Thus, the peptide backbone at the proline(s) may hydrogen-bond with water; i.e., with its higher relative proline content, a protein like elastin is more hydrated and flexible than its counterparts with fewer of these residues. The vast majority of peptide bonds in proteins are trans. However, if proline is present (XaaPro), the propensity for the cis isomer is greatly increased. About 90% of the cis rotamers in the Brookhaven Protein Data Bank19 are XaaPro. The trans and cis isomers of the XaaPro have a enthalpic difference of 2 kJ/mol, in contrast to the 10 kJ/mol difference for the XaaYaa (Yaa≠Pro) isomers.20 Due to the lower thermodynamic barrier, cis-to-trans isomerization (CTI) plays a key role in the folding of proteins.21 For example, the conversion to the cis configuration at a prolyl amide bond opens an ion channel in a hydroxytryptamine receptor, and isomerization to the trans state closes the channel.22


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Biochemistry

CTI is a viable mechanism for elastin’s extensibility, such that the trans population increases as the system is elongated or extended. In contrast, the more compact states are characterized by a greater percentage of cis isomers. (The distance from C-Xaa to C-Pro in the cis isomer is approximately 1 Å shorter than the trans.23) CTI was proposed as the mechanism for extension in the homopolypeptide Pro260.24 Other systems that contain Xaa-(cis-Pro) (simply represented as cis-Pro) populations include the elastomeric protein titin25 and its PEVK mimetic.26 Cis-Pro has also been observed in a recombinant (spider) silk protein, and when in the supercontracted state, the amount of this rotamer increases.27 This rotamer has also been observed in elastin mimetics,8,28 but never directly in native elastin, until the current study. Notably, CTI does not occur, if prolyl bonds are restricted to the trans configuration, such as the case for collagen.29 Young’s modulus for collagen30 is about two orders in magnitude higher than elastin31; i.e., collagen is non-extensible. The strategy of the current study employs selective amino acid labelling and solid-state NMR measurements to target and characterize the proline residues in elastin. Isotopic enrichment dramatically increases the signal-to-noise (S/N) of the targeted residue type and allows two-dimensional NMR experiments for this insoluble and extensive biopolymer, which is inaccessible to other high-resolution methods like X-ray crystallography and solution NMR spectroscopy. This general approach led to the assignments of multiple populations of elastin’s glycines32 and alanines,9 as well as preliminary information on the prolines.4 The NMR spectra are reflective of the target population’s secondary structure(s), (distributions of) sequence, and, in the case of proline, cis-trans isomerism. Specifically, isotropic 13CO and 13Cα chemical shifts are dependent on their local environment, which may include structural considerations such as the backbone conformation, or secondary structure. The interconversion between the cis-trans 5 ACS Paragon Plus Environment

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isomers is slow on the NMR timescale and can be conveniently monitored via the 13Cβ- and 13Cγ-Pro

chemical shifts. Finally, the 13Cδ-Pro and 15N-Pro resonances are sensitive to

neighboring residue effects. Thus, with full enrichment of the carbons and nitrogens of prolines, a detailed assessment of its structural features may be undertaken. Recently, we reported that the successful incorporation of 15N-Pro in elastin was accomplished by feedback-by-inhibition.4 In turn, a broad range of 15N solid-state NMR spectroscopic methods was employed for the characterization of the proline-rich domains of elastin.4 In the current study, elastin is enriched with uniformly-13C-labeled prolines, [U-13C-Pro], for a more comprehensive evaluation of the conformational ensemble that characterizes this key amino acid. Heteronuclear and homonuclear NMR strategies, together with isotope-edited FTIR spectroscopy, provide more support for models with structural heterogeneity; i.e., the prolines of hydrated, native elastin are detected in Type II β-turns and random coils. Our study also shows the coexistence of both trans- and cis-Pro isomers in native elastin. Tentative structural assignments from isotropic 13C chemical shifts of the hydrated protein at physiological temperatures are complemented by chemical shift anisotropy (CSA) data for the frozen elastin, which provides evidence for the possibility of PPII, as well. These structural details are then discussed in the context of elastin structure-function, as the role of prolines in biological elasticity is further examined.


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Biochemistry

Experimental Procedures

Preparation of enriched elastin and unenriched polypeptides Previous publications provide details of the preparation and purification of Pro-labeled elastin from the neonatal rat smooth muscle cell (NRSMC) line.4,32 Briefly, the smooth muscle cells were isolated from the aorta of 2-3 day old rat pups and were incubated in a growth media that includes Dulbecco’s modified Eagle’s medium (DMEM) and minimum essential medium (MEM) non-essential amino acids. All growth media components for the cell cultures were purchased from ThermoFisher (Hanover Park, IL). Upon reaching confluency, the cells initiate elastin synthesis. The elastin was enriched at the prolines using a MEM non-essential amino acid solution containing a 10-fold excess (100 mM) of [U-13C, 99%]Pro (Cambridge Isotope Laboratories, Andover, MA). The ten-fold excess in proline concentration (compared to standard growth media without isotopic enrichment) is necessary for high isotopic enrichment through the feedback-by-inhibition mechanism.4 The elastin-rich matrix was harvested after 6-8 weeks of incubation. Elastin was purified by the cyanogen bromide method,33 which cleaves proteins at the methionines, of which elastin has none.15 The incorporation of [U-13C]Pro was verified by 13C solution NMR of the elastin hydrolysate and was consistent with the LCMS study from our previous work.4 The 1JCC of the prolines in the elastin hydrolysate (57.7, 32.9, 32.7, and 31.8 Hz) are nearly identical with those of the [U-13C-Pro] thyrotropin releasing factor (Table S1),34 indicating that the enriched proline was not metabolized by the cell. The protein is hydrated with ultrapure water, and the approximate water content is ~70% (w/w). The wet weight of the elastin sample for this ssNMR study is ~60 mg. The enriched elastin was packed into a 4 mm rotor that 7 ACS Paragon Plus Environment

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was sealed to preserve water content using a Kel-F spacer (Revolution NMR, Ft. Collins, CO) fitted with fluorosilicone micro-o-rings (Apple Rubber Products Inc., Lancaster, NY).35 The preparation of model peptides poly(GlyPro) and poly(ProGlyGly) have been described previously.4,36 Briefly, the protected tetrapeptide BocGPGPOBzl (Bzl = benzyl) was prepared. Hydrogenolysis followed by coupling with 4-nitrophenol afforded the nitrophenyl ester. Removal of the Boc group with subsequent base-catalyzed polymerization gave poly(GlyPro). The synthesis of poly(ProGlyGly) begins with the coupling of BocPro and GlyGlyOMe to give the protected tripeptide BocPGGOMe. Deprotection took place over two steps. First, base-catalyzed hydrolysis of the C-terminal methyl group followed by coupling with 4-nitrophenol afforded the nitrophenyl ester. The IR spectrum showed an intense band at 1767 cm-1. Second, Boc was removed by reaction with trifluoroacetic acid. Upon removal of the solvent and excess acid, polymerization was initiated with the addition of triethylamine. The product precipitated from the reaction mixture and was filtered. The residue was dissolved in formic acid and allowed to dry as a thin film. IR spectrum of the product showed intense bands at 3093 (NH) and 1649 (CO) cm-1.

Solid-state NMR spectroscopy Data were acquired on an Agilent DD2 NMR spectrometer (or a Varian Inova 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 magic angle spinning (MAS) probe (Chemagnetics/Varian NMR, Ft. Collins, CO). The sample 8 ACS Paragon Plus Environment

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Biochemistry

temperatures were calibrated using lead nitrate, Pb(NO3)2,37 under MAS with an 8000 Hz spinning rate, unless otherwise specified. 1H chemical shifts were externally referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) in D2O [δ(1H) = 0.0 ppm at 25 °C]. 13C chemical shifts were referenced to the tetramethylsilane scale, using hexamethylbenzene as an external standard [δ(13C) = 17.0 ppm at 25 C]. The 13C direct polarization (DP) measurements at 37 °C for the labelled and unenriched elastin samples were acquired with a 4.0 μs 90° pulse followed by a Hahn echo (τ = 10 μs). The recycle delay was set to 5 s. Two-pulse phase modulation (TPPM) 1H decoupling38 was applied during acquisition for the experiments, using an applied field strength of HB1H/2 ~ 60 kHz. The spectra were processed with 30 Hz line broadening using MestReNova (MestReLab Research, Escondido, CA). Deconvolution and peak fitting for 1D spectra were performed with MATLAB (MathWorks Inc., Natick, MA). The T1 values for the proline peaks in elastin at temperatures ranging from 42 °C to -5 °C were determined from 13C inversion recovery experiments. A 4.5 μs 13C 90° pulse was used. The peak intensities were plotted against the delays and fitted to the equation: I(τ) = Mz∞[1 – 2 exp(τ∕T1)] with Origin (Origin Lab Corporation, Northampton, MA). The 1H-13C refocused insensitive nuclei enhanced by polarization transfer heteronuclear correlation (rINEPT-HETCOR) spectrum was taken at 37 °C. The 90° pulse lengths of 4.0 μs and 5.0 μs for 1H and 13C excitation were used, respectively. A 3 s recycle delay was used. Delays for the first spin-echo ( -  - ) and the second (’ -  - ’) of rINEPT were set to  = 1.5 ms and ’ = 0.75 ms, respectively. The 13C and 1H transmitter frequencies were set to 41 ppm and 3 ppm, respectively. The spectral width in the indirect dimension was 2000 Hz, with a 9 ACS Paragon Plus Environment

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maximum t1 evolution time of 17.5 ms over 36 increments. Each t1 point required 256 scans. “Semi-constant time” 1H chemical shift evolution in the indirect dimension39 was used. The spectrum was processed by applying 30 Hz line broadening in the direct dimension and 1 Hz in the indirect dimension. The 13C-13C R-TOBSY correlation for the aliphatic region was taken at 42 °C. The spinning speed was set to 8065 Hz. Low-power 1H irradiation (HB1H/2 ~ 1 kHz) for nuclear Overhauser enhancement (NOE) was applied during the entire 3 s recycle delay. A 4.0 μs 13C 90° pulse was used for excitation. The 13C transmitter frequency was placed at 45.7 ppm. The spectral width in the indirect dimension was 5250 Hz, with a maximum t1 evolution time of 9.3 ms over 50 increments. Each t1 point required 256 scans. The t1 evolution took place under TPPM decoupling with a field strength 𝛾HB1H/2𝜋 ~ 41 kHz. The mixing utilized the 𝑅3014 6 sequence40-41 (Figure S1) with a mixing time of 7.4 ms. During mixing, CW decoupling was set at 𝛾HB1H/2π~ 44 kHz with an offset of -32 kHz. The spectrum was processed by applying 10 Hz line broadening in both dimensions. The 13CO-13C R-TOBSY correlation experiment was done at 42 °C. The spinning speed was set to 8065 Hz. A 1.7 s recycle delay was used with low power 1H irradiation (HB1H/2 ~ 1 kHz) for NOE enhancement during the entire delay. A 90° pulse length of 4.5 μs was used for excitation. The 13C transmitter frequencies were set to 60 ppm, 118 ppm, and 175 ppm during t1, mixing, and t2 periods, respectively. The spectral width in the indirect dimension was 2500 Hz, with a maximum t1 evolution time of 18.8 ms over 48 increments. Each t1 point required 256 scans. The t1 evolution took place under TPPM decoupling with a field strength 𝛾HB1H/2𝜋 ~ 50 40-41 with a mixing time of 5.2 ms. LongkHz. The mixing utilized the 𝑅3014 6 sequence


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Biochemistry

observation-window band-selective homonuclear decoupling (LOW-BASHD) decoupling42 was implemented for 13C=O detection without 13C -13C=O J-coupling. Selective 200 s Gaussian  pulses were applied every 8 ms during the acquisition time. The spectrum was processed by applying 5 Hz line broadening on both dimensions. A scaled 13C-Pro CSA powder pattern of the labelled elastin was obtained from a ROCSA experiment43 at -20 °C. The spinning speed was set at 8104 Hz. 1H-13C crosspolarization (CP) was used for the excitation. A 5.0 μs ¹H 90° pulse was followed by 1 ms contact time with a 5 s recycle delay. The 13C transmitter frequency was set to 62.0 ppm. The CP field strength was set to γCB1C /2π = 𝛾HB1H/2π = 50 kHz. Following CP, ROCSA with the 𝐶212 symmetry class was applied for the t1 sampling, with the ROCSA coefficients of (a, b) = (0.032, 0.467). The CB1C/2 for ROCSA was set at 32.4 kHz corresponding to 4.00*r, as suggested by Chan and Tycko.43 The 1H decoupling field strength during ROCSA was HB1H/2 ~ 96 kHz to keep 1H/1C ~ 3.0.44 The spectral width in the indirect dimension was 4052 Hz with a maximum t1 evolution time of 4.9 ms over 21 increments. Each t1 point required 64 scans. The t1 evolution took place under CW decoupling with 𝛾HB1H/2𝜋 ~ 96 kHz. After the short Z-filter (~ 100 s) with 4.2 s 13C 90° pulses, 1H decoupling TPPM38 was applied during acquisition with an applied field strength of HB1H/2 ~ 65 kHz. The spectrum was processed without line broadening. Numerical simulations of the ROCSA spectra were carried out using the SIMPSON environment (version 4.1.1).45 For powder averaging, 320 pairs of  and  crystallite angles with the REPULSION scheme46 were used, together with 36  angles. The maximum time step was set as 2.5 s, during which time the Hamiltonian is assumed to be time-independent. In the 11 ACS Paragon Plus Environment

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simulation, the 13C CSA and the dipolar coupling between 14N and 13C were considered for this 13C-enriched

sample. The principal values of the CSA and the spatial orientation between the

CSA tensor and 14N-13C dipolar vector were obtained from the ab initio study of proline by Oldfield and coworkers.47 The dipolar strength was assumed as 700 Hz corresponding to C-N bond length of 1.46 Å. The root-mean-square-deviation (RMSD) between the observed and simulated spectra was minimized by scaling the intensity of each simulated spectrum with the SIMPLEX method in the MATLAB environment.

Isotope-edited Fourier-transformed infrared (FTIR) spectroscopy [U-13C-Pro] elastin and unenriched elastin were lyophilized and soaked in D2O overnight. The deuterated solvent was replaced with fresh D2O. The suspension was heated at 45 °C for 30 minutes to ensure complete exchange and then cooled to room temperature for measurement. All IR spectra were recorded using a Thermo Scientific iS10 Nicolet FT-IR spectrometer (ATR mode) equipped with a deuterated triglyceride sulfate detector. For each spectrum, 512 scans were collected using a resolution of 4 cm-1. Fourier transformation was done with the Happ-Genzel apodization function and zero-filled twice. Data were processed using Omnic software (ThermoFisher Scientific, Waltham, MA).

Prediction of Pro 13C chemical shifts using semi-empirical and database-based approaches Coil: Recent papers describe the prediction of chemical shifts of Ala and Gly in random coils by a semi-empirical approach.9,48 This methodology was extended to 13CO-Pro and 13C-Pro in elastin. Briefly, random coil chemical shifts were previously reported for an extensive series of 12 ACS Paragon Plus Environment

Biochemistry

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peptides, Ac-GGXGG-NH2, in denaturing conditions (8 M urea), where X is one of the proteinogenic amino acids.49 The shifts reflect the effect of neighboring residues, such that random coil shifts are reflective of sequence, in this case, R-1ProR+1. PPII and -turn: The chemical shifts of 13CO-Pro and 13C-Pro in PPII and -turns were extracted from the RefDB-C.db file in the RefDB website (http://refdb.wishartlab.com/RefDBC.db; downloaded on September 27, 2016). The chemical shifts assigned as coil in this file were chosen for this analysis, and the α-helix and β-sheet datasets were excluded. I.e., the information in the coil subset excludes helix and sheet but may include other secondary structures.50 The torsion angles of prolines and the next residue (R+1), ((Pro, Pro), (R+1, R+1)), were extracted from PDB structures with corresponding RefDB entries. Only the trans isomer (Pro ~ 180) of Pro was included in the data set. In the aggregate data set {ProCα, ProCO, Pro, Pro, R+1, R+1}, 13C chemical shifts of Pro with the conditions that satisfy (Pro - structPro)2 + (Pro -  structPro)2  (30)2 and (R+1 - struct R+1)2 + (R+1 -  struct R+1)2  (30)2 were further classified as PPII or Type II -turn. The criteria set for PPII was (structPro,  structPro) = (-70, 150) and (structR+1,  struct R+1) = (-70, 150). For Type II -turn, (structPro,  structPro) = (-70, 120) and (structR+1,  struct R+1) = (90, 0). Hydrogen-bonding was not considered in defining the local structure. Simulation of spectra: A peak with random coil chemical shift of the Pro in each of the 3-aminoacid sequences in elastin was plotted as a Gaussian function (fwhm~0.17 ppm) with relative intensity that reflects the number of occurrences (of that sequence) in elastin, as summarized in Table S2. For the -turn spectrum, the information from the PDB/RefDB (above) was selected 13 ACS Paragon Plus Environment

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for the 3-amino-acid sequences found in tropoelastin. Then, these distributions are plotted with a smoothing Gaussian function (fwhm~0.50 ppm).


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Biochemistry

Results and Discussion

Isotropic 13C chemical shifts reflect a minimum of two proline populations in elastin Isotropic chemical shifts reflect the local environment of the nuclei and provide a basis for the identification of secondary structures. For unambiguous assignments, however, isotopic enrichment at a targeted site or, in the case of elastin, a targeted amino acid type (e.g., Pro) is essential. [U-13C]Pro was incorporated into elastin by the feedback-by-inhibition strategy.4 This method yielded ~80% isotopic enrichment at the prolines. 13C solution NMR spectra of the [U13C-Pro]

elastin hydrolysate confirmed that there was no metabolic scrambling (Figure S2); i.e.,

only prolines in elastin are enriched. Figure 1 illustrates the 1D 13C DPMAS NMR spectrum of the aliphatic region of [U-13CPro] elastin. This difference spectrum was obtained by subtracting the spectrum of the unenriched elastin from that of the labeled protein (Figure 1a, Figures S3a-c). Five prominent peaks with center-of-masses at 175.0, 61.5, 48.4, 29.9, and 25.5 ppm correspond to 13C=O-, 13C-, 13Cδ-, 13Cβ-,

and 13Cγ-Pro, respectively (Table 1). Deconvolution was used to identify

minor populations of cis-Pro and 4-hydroxyproline (4-Hyp or Hyp) (Figure 1b). The 13Cβ and 13Cγ

of the cis-Pro conformer were observed at 32.6 and 22.7 ppm. Minor signals at 70.5, 55.8

and 37.8 ppm were attributed to the 4-Hyp residues.


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Figure 1. Observed and fitted 13C DPMAS spectra of [U-13C-Pro] elastin (a) Black trace is the difference between 13C DPMAS spectra of [U-13C-Pro] elastin and unenriched elastin. Blue trace is the sum of individual peaks from deconvolution of the difference spectrum (see part (b)). The difference of the observed (black) and fitted (blue) lineshapes is shown in red. The baseline (gray) is shown for comparison. (b) Individual contributions from the major Pro population (black), Hyp (green), and cis-Pro (magenta), as determined by deconvolution. The 13C-Pro and 13C=O-Pro chemical shifts were compared to mimetics of elastin and collagen, silk protein and an unstructured peptide (Table 1, Figure 2). Elastin’s 13Cα-Pro shift of 61.5 ppm is similar to the 13Cα-Pro shifts of (VPGVG)18 at 62.1 ppm51 and flagelliform silk’s (GPGGX) at 61.6 ppm.52 Both mimetics adopt β-turns, as confirmed by CD and NMR spectroscopies.51-52 The (13Cα-Pro) is similar to that of the denatured peptide, Ac-GGPGG-NH2, at 61.7 ppm.49 In contrast, elastin’s (13Cα –Pro) is significantly different (Δ=2.1 ppm) from polyPGG (Figure 2a, 2c), which is known to form PPII.53 Also, RefDB gives average shifts for 13Cα-Pro shift

in an α-helix (63.5 ppm) and β-sheet (60.6 ppm), respectively,54 and these

structures are also less probable than the -turn (Table S3). Again, prolines in elastin are most likely present in a β-turn or coil environment, corroborating our observations from [15N-Pro] elastin.4 This finding supports the propensity of Pro in a turn to be higher (than other amino 16 ACS Paragon Plus Environment

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Biochemistry

acids), especially when followed by Gly.55 Notably, about three-fourths of the prolines in elastin neighbor glycines, as ProGly. Figure 2. Comparison of 13Cα-Pro chemical shifts from experiment and databases. (a) 13C DPMAS

NMR spectrum of 13Cα-Pro of

[U-13C-Pro] elastin at 37 C, obtained by subtracting the natural-abundance 13C spectrum from the one obtained for the enriched protein; (b) 13Cα-Pro chemical shift distributions from RefDB and PDB database for Type II β-turn (ψPro=120°); (c) 13Cα-Pro region of 13C CPMAS spectra of pPGG, a model PPII polypeptide; and, (d) 13Cα-Pro chemical shift distributions from database for PPII (ψPro=150°). For (b) and (d), the 13CPro resonances were sorted into 0.5 ppm intervals. The center-of-masses of the distributions from the databases are indicated by dashed orange lines.


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Here we note that the single 13C=O-Pro peak (Figure S3c) was observed at 175.0 ppm. This site appears less sensitive to environment; e.g., the chemical shifts of this site in the -turn and random coil are nearly identical. Thus, an alternate approach and the corresponding analysis on the 13C=O site are described in a later section. Table 1. Isotropic 13C chemical shifts (ppm) of proline in elastin and selected proteins and polypeptides. All shifts are referenced to TMS. Chemical shifts in elastin are compared to (VPGVG)18,51 -GPGGX- in recombinant flagelliform silk,52 AcGGPGGNH2 in denaturing conditions,49 polyPGG, and polyGP. elastin

C=O

175.0

assignment

d

(VPGVG)18a

GPGGXb

AcGGPGGNH2c

polyPGG

β-turn

β-turn

random coil

PPII

175.9

175.4

175.8

174.2

175.0

61.6

61.7

59.4

61.0

47.4

47.5

Cα

61.5

β-turn or coil

62.1

Cδ

49.0

ValPro

49.6

47.9

XaaPro

32.6

cis

29.9

trans

30.7

30.

25.5

trans

26.0

25.3

22.7

cis

Cβ

Cγ a

47.9

polyGP

32.2 (cis) 30.2

30.1

30.0 (trans)

25.2

24.6 (trans) 22.4 (cis)

Ref. 51,b Ref. 52, c Ref. 49, d multiple components (will be resolved in a later subsection) The relationship of 13C chemical shifts of prolines to secondary structure is not as well-

defined as other amino acids. Therefore, a new approach that merges the structural information from the PDB with the chemical shifts from the NMR database RefDB is utilized here. A proline with its next-neighbor-residue R+1 is described with a given set of torsion angles (Pro, Pro, R+1, R+1). Each of the secondary structures may be defined by a range of torsion angles, e.g., (Pro=120, R+1=90, R+1=0) for a Pro in a type II -turn, with invariant Pro. Thus, the PDB structures are selected using these torsion angles, and the corresponding 13C chemical shifts are extracted from the NMR database RefDB, yielding a combined parameter set {ProCα, Pro, Pro, 18 ACS Paragon Plus Environment

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Biochemistry

R+1, R+1} for this analysis. (While this approach may be used to confirm or exclude one or more secondary structures, it cannot be used in the same manner for the random coil, i.e., by its very nature, the random coil does not have a preferred  (and/or ) value, so the database structures (and corresponding chemical shifts) cannot be selected in the same fashion.) The database information strongly suggests that the predominant conformation of the prolines in elastin (other than the random coil) is the Type II -turn, and it is more highly populated than the PPII conformation. The 13Cα-Pro shifts corresponding to PPII and Type II βturn were extracted from the RefDB and PDB databases and compared to observed peaks in the spectra of enriched elastin and the model PPII polymer, polyPGG (Figure 2). The 61.5 ppm peak of 13Cα-Pro in elastin has a difference of 0.5 ppm to the center-of-mass (COM) of the distribution of chemical shifts for the Type II β-turn (ψPro=120°, COM=62.0). The COM for the 13Cα-Pro resonances of the PPII conformation (ψPro=150°) is 60.5 ppm, or 1.5 ppm upfield relative to that of Type II β-turn. Sequences like ProPro and AlaPro have high propensity of the PPII conformation,56-57 but they are not abundant in elastin. The peak positions of the proline sidechain carbons reflect the configuration of the XaaPro peptide bond,58-59 as well as the identity of Xaa. The 13Cβ and 13Cγ shifts distinguish the trans- and cis-prolyl isomers. The major populations (85-90%) at 29.9 and 25.5 ppm were assigned to the 13Cβ- and 13Cγ of trans-Pro. The minor 13Cβ and 13Cγ peaks, at 32.6 and 22.7 ppm, were assigned to the cis-Pro isomers and accounts for 10-15% of all 13Cβ and 13Cγ signals (Table S4). The broad 13Cδ signal was deconvolved into two components at 49.0 and 47.9 ppm that were assigned as a function of the amino acid Xaa that precedes Pro, i.e., the 13Cδ-Pro shift in ValPro (49.0 ppm) is more downfield than in XaaPro with Xaa≠Val (47.9 ppm), analogous to 19 ACS Paragon Plus Environment

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the observed neighboring residue-dependence for Val[15N-Pro] versus Xaa[15N-Pro].4 This assignment is also consistent with 13Cδ-Pro shifts reported for the Type II -turn peptides, (VPGVG)18,51 polyLys-25,52 (GPGGSGPGGY)2GPGGK,60 and (GPGGA)6G.61 This study provides the first estimate of cis-Pro in hydrated elastin (at 37C). Cis-totrans ratio is estimated at 1:6 (upper limit) to 1:9 (lower limit), or roughly 2-3 times that of “nonelastin” (or general) protein populations. A 1990 reference states that 0.05% of ~31000 amide (Xaa-Yaa, YaaPro) bonds in the Brookhaven PDB are cis.20 The cis-to-trans ratio is 1:2000 for peptide bonds, generally, and reflects the “non-elastin sequences”. This study also reports that 6.5% of the Xaa-Pro bonds are cis. The cis-to-trans ratio in Xaa-Pro (general, “non-elastin sequences”) is ~1:15. This value is similar to a 2010 paper that identifies the cis rotamer in 5% (5% cis, 95% trans) of Xaa-Pro bonds in ~580 proteins in the BMRB (NMR) database,59 which gives a cis-to-trans ratio of 1:19 (general, “non-elastin”). The higher percentages of the cis-Pro rotamer in hydrated elastin at 37 C are somewhat analogous to the cis-Pro populations reported for a recombinant silk protein that undergoes supercontraction when hydrated.27 I.e., higher cisto-trans ratios are observed in Pro-rich elastic proteins.

Homonuclear 13C and heteronuclear 1H-13C 2D NMR experiments target the aliphatic 13C region to provide structural information on Pro and to confirm the presence of Hyp Assignments of the multiple populations of proline were confirmed with two-dimensional NMR experiments. A combination of strategies that distinguished cis- and trans-Pro isomers, identified the Hyp, and relied on the roles of sequence and secondary structure were employed for the characterization of the aliphatic 13C sites in [U-13C]Pro elastin. 20 ACS Paragon Plus Environment

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Biochemistry

Proline is unique among all amino acids for its high propensity to form cis peptide bonds. The C- and C-Pro sites experience different shielding effects in the two conformers, due to their relative orientations with the preceding residue’s C=O.58 The average 13C chemical shifts in trans-Pro are 29.8 ± 1.0 ppm (13Cβ) and 25.4 ± 0.9 ppm (13Cγ) versus 31.8 ± 1.2 ppm (13Cβ) and 22.4 ± 0.7 ppm (13Cγ) for the cis-Pro.59 Typically, the 13Cγ -Pro shift is considered the more reliable indicator for cis-trans isomerism, because the 13Cβ-Pro resonance is also dependent on the effects of the γ-turn, if present.62 The deconvolution of the 1D NMR data (Figure 1b) indicate that populations of trans- and cis-Pro may be present in hydrated elastin, but resolution and sensitivity in a single dimension impose limits on confidence levels. The major and minor populations in hydrated elastin at 37 C were resolved using the rINEPT-HETCOR experiment (Figure 3). The major population is observed at ((1H), (13C)) = (4.40, 61.5), (3.62, 48.2), (2.25 and 1.91, 29.9) and (2.01, 25.1) ppm for -, δ-, β-, and γ-Pro sites respectively (Table S5). The 13Cβ and 13Cγ signals at 29.9 and 25.1 ppm arise from the trans isomers. A minor contribution from cis-Pro (((1H), (13C)) = (1.91, 22.7 ppm) is also observed. It is resolved from the methyl 1Hδ-Leu resonance (0.88 ppm) in the 1H dimension.


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Figure 3. Two-dimensional 1H-13C rINEPT-HETCOR spectrum of [U-¹³C-Pro] elastin at 37 °C. The spectrum was taken with 256 scans per t1 point. The spinning speed was 8 kHz. The spectrum was processed with 30 Hz line broadening in the direct dimension and 1 Hz in the indirect dimension. Insets show expansions of Cα/Hα, C/H, Cβ/Hβ, and Cγ/Hγ regions.


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Biochemistry

Whereas the 13Cβ- and 13Cγ-Pro shifts encode cis-trans isomerism, the 13Cδ-Pro shifts are sensitive to neighboring residues. The 2D 1H-13C rINEPT-HETCOR experiment resolves at least two populations of 13Cδ at 49.0 and 47.9 ppm. The assignment of the downfield (13C) = 49.0 ppm peak to ValPro is consistent with the 1Hδ shifts of 3.65 and 3.84 ppm. These 1H peaks are nearly identical to ValPro in model peptides of elastin (3.70 and 3.90 ppm)60 and collagen (3.67 and 3.81 ppm).63 All other prolines (XaaPro, XaaPro) are detected at (13Cδ, 1Hδ) = (47.9, ~3.6) ppm. The homonuclear R-TOBSY experiment at 42 °C (Figure 4) provides additional resolution of the aliphatic populations, while it also correlates the telltale cis and trans 13C- and 13C-Pro 13C-Pro

peaks with the sequence information inherent in the 13C-Pro features. First, a single peak is observed at 61.5 ppm, as in 1D and rINEPT-HETCOR experiments. And,

similar to the rINEPT-HETCOR results, major and minor populations of 13C-Pro (29.9 and 32.6 ppm) and 13C-Pro (25.5 and 22.7 ppm) correspond to the proline in trans (Figure 4, black peaks) and cis isomers (Figure 4, magenta peaks). Finally, the 13C-Pro resonances for the ValPro and the XaaPro are 49.0 and 47.9 ppm, respectively. The  cross-peak shows alignment of the 13C of cis-Pro with 13C-Pro of XaaPro, where XaaVal. I.e., the homonuclear R-TOBSY experiment provides strong evidence that the ValPro population is exclusively trans, consistent with our previous assignment of this motif to a Type II β-turn.4 The cis rotamer is favored for Xaa=Gly, Phe, and Tyr.64 The aromatic sidechains stabilize the cis configuration via ring-ring stacking interactions.65 However, there are very few PhePro or TyrPro in elastin, relative to the amount of GlyPro. Additionally, the characteristic upfield 1H shifts of the stacked rings of cis PhePro and cis TyrPro are not observed. Thus, the 23 ACS Paragon Plus Environment

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Page 24 of 46

most likely sequence for the cis isomers in elastin is GlyPro The more compact cis can accommodate a sterically small residue like Gly. Comparison to published 13C-, 1H-, and 15N-Pro shifts of polyGP also support this assignment.4,66-67 Figure 4. Two-dimensional 13C-13C RTOBSY spectrum of [U-13C-Pro] elastin at 42 °C. The cis correlations are highlighted in magenta while trans are shown in black. The correlations for 4-Hyp signals (green) are indicated by dashed lines. Slices of the cis (magenta) and trans(black) isomers along the F1 dimension are also illustrated. The spectrum was taken with 256 scans per t1 point. The spectrum was processed by applying 10 Hz line broadening in both dimensions.

The 13C-13C experiment also unambiguously identifies the 4-Hyp (Figure 4, green peaks) at 70.5, 60.4, 56.6 and 37.8 ppm, consistent with the deconvolution of the DPMAS difference 24 ACS Paragon Plus Environment

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Biochemistry

spectrum (Figure 1b). The 60.4 ppm component of the 13Cα region in Figure 3 is assigned to 13Cα-(4-Hyp),

not 13Cα-Pro in a PPII conformation. The 13Cβ- and 13Cγ-Hyp shifts indicate that

the hydroxyprolines adopt the trans isomer, 68 as expected. Characterization of the conformational ensemble at the backbone with 2D NMR and isotopeedited IR spectra Four 13C=O-Pro populations were resolved using a two-dimensional R-TOBSY experiment41 with LOW-BASHD homonuclear (13C=O-13Cα) J-decoupling42 in the direct dimension for narrower linewidths. Two major 13C=O-Pro peaks at 175.3 (peak 1) and 174.9 ppm (peak 2) are tentatively assigned as β-turn and coil, respectively (Figure 5a). Peak 3 has low intensity at (13C=O, 13C) = (174.6 ppm, 60.5 ppm) (peak 3). The aliphatic R-TOBSY experiment (Figure 4) was used to assign this population to the hydroxyprolines in elastin. Finally, peak 4 at 174.4 ppm represents (unresolved) contributions from [13C=O-Pro]-Tyr, [13C=O-Pro]-Phe and [13C=O-Pro]-Ala in elastin.


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Figure 5. Experimental and calculated spectra of the [13C=O-Pro] in elastin. (a) Two-dimensional 13C-13C R-TOBSY spectrum of [U-13C-Pro] elastin at 42 °C, with skyline projections. The spectrum was collected with 256 scans per t1 point. The R-TOBSY experiment was acquired with LOW-BASHD decoupling, yielding a lineshape without 1J COCα

in the F2 dimension. The thresholds in

both dimensions are marked with broken red lines. (b, top) Predicted random coil spectrum using semi-empirical approach. The intensities were weighted by the number of occurrences in the primary sequence of tropoelastin. (b, bottom) 13C NMR DPMAS spectrum, acquired with LOW-BASHD decoupling, of a denatured elastin sample (8M urea). (c) Predicted Type II β-turn (ψPro=120°) using integrated PDB/RefDB-based approach. Only chemical shifts for XPZ sequences in tropoelastin are shown here. The intensities reflect the number of occurrences of the selected XPZ sequences in the databases. For (b, top) and (c), each peak was plotted with a Gaussian smoothing function. Dashed and continuous vertical lines mark the positions of peaks 1 and 2, respectively. 26 ACS Paragon Plus Environment

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Biochemistry

The random coil shifts of prolines in elastin were calculated using a semi-empirical approach (Figure 5b). Proline is found in a limited number of sequence variations XPZ in rat tropoelastin. The [13C=O-Pro] peak position for each XPZ sequence is calculated with values obtained from a comprehensive study of denatured GGXGG peptides49 and weighted by the number of occurrences in tropoelastin. Three resolved populations are observed in the calculated spectrum, corresponding to GPG, XPG (X≠G) and XPZ (Z=Y, F, A) at 175.2, 175.0, and 174.4 ppm, respectively (Figure 5b). These values show good agreement with the (13C)=175.0 ppm peak that is observed with an enriched elastin in 8 M urea (Figure 5b). The calculated spectrum also provides support for the assignments of peaks 2 and 4 to random coil prolines in XPG (X≠G) and XPZ (Z=Y, F, A) sequences, respectively (Figure S4). The chemical shift distributions of 13C=O and 13C in Pro in Type II β-turn (ψ=120°) were also calculated. (Plots for the other secondary structures are shown in Figure S5.) Tropoelastin has a limited number of sequences with Pro, i.e., R-1PR+1. These R-1PR+1 sequences are used as one of the filters to select the (corresponding) 13C=O-Pro and 13C-Pro chemical shifts. The second filter is the conformation; i.e., only shifts that correspond to sequences in tropoelastin and are correlated with the -turn conformation are used. A peak is simulated for each entry that meets both criteria (sequence and conformation) from RefDB, and the composite is illustrated in the spectrum of Figure 5c. The peak intensities reflect the number of occurrences of a given R-1PR+1 sequence with ψ=120° in RefDB. The aggregate database information supports the assignment of peak 1 to a -turn. It is also consistent with the observed 13C=O-Pro shift of the elastin mimetic poly(Lys-25) in solution.60 The bimodal distribution of β-turn and


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coil in the prolines was previously reported in a variable-temperature study of enriched 15N-Pro and [1-13C-Val, 15N-Pro] elastin.4 To complement the chemical shift information, isotope-edited FTIR spectroscopy was also employed. The difference (spectrum) between the unenriched and the isotopically-labelled samples in the Amide I (carbonyl stretch) region is reflective of conformation at the site of enrichment. The average Amide I band positions for a protein sample in D2O for β-turn and random coil are 1671 and 1645 cm-1, respectively.69 In additional to conformational effects, the IR properties of different isotopes may vary. The effects of both chemical environment and isotope may be exploited in protein structure determination. For example, the absorption band for the natural-abundance 12C=16O is less than 13C=16O by ~40 cm-1.69-72 This approach was used to confirm the antiparallel β-sheets and irregular structures of the Pro residues in ribonuclease.73 A red Amide I shift is observed in the comparison of the IR spectra for [U-13C-Pro]- and unenriched elastin (Figures 6a-6b); peak-fitting of the Amide I difference spectrum (Figure 6c) yields two components centered at 1667 and 1641 cm-1, which are assigned to β-turn and coil, roughly in a 1:1 ratio. The determination of two populations in the IR spectra are consistent with the assignments of the peaks in the 13CC=O-13Cα TOBSY-LOWBASHD data.


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Biochemistry

Figure 6. Red shift of amide I IR band of [U-13C-Pro] elastin compared to unenriched elastin at 25 °C. (a) IR spectra of labelled (black trace) and unlabelled (orange trace) elastin. The intensities of the IR peaks of the enriched and unenriched elastin samples were normalized to the Tyr band at 1515 cm-1. (b) Difference spectrum (7) of [U-13C-Pro] elastin and unenriched elastin. (c) Expansion of the 1620-1750 cm-1 region in (b). The lineshape was deconvolved into two components (gray). The sum (blue) shows good agreement with the difference spectrum.

13C

T1 values are consistent with liquid-like motion of the prolines in hydrated elastin Narrow linewidths are routinely observed with hydrated elastin at physiological

temperatures, suggesting fast isotropic motion. The full-widths at half-maximum (FWHM) of elastin’s proline peaks in 13C DPMAS spectra are 200-300 Hz (Table S4), which are relatively narrow for a large biopolymer in the solid state. Linewidths in a static DP spectrum (not shown) are not significantly broader than the MAS lineshapes. Furthermore, the NOE enhancement of prolines in elastin is 1.5-1.7 (data not shown), similar to rapidly reorienting Gly residues in collagen fibrils.74 29 ACS Paragon Plus Environment

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To probe the high-frequency fluctuations of the prolines, 13C T1 relaxation was measured at 37 °C (Table 2). The T1 values for the resolved 13C sites of Pro in hydrated elastin range from 0.4-1.1 s, consistent with previously reported 13C T1 constants of hydrated elastin above the glass transition temperature.33 These values remain nearly unchanged for the range of -5C to 42C (data not shown). Elastin’s short ¹³C T1 times are indicative of high-amplitude motion, similar to those observed in hydrated silk.75 In contrast, 13C T1 relaxation times range from 100-101 s for typical (solid) protein or polymers in a similar static field.76 The relationship of the 13C T1 values (to each other) is also reflective of the fast, liquidlike motion of the prolines in hydrated elastin. Typical solids and liquids have contrasting relationships among proline’s 13C T1 values. In lyophilized peptides, the 13C-Pro backbone is less mobile than the sidechain carbons, as 13C- and 13C-Pro undergo ring-puckering motion, which has frequencies similar to the Larmor frequency.77 These differences in dynamics are reflected in the nT1 gradient, nT1γ < nT1β < nT1δ < nT1 , where n as the number of covalently bonded protons76. In solution, however, the rapid segmental reorientation of the backbone approaches the timescale of ring motion, which is ~3 to 4 orders of magnitude faster than observed in rigid solids.66,78 Thus, the nT1 gradient is reversed in proline-containing peptides in solution, i.e., nT1γ > nT1β > nT1δ > nT1. Table 2 shows the nT1 = 0.65 s, 0.96 s, 0.94 s, and 0.88 s for Cα, Cβ, Cγ, and Cδ, respectively. I.e., the nT1 gradient of Pro in elastin, nT1γ ≈ nT1β > nT1δ > nT1 , mirrors the trend for proline-containing peptides in solution.


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Biochemistry

Table 2. Spin-lattice relaxation times, 13C T1 (s) and nT1 (s) for the prolines in elastin at 37 °C. n is the number of protons that are directly bonded to the carbon atom.

C=O C C C C

T₁ ± 0.01(sec) 1.1 0.65 0.48 0.47 0.44

n, number of H's 0 1 2 2 2

nT₁ (sec) NA 0.65 0.96 0.94 0.88

The rapid motions are consistent with models for proteins that do not have extensive (intramolecular) hydrogen-bonding networks at physiological temperatures. Results of our previous study showed that only a fraction of the Gly-rich regions in elastin are hydrogenbonded to other segments of the protein.4 Proline increases backbone hydration, which is anticorrelated to hydrogen-bond content.17 The 13C T1 measurements point to highly mobile prolines, most likely in an environment that is a mixture of -turn and coil.

CSA of 13C-Pro is consistent with extended conformations The high mobility of the proline residues in hydrated elastin at ambient or physiological temperatures precludes experiments that elucidate structural information from dipolar couplings and CSA. However, the internal motions in this biopolymer, including rapid conformational exchange, slow considerably upon cooling. PPII has been observed in cooled samples of numerous model peptides of elastic proteins, including mimetics from the hydrophobic domains of tropoelastin,7 the peptide encoded by exon 172 of human titin,26 and high-molecular weight


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subunits of wheat glutenin.79 Notably, intrinsically disordered proteins (IDPs) have increased propensity for PPII conformation at low temperatures.80 The principal values of the CSA tensor and the corresponding lineshape are useful for structural elucidation in peptides and proteins.81 Experimentally, the 13Cα-Gly of an elastin mimetic was observed and then compared to simulated CSA lineshapes.82 The α-helix and βsheet were excluded as possible local structures for Gly, due to significant differences between the observed pattern and the simulated lineshapes for these conformations. Here, a similar approach was applied to determine the 13Cα-Pro CSA in frozen elastin. The scaled CSA powder pattern of 13Cα-Pro in elastin was first extracted from the ROCSA experiment.43 The ROCSA sequence recouples CSA from an MAS experiment and has been successfully applied to determine CSA’s at multiple (enriched) sites in a microcrystalline protein.83 The experimental CSA lineshape for 13Cα-Pro in frozen elastin was compared with the simulated lineshape of four conformations (Figure 7). The principal values of the trans 13Cα-Pro tensor over all ψ torsion angles were previously calculated by ab initio methods.47 For 13Cα-Pro in frozen elastin, smaller deviations are observed for PPII and Type II β-turn (Figures 7a-7b). Although the fit for the former appears slightly better than the latter, the possibility of the β-turn cannot be completely ruled out. Notably, relatively small differences exist between the Type II βturn (ψ=135°) and PPII (ψ=150°). The noticeable disparity for the α-helix is consistent with the 13Cα-Pro

shift analysis, eliminating the possibility of this secondary structure. (Figure 7c). The γ-

turn was also explored, as it was determined to exist in equilibrium with the β-turn in depsipeptide elastin mimetics.84 However, it also appears to be a poor fit for this system (Figure 32 ACS Paragon Plus Environment

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Biochemistry

7d). This CSA result is also consistent with the 1H- 13C rINEPT-HETCOR data; i.e., the characteristic (-turn) 1Hβ-Pro resonance at 1.75 ppm85 is absent. Figure 7. Comparison of experimental and simulated Observed

13Cα-Pro 13Cα-Pro

CSA

lineshapes.

CSA lineshape from

elastin at -20 C (black) was compared to simulations for four secondary structures (blue). The spectrum was taken with 64 scans per t1 point.: (a) PPII (ψ =150°), (b) Type II βturn (ψ=135°), (c) α-helix (ψ = -45°), and (d) γ-turn (ψ =60°). The scaled

13C-Pro

CSA

powder pattern was obtained from a ROCSA experiment. The simulated spectra were processed with 150 Hz Gaussian broadening.

Conclusion The current study provides new structural and dynamical information on the prolines of native, hydrated elastin, using a combination of strategic isotopic enrichment, solid-state NMR spectroscopy, and FTIR spectroscopy. All of the 13C chemical shifts for the prolines in hydrated elastin are resolved by 1D and 2D NMR methods. At physiological temperatures, the prolines – long believed to be an essential element in elastomeric proteins – are found in -turn and random coil. The CSA of 13C-Pro in frozen elastin also supports the possibility of PPII in frozen elastin. The distribution of -turn, PPII and random coil has been observed in single hydrophobic domains of elastin,7 as well as other elastomeric proteins like titin26 and glutelin.79 The absence of long-range order, such as found in -helices and β-sheets, is compatible with the high degree 33 ACS Paragon Plus Environment

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of flexibility and elasticity that are characteristic of elastin. And, the absolute and relative values of 13C T1’s indicate that the motion at the prolines is fast, near-isotropic, and liquid-like at temperatures above Tg. In addition to the ensemble of conformations (and coil) in the Pro-rich regions of elastin, this study is the first to directly observe both cis and trans isomers at the prolines in this protein. The compact (cis) and extended (trans) states of XaaPro (Figure 8) may be a molecular-scale analogue of elongation and restoration of fiber or tissue. This study is also the first to correlate the trans and cis isomers with discrete secondary structures at the prolines in elastin. For example, ValPro is predominantly trans, and -turns are observed at this proline (ValPro).4 The 13C-Pro

CSA measurement provides support for -turns and PPII, both extended conformations

which are also found in the trans population. The trans isomer is strongly favored over the cis and is found in all XaaPro sequences. However, small populations of the cis isomer have been observed in the poly(VPGVG) elastin mimetic28 and in the elastin-like polypeptide with multiple hydrophobic and crosslinking domains.8 And, a minor population of the cis-Pro isomer is detected in the current study. Cis-Pro is likely found in (some) GlyPro subunits of native elastin, based on steric considerations and previously published NMR data of the poly(GP).4,66-67 Hydrogen-bonds are not broken in CTI, making the conversion relatively facile and reversible under physiological conditions. Figure 8. Proposed cis-to-trans isomerization (CTI) at XaaPro upon stretching. Xaa = Gly is used for illustration.


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These observations highlight the critical role of the multiple populations of proline in the extensibility of elastin and elastomeric proteins in general. Proline promotes the formation of labile structures with a minimum number of hydrogen-bonds, allowing stretch and recoil cycles to occur more readily. Seemingly, the prolines in elastomeric proteins, including elastin, have a universal feature, namely, a distribution of local structures with shallow energy minima. By expanding this approach to other key amino acids in elastin, a consensus on the molecular basis of the structure-function of this biopolymer will soon emerge, leading to a more unified model among elastomers.

Accession ID ELN_RAT Q99372

Acknowledgement The authors thank Dr. Mike Cooney and Ms. Catherine Wong for access to the FTIR and associated software for data analysis. This work was partially supported by grants to KKK from the National Science Foundation (MCB-1022526).


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Supporting Information Three-amino-acid sequences (containing proline) in rat tropoelastin, information on NMR spectrum of sample (chemical shift, line widths, peak area), chemical shift comparisons and data on the hydrolyzed sample.

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