Elastin is an extracellular matrix material found in all vertebrates. Its reversible elasticity, robustness, and low stiffness are essential for the function of arteries, lungs, and skin. It is among the most resilient elastic materials known: During a human lifetime, arterial elastin undergoes in excess of 2 × 109 stretching/contracting cycles without replacement, and slow oxidative hardening has been identified as a limiting factor on human lifespan. For over 50 y, the mechanism of entropic recoil has been controversial. Herein, we report a combined NMR and thermomechanical study that establishes the hydrophobic effect as the primary driver of elastin function. Water ordering at the solvent:protein interface was observed as a function of stretch using double quantum 2H NMR, and the most extensive thermodynamic analysis performed to date was obtained by measuring elastin length and volume as a function of force and temperature in normal water, heavy water and with cosolvents. When stretched, elastin's heat capacity increases, water is ordered proportional to the degree of stretching, the internal energy decreases, and heat is released in excess of the work performed. These properties show that recoil in elastin under physiological conditions is primarily driven by the hydrophobic effect rather than by configurational entropy as is the case for rubber. Consistent with this conclusion are decreases in the thermodynamic signatures when cosolvents that alter the hydrophobic effect are introduced. We propose that hydrophobic effect-driven recoil, as opposed to a configurational entropy mechanism where hardening from crystallization can occur, is the origin of elastin's unusual resilience.

Elastin, the protein material responsible for the elasticity of blood vessels, is the best known member of a class we term elastic protein materials (EPMs). A hallmark of EPMs is their entropically driven recoil. Soluble monomeric precursors to EPMs have sequence features of intrinsically disordered proteins, including low sequence diversity and a high frequency of proline and small hydrophobic residues. The human genome contains more than 100 such proteins, primarily expressed in skin, lung, and connective tissues.

Elastin fibers assemble in the extracellular matrix from the precursor protein tropoelastin and provide the flexibility and spontaneous recoil required for arterial function. Unlike many proteins, a structure-function mechanism for elastin has been elusive. We have performed detailed NMR relaxation studies of the dynamics of the minielastins 24x' and 20x' using solution NMR, and of purified bovine elastin fibers in the presence and absence of mechanical stress using solid state NMR. The low sequence complexity of the minielastins enables us to determine average dynamical timescales and degrees of local ordering in the cross-link and hydrophobic modules separately using NMR relaxation by taking advantage of their residue-specific resolution. We find an extremely high degree of disorder, with order parameters for the entirety of the hydrophobic domains near zero, resembling that of simple chemical polymers and less than the order parameters that have been observed in other intrinsically disordered proteins. We find that average backbone order parameters in natural, purified elastin fibers are comparable to those found in 24x' and 20x' in solution. The difference in dynamics, compared with the minielastins, is that backbone correlation times are significantly slowed in purified elastin. Moreover, when elastin is mechanically stretched, the high chain disorder in purified elastin is retained, showing that any change in local ordering is below that detectable in our experiment. Combined with our previous finding of a 10-fold increase in the ordering of water when fully hydrated elastin fibers are stretched by 50%, these results support the hypothesis that stretch induced solvent ordering, i.e., the hydrophobic effect, is a key player in the elastic recoil of elastin as opposed to configurational entropy loss.

Elastin is the most abundant elastomer in nature. The recoil mechanism is believed to be entropically driven for which there re two molecular level theories. Recoil in one theory is a rubber-like mechanism in which the decrease in entropy with stretch is due to a decrease in conformational entropy from backbone ordering. The alternative is a decrease in solvent entropy with stretch from solvent ordering, i.e., the hydrophobic effect. Using a novel double quantum (2Q) pulse sequence in solid state NMR; water ordering at elastin fiber surface was studied quantitatively. We find that water is weakly oriented at the Elastin-water surface when the fiber is relaxed and stretching the fiber or increasing the temperature significantly increases the 2Q signal, i.e., increases the ordered water fraction. Furthermore, the 2Q signal decreases with the addition of high molecular weight polyethylene glycol (20kDa or 6kDa PEG) that is known to precipitate proteins and decrease their hydration in solution. We believe PEG causes less water to be available on the fibers surface and thus less ordered water. Also, this is likely the reason why the fibers become less flexible. Our preliminary results in both water and high molecular weight PEG experiments directly correlate the recoil mechanism with the hydrophobic effect.

Artificial minielastin constructs have been designed that replicate the structure and function of natural elastins in a simpler context, allowing the NMR observation of structure and dynamics of elastin-like proteins with complete residue-specific resolution. We find that the alanine-rich cross-linking domains of elastin have a partially helical structure, but only when capped by proline-rich hydrophobic domains. We also find that the hydrophobic domains, composed of prominent 6-residue repeats VPGVGG and APGVGV found in natural elastins, appear random coil by both NMR chemical shift analysis and circular dichroism. However, these elastin hydrophobic domains exhibit structural bias for a dynamically disordered conformation that is neither helical nor β sheet with a degree of nonrandom structural bias which is dependent on residue type and position in the sequence. Another nonrandom-coil aspect of hydrophobic domain structure lies in the fact that, in contrast to other intrinsically disordered proteins, these hydrophobic domains retain a relatively condensed conformation whether attached to cross-linking domains or not. Importantly, these domains and the proteins containing them constrict with increasing temperature by up to 30% in volume without becoming more ordered. This property is often observed in nonbiological polymers and suggests that temperature-driven constriction is a new type of protein structural change that is linked to elastin's biological functions of coacervation-driven assembly and elastic recoil.

Tropoelastin, the soluble precursor of elastin, is highly flexible. Thus, structure elucidation has been and remains a challenge. Minielastin constructs mimic the structure of tropoelastin with alternating hydrophobic and cross-link modules. Simpler minielastin constructs allow elucidation of the secondary structure with residue-specific resolution using NMR spectroscopy. Complete chemical shift assignments of the backbone and side chain were obtained via 2D and 3D NMR experiments. Secondary shifts revealed disorder in the hydrophobic domain and weakly helical structure of the cross-link domain. Amide proton NOE suggested compaction in the cross-link module. Furthermore, using PFGNMR we have shown that this compaction increases at increasing temperature. These findings will help in understanding the coacervation process of tropoelastin. Coacervation is important in the synthesis of insoluble elastin and is thought to be favored by hydrophobic effect.

Solvent ordering at an interface can be studied by multiple-quantum NMR. Quantitative studies of (2)H2O ordering require clean double-quantum (2Q) filtration and an analysis of 2Q buildup curves that accounts for relaxation and, if randomly oriented samples are used, the distribution of residual couplings. A pulse sequence with absorption mode detection is extended for separating coherences by order and measuring relaxation times such as the 2Q filtered T2. Coherence separation is used to verify 2Q filtration and the 2Q filtered T2 is required to extract the coupling from the 2Q buildup curve when it is unresolved. With our analysis, the coupling extracted from the buildup curve in (2)H2O hydrated collagen was equivalent to the resolved coupling measured in the usual 1D experiment and the 2Q to 1Q signal ratio was in accord with theory. Application to buildup curves from (2)H2O hydrated elastin, which has an unresolved coupling, revealed a large increase in the 2Q signal upon mechanical stretch that is due to an increase in the ordered water fraction while changes in the residual coupling and T2 are small.

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 domains, a protein kinase domain and several long intrinsically disordered regions containing tandem short repeats. The 1500 residue BLUE region consists of 92 copies of 16-residue repeats, each with two octads of 8 residues. The octads in BLUE motifs form unstable α-helix-like coils in aqueous solution and negligible heptad-based, α-helical coiled-coils. The α-helix-like conformation is highly temperature dependent. The α-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface( Forbes et al., (2010) J. Mol. Biol., 398, 672).The conformation of BLUE octads was investigated further by NMR and molecular dynamics simulations. Molecular dynamics simulations of a 16-residue repeat indicated different behaviors of the helical state depending upon the force field used and the charge state of the peptide termini. Multidimensional NMR of the same peptide sequence indicated a helical conformation with a break in the helicity in the middle of the peptide consistent with one of the simulations. Interestingly, some of the simulations indicate the transient formation of 4.4 residue per turn left-handed π-helices, at the expense of α helices in the octads of BLUE. These observations suggest an intriguing possibility of an α- π conversion that alters reversibly the net contour length of the BLUE peptides, effecting protein elasticity in a heretofore unexpected mechanism.

The elasticity of native full length nebulin, demonstrated recently via atomic force microscopy with site-specific antibody pairs as force handles (Langmuir, 2009, 25, 7496), suggests that in the thin filaments, nebulin is stretched to cope with the actin length and imposes significant force and influences the functions of the underlying actins. This pre-stressed mechanical state of thin filaments may have important implications for the role of nebulin as a length ruler and as a regulator of actomyosin interaction. The structural basis of nebulin elasticity remains open. We report here the structural characterization of modules from the super-repeat and single repeat regions by a combination of circular dichroism (CD), NMR, SAXS, AFM, structural predictions and steered molecular dynamics simulations. In aqueous solutions of common buffers, these modules are intrinsically disordered, but are poised to form alpha-helices, especially in the presence of trifluoroethanol. SAXS analysis of a four-module construct indicates an elongated structure with a radius of gyration of 3.6 nm and, as modeled with DAMMIN, shows a contour length of ∼15 nm. Interestingly, this extended structure is also evident in a small population of the structural models as predicted by ROSETTA++. AFM images of the modules on an inert surface are predominantly compact with an average height of ∼ 2.5 nm, consistent with the bulk of the ROSETTA predictions. These structural ensembles of compact and extended structures are significantly shorter than what it would take for nebulin modules to wrap around the perimeter of actin filaments (∼6 nm per module). We propose that nebulin modules’ disorder-order transition of alpha helices, contributes to its elasticity and how nebulin juxtapositions itself onto the actin to form a pre-stressed thin filaments in the muscle sarcomere.

It is increasingly recognized that many proteins are intrinsically disordered and do not have a unique compact structure as those found in globular proteins. Titin is a giant modular protein (3-4 MDa) found in the muscle sarcomere that is comprised of both globular and disordered modules. The elastic titin PEVK segment, with tandem repeats of ∼28 residue modules, plays a major role in the passive tension of skeletal and heart tissues. We have proposed based on AFM studies of a cloned titin PEVK fragment, that salt-bridges play a central role in the elasticity of this PEVK polyelectrolyte. We have engineered a construct of 15 repeats of a single titin 28-residue PEVK module (human exon 172). The 50 kDa polyprotein shows well-resolved NMR spectra in dilute solution and in highly concentrated gels. Both chemical shifts and sequential NOE's indicate the presence of polyproline II helices. From long-range NOE's, we observed, for the first time, stable K to E salt-bridges with non-random pairings. Simulated annealing with NMR restraints yielded a manifold of plausible structures for an exon 172 trimer showing many salt-bridges. Steered molecular dynamics simulations (SMD) were done to study how the manifold of salt-bridges evolves during the stretching experiment. Repeated SMD simulations at slow velocity (0.0005 nm/ps) show force spectra consistent with experimental AFM force spectra of the polyprotein. SMD shows that salt-bridges occur even at high degrees of stretch and that these short range interactions are in integral part of the mechanical properties of PEVK. We propose that the long-range, non-stereospecific nature of electrostatic interactions provide a facile mechanism to tether and untether the flexible chains, which in turn affect elasticity as well as control the accessibility of protein-protein interaction to these nanogel-like proteins.

Key players in the contractile machineries of muscle and nonmuscle cells possess intrinsically disordered domains that perform essential force and signal transduction functions. These disordered domains exist as ensembles of rapidly equilibrating conformations that manifest as unique elasticity when subjected to external force that in turn regulates protein interactions. We are performing systematic structural, nanomechanical and protein interaction analysis of intrinsically disordered domains in the force generating and bearing molecules with NMR, CD, atomic force microscopy of single molecules and the identification of force‐relevant structural transitions by Steered Molecular Dynamics simulations. For the giant elastic protein titin, the dynamic breaking and rejoining of salt bridges are major force generating events that modulate PEVK elasticity and binding to SH3 domain. For the giant ruler protein nebulin, mechanical unfolding of a helices and re‐orientation of salt bridges accompanied its stretch to full length (1µ) that compresses and stiffens actin filaments. For the motor protein myosin, the unfolding of the disordered coiled coil of S2 appears to be an integral part of the active force generating events. We propose that the shuffling of long‐range, non‐stereospecific salt bridges provide a facile mechanism to tether and untether the flexible chains of intrinsically disordered proteins, which in turn affect elasticity as well as control the accessibility of protein‐protein interaction to these "nanogel" in signaling pathways.

Flavins are central to the reactivity of a wide variety of enzymes and electron transport proteins. There is great interest in understanding the basis for the different reactivities displayed by flavins in different protein contexts. We propose solid-state nuclear magnetic resonance (SS-NMR) as a tool for directly observing reactive positions of the flavin ring and thereby obtaining information on their frontier orbitals. We now report the SS-NMR signals of the redox-active nitrogens N1 and N5, as well as that of N3. The chemical shift tensor of N5 is over 720 ppm wide, in accordance with the predictions of theory and our calculations. The signal of N3 can be distinguished on the basis of coupling to 1H absent for N1 and N5, as well as the shift tensor span of only 170 ppm, consistent with N3's lower aromaticity and lack of a nonbonding lone pair. The isotropic shifts and spans of N5 and N1 reflect two opposite extremes of the chemical shift range for "pyridine-type" N's, consistent with their electrophilic and nucleophilic chemical reactivities, respectively. Upon flavin reduction, N5's chemical shift tensor contracts dramatically to a span of less than 110 ppm, and the isotropic chemical shift changes by approximately 300 ppm. Both are consistent with loss of N5's nonbonding lone pair and decreased aromaticity, and illustrate the responsiveness of the 15N chemical shift principal values to electronic structure. Thus. 15N chemical shift principal values promise to be valuable tools for understanding electronic differences that underlie variations in flavin reactivity, as well as the reactivities of other heterocyclic cofactors.

Li(+) and Ca(2+) binding to the carbonyl oxygen sites of a model peptide system has been studied by (17)O solid-state NMR spectroscopy. (17)O chemical shift (CS) and quadrupole coupling (QC) tensors are determined in four Gly-(Gly-(17)O)-Gly polymorphs by a combination of stationary and fast magic-angle spinning (MAS) methods at high magnetic field, 19.6 T. In the crystal lattice, the carbonyl oxygen of the central glycyl residue in two gly-gly-gly polymorphs form intermolecular hydrogen bonds with amides, whereas the corresponding carbonyl oxygens of the other two polymorphs form interactions with Li(+) and Ca(2+) ions. This permits a comparison of perturbations on (17)O NMR properties by ion binding and intermolecular hydrogen bonding. High quality spectra are augmented by density functional theory (DFT) calculations on large molecular clusters to gain additional theoretical insights and to aid in the spectral simulations. Ion binding significantly decreases the two (17)O chemical shift tensor components in the peptide plane, delta(11) and delta(22), and, thus, a substantial change in the isotropic chemical shift. In addition, quadrupole coupling constants are decreased by up to 1 MHz. The effects of ion binding are found to be almost an order of magnitude greater than those induced by hydrogen bonding.

Complete (17)O chemical shielding (CS) and quadrupole coupling (QC) tensors and their molecular orientations were determined for the central residues in two tripeptides Gly-Gly-Val (GGV) and Ala-Gly-Gly (AGG) by single-crystal NMR methods. Tensor orientations in the two peptides are very similar, however, principal components are different. The most shielded CS and smallest magnitude QC components are normal to the peptide plane, while the most deshielded CS and largest QC components are in the peptide plane either at an angle of 17 degrees (CS) or perpendicular (QC) to the C=O bond. Comparisons of principal components from experiment and DFT calculations indicate that the smaller shielding tensor span in GGV (549 ppm) compared to AGG (606 ppm) is likely due to two factors: a shorter "direct" H-bond distance to the peptide carbonyl oxygen and an "indirect" H bond of the peptide NH to a carboxylate rather than a carbonyl. We anticipate that (17)O NMR should be generally useful for probing H-bonding and local electrostatic interactions in proteins and polypeptides. Using the single-crystal data as an accurate reference, we show that a useful subset of the NMR parameters, QC and CS principal components and their relative orientation, can be obtained with reasonable accuracy from a very high-field (21.2 T), stationary sample powder spectrum.

15N shielding tensors were determined for the central peptide groups in GGV, AGG, and APG by single-crystal NMR. We find that the angle between the downfield component (delta11) and the N-H or the N-C(delta) (pro) bonds is in the range of 20-23 degrees and in accord with previous solid-state NMR measurements. However, AGG, unlike APG or GGV, has a distorted peptide plane, and delta11 lies approximately in the plane of N, C(alpha), and H rather than in the peptide plane defined by heavy atoms. Accurate orientations of delta22 and delta33 were determined, and the usual assumption that delta22 is along the peptide normal was found only in APG which has a highly nonaxial tensor. More generally, delta22 and delta33 are rotated about the delta11 axis (36 degrees in GGV). These results are compared with DFT calculations to gain a structural understanding of the effects of intermolecular interactions on shielding tensor principal components and orientations. Trimeric clusters containing H-bonded neighbors predict the orientations of the principal components within 2-3 degrees, but calculated principal components are less quantitative. Possible reasons for this disagreement are explored.

Elastin is a key protein in soft tissue function and pathology. Establishing a structural basis for understanding its reversible elasticity has proven to be difficult. Complementary to structure is the important aspect of flexibility and disorder in elastin. We have used solid-state NMR methods to examine polypeptide and hydrate ordering in both elastic ( hydrated) and brittle (dry) elastin fibers and conclude (i) that tightly bound waters are absent in both dry and hydrated elastin and (ii) that the backbone in the hydrated protein is highly disordered with large amplitude motions. The hydrate was studied by H-2 and O-17 NMR, and the polypeptide by C-13 and H-2 NMR. Using a two-dimensional C-13 MAS method, an upper limit of S < 0.1 was determined for the backbone carbonyl group order parameter in hydrated elastin. For comparison, S similar to 0.9 in most proteins. The former result is substantiated by two additional observations: the absence of the characteristic 2H spectrum for stationary amides and "solution-like" C-13 magic angle spinning spectra at 75 degrees C, at which the material retains elasticity. Comparison of the observed shifts with accepted values for alpha-helices, beta-sheets, or random coils indicates a random coil structure at all carbons. These conclusions are discussed in the context of known thermodynamic properties of elastin and, more generally, protein folding. Because coacervation is an entropy-driven process, it is enhanced by the observed backbone disorder, which, we suggest, is the result of high proline content. This view is supported by recent studies of recombinant elastin polypeptides with systematic proline substitutions.

Soil humic substances (HS) are heterologous, polydispersive, and multi-functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with C-13-glucose or N-15-nitrate for 8.5 months. Evidence for random-coil peptidic structures in the labeled HS was obtained from 2-D nuclear magnetic resonance (NMR), pyrolysis gas chromatography-mass spectrometry (pyro-GC-MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid-state NMR data. Detailed C-13 and N-15 labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC-MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre-existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.

Protonenleiter: Ketten aus Wassermolekülen spielen eine wichtige Rolle bei der Permeation von Protonen durch Transmembranproteine. Berichtet wird über die Kristallstrukturen, 2H- und 17O-Festkörper-NMR-Spektren und kalorimetrische Untersuchungen zweier Imidazolhydrate, die endlose eindimensionale Ketten aus Wassermolekülen stabilisieren (siehe Bild; N blau, O rot, H weiß). Die 2H-NMR-Spektren weisen auf eine unterschiedliche Umorientierungsdynamik der Wassermoleküle in beiden Verbindungen hin. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2003/z52157_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.