Static Solid Relaxation Ordered Spectroscopy: SS-ROSY.

A two-dimensional pulse sequence is introduced for correlating nuclear magnetic resonance anisotropic chemical shifts to a relaxation time (e.g., T1) in solids under static conditions. The sequence begins with a preparatory stage for measuring relaxation times, and is followed by a multiple pulse sequence for homonuclear dipolar decoupling. Data analysis involves the use of Fourier transform, followed by a one-dimensional inverse Laplace transform for each frequency index. Experimental results acquired on solid samples demonstrate the general approach, and additional variations involving heteronuclear decoupling and magic angle spinning are discussed.

Alterations of elastin in female reproductive tissues arising from advancing parity.

Female reproductive tissues undergo significant alterations during pregnancy, which may compromise the structural integrity of extracellular matrix proteins. Here, we report on modifications of elastic fibers, which are primarily composed of elastin and believed to provide a scaffold to the reproductive tissues, due to parity and parturition. Elastic fibers from the upper vaginal wall of virgin Sprague Dawley rats were investigated and compared to rats having undergone one, three, or more than five pregnancies. Optical microscopy was used to study fiber level changes. Mass spectrometry, 13C and 2H NMR, was applied to study alterations of elastin from the uterine horns. Spectrophotometry was used to measure matrix metalloproteinases-2,9 and tissue inhibitor of metalloproteinase-1 concentration changes in the uterine horns. Elastic fibers were found to exhibit increase in tortuosity and fragmentation with increased pregnancies. Surprisingly, secondary structure, dynamics, and crosslinking of elastin from multiparous cohorts appear similar to healthy mammalian tissues, despite fragmentation observed at the fiber level. In contrast, elastic fibers from virgin and single pregnancy cohorts are less fragmented and comprised of elastin exhibiting structure and dynamics distinguishable from multiparous groups, with reduced crosslinking. These alterations were correlated to matrix metalloproteinases-2,9 and tissue inhibitor of metalloproteinase-1 concentrations. This work indicates that fiber level alterations resulting from pregnancy and/or parturition, such as fragmentation, rather than secondary structure (e.g. elastin crosslinking density), appear to govern scaffolding characteristics in the female reproductive tissues.

Quantification of desmosine and isodesmosine using MALDI-ion trap tandem mass spectrometry.

Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS2 analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d4 (labeled-Des) as an internal standard in different biological fluids, such as urine and serum. The method demonstrated linearity over two orders of magnitude with a detection limit of 0.02 ng/µL in both urine and serum without enrichment prior to mass spectrometry, and relative standard deviation of < 5%. The method is used to evaluate the time-dependent degradation of Des upon UV irradiation (254 nm) and found to be consistent with quantification by 1H NMR. This is the first characterized MALDI-MS2 method for quantification of Des and Isodes and illustrates the potential of MALDI-ion trap MS2 for effective quantification of biomolecules. The reported method represents improvement over current liquid chromatography-based methods with respect to analysis time and solvent consumption, while maintaining similar analytical characteristics. Graphical abstract ᅟ.

Quantitative Analysis of Solid-State Homonuclear Correlation Spectra of Antiparallel ß-Sheet Alanine Tetramers.

Poly-l-alanine (PLA) sequences are a key element in the structure of the crystalline domains of spider dragline silks, wild silkworm silks, antifreeze proteins, and amyloids. To date, no atomic-level structures of antiparallel (AP)-PLA longer than Ala4 have been reported using the single-crystal X-ray diffraction analysis. In this work, dipolar-assisted rotational resonance solid-state NMR spectra were observed to determine the effective internuclear distances of 13C uniformly labeled alanine tetramer with antiparallel (AP) ß-sheet structure whose atomic coordinates are determined from the X-ray crystallographic analysis. Initial build-up rates, R j, k, were obtained from the build-up curves of the cross peaks by considering the internuclear distances arising in the master equation. Subsequently, experimentally obtained effective internuclear distances, reffj, k(obs), were compared with the calculated reffj, k(calc) values obtained from the X-ray crystallographic data. Fairly good correlation between reffj, k(obs) and reffj, k(calc) was obtained in the range of 1.0-6.0 Å, with the standard deviation of 0.244 Å, without considering the zero-quantum line-shape functions. It was further noted that the internuclear distances of intermolecular contributions provide details relating to the molecular packing in solid-state samples. Thus, the present data agree well with AP-ß-sheet packing but do not agree with P-ß-sheet packing.

Comparing the efficacy of solid and magic-echo refocusing sequences: Applications to 1H NMR echo spectroscopy of shale rock.

Quantitative evaluation of the solid and viscous components of unconventional shale rock, namely kerogen and bitumen, is important for understanding reservoir quality. Short transverse coherence times, due to strong 1H-1H dipolar interactions, motivates the application of solid state refocusing pulse sequences that allow for investigating components of the free-induction decay that are otherwise obscured by instrumental effects such as probe ringdown. This work reports on static, wide-line 1H spectroscopy of shale rock and their extracted components, which include kerogen and bitumen, by the application of solid echo and magic echo pulse sequences. We characterize the efficiency of these cycles as a function of the radio frequency power and inter-pulse spacing. Magic echos are shown to provide superior refocusing in comparison to solid echo based experiments, as can be understood from the truncation of the Magnus expansion and ability to also refocus any Iz Hamiltonians (e.g. static field inhomogeneity). We characterize the optimal echo spacing and RF power for two shale samples of different maturity, motivating routine core and cuttings analysis and applications.

The Coupled Bio-Chemo-Electro-Mechanical Behavior of Glucose Exposed Arterial Elastin.

Elastin, the principle protein component of the elastic fiber, is a critical extracellular matrix (ECM) component of the arterial wall providing structural resilience and biological signaling essential in vascular morphogenesis and maintenance of mechanical homeostasis. Pathogenesis of many cardiovascular diseases have been associated with alterations of elastin. As a long-lived ECM protein that is deposited and organized before adulthood, elastic fibers can suffer from cumulative effects of biochemical exposure encountered during aging and/or disease, which greatly compromise their mechanical function. This review article covers findings from recent studies of the mechanical and structural contribution of elastin to vascular function, and the effects of biochemical degradation. Results from diverse experimental methods including tissue-level mechanical characterization, fiber-level nonlinear optical imaging, piezoelectric force microscopy, and nuclear magnetic resonance are reviewed. The intriguing coupled bio-chemo-electro-mechanical behavior of elastin calls for a multi-scale and multi-physical understanding of ECM mechanics and mechanobiology in vascular remodeling.

Ultraviolet radiation reduces desmosine cross-links in elastin.

Elastic fibers, a major component of the extracellular matrix of the skin, are often exposed to ultraviolet (UV) radiation throughout mammalian life. We report on an in vitro study of the alterations in bovine nuchal ligament elastic fibers resulting from continuous UV-A exposure by the use of transmission electron microscopy (TEM), histology, mass spectrometry, and solid state 13C NMR methodologies. TEM images reveal distinct cracks in elastic fibers as a result of UV-A irradiation and histological measurements show a disruption in the regular array of elastic fibers present in unirradiated samples; elastic fibers appear shorter, highly fragmented, and thinner after UV-A treatment. Magic angle spinning 13C NMR was applied to investigate possible secondary structural changes or dynamics in the irradiated samples; our spectra reveal no differences between UV-A irradiated and non-irradiated samples. Lastly, MALDI mass spectrometry indicates that the concentration of desmosine, which forms cross-links in elastin, is observed to decrease by 11 [Formula: see text] following 9 days of continuous UV-A irradiation, in comparison to unirradiated samples. These alterations presumably play a significant role in the loss of elasticity observed in UV exposed skin.

Characterization of water in hydrated Bombyx mori silk fibroin fiber and films by 2H NMR relaxation and 13C solid state NMR.

The mechanical properties of Bombyx mori silk fibroin (SF), such as elasticity and tensile strength, change remarkably upon hydration. However, the microscopic interaction with water is not currently well understood on a molecular level. In this work, the dynamics of water molecules interacting with SF was studied by 2H solution NMR relaxation and exchange measurements. Additionally, the conformations of hydrated [3-13C]Ala-, [3-13C]Ser-, and [3-13C]Tyr-SF fibers and films were investigated by 13C DD/MAS NMR. Using an inverse Laplace transform algorithm, we were able to identify four distinct components in the relaxation times for water in SF fiber. Namely, A: bulk water outside the fiber, B: water molecules trapped weakly on the surface of the fiber, C: bound water molecules located in the inner surface of the fiber, and D: bound water molecules located in the inner part of the fiber were distinguishable. In addition, four components were also observed for water in the SF film immersed in methanol for 30s, while only two components for the film immersed in methanol for 24h. The effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and non-crystalline domains of 13C selectively labeled SF, respectively, could be determined independently. Our measurements provide new insight relating the characteristics of water and the hydration structure of silk, which are relevant in light of current interest in the design of novel silk-based biomaterials. STATEMENTS OF SIGNIFICANCE: The mechanical properties of Bombyx mori silk fibroin (SF) change remarkably upon hydration. However, the microscopic interaction between SF and water is not currently well understood on a molecular level. We were able to identify four distinct components in the relaxation times for water in SF fiber by 2H solution NMR relaxation and exchange measurements. In addition, the effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and non-crystalline domains of 13C selectively labeled SF, respectively, could be determined independently. Thus, our measurements provide new insight relating the characteristics of water and the hydration structure of silk, which are relevant in light of current interest in the design of novel silk-based biomaterials.

Observation of structural universality in disordered systems using bulk diffusion measurement.

We report on an experimental observation of classical diffusion distinguishing between structural universality classes of disordered systems in one dimension. Samples of hyperuniform and short-range disorder were designed, characterized by the statistics of the placement of micrometer-thin parallel permeable barriers, and the time-dependent diffusion coefficient was measured by NMR methods over three orders of magnitude in time. The relation between the structural exponent, characterizing disorder universality class, and the dynamical exponent of the diffusion coefficient is experimentally verified. The experimentally established relation between structure and transport exemplifies the hierarchical nature of structural complexity-dynamics are mainly determined by the universality class, whereas microscopic parameters affect the nonuniversal coefficients. These results open the way for noninvasive characterization of structural correlations in porous media, complex materials, and biological tissues via a bulk diffusion measurement.

Mechanical, structural, and dynamical modifications of cholesterol exposed porcine aortic elastin.

Elastin is a protein of the extracellular matrix that contributes significantly to the elasticity of connective tissues. In this study, we examine dynamical and structural modifications of aortic elastin exposed to cholesterol by NMR spectroscopic and relaxation methodologies. Macroscopic measurements are also presented and reveal that cholesterol treatment may cause a decrease in the stiffness of tissue. 2H NMR relaxation techniques revealed differences between the relative populations of water that correlate with the swelling of the tissue following cholesterol exposure. 13C magic-angle-spinning NMR spectroscopy and relaxation methods indicate that cholesterol treated aortic elastin is more mobile than control samples. Molecular dynamics simulations on a short elastin repeat VPGVG in the presence of cholesterol are used to investigate the energetic and entropic contributions to the retractive force, in comparison to the same peptide in water. Peptide stiffness is observed to reduce following cholesterol exposure due to a decrease in the entropic force.

Pregnancy-Induced Dynamical and Structural Changes of Reproductive Tract Collagen.

The tissues and organs of the female reproductive tract and pelvic floor undergo significant remodeling and alterations to allow for fetal growth and birth. In this work, we report on a study of the alterations of murine reproductive tract collagen resulting from pregnancy and parturition by spectrophotometry, histology, and (13)C, (2)H nuclear magnetic resonance (NMR). Four different cohorts of rats were investigated that included virgin, multiparous, two- and fourteen-day postpartum primiparous rats. (13)C CPMAS NMR revealed small chemical shift differences across the cohorts. The measured H-C internuclear correlation times indicated differences in dynamics of some motifs. However, the dynamics of the major amino acids, e.g., Gly, remained unaltered with respect to parity. (2)H NMR relaxation measurements revealed an additional water reservoir in the postpartum and multiparous cohorts pointing to redistribution of water due to pregnancy and/or parturition. Spectrophotometric measurements indicated that the collagen content in virgin rats was highest. Histological analysis of the upper vaginal wall indicated a signature of collagen fiber dissociation with smooth muscle and a change in the density of collagen fibers in multiparous rats.

Theoretical calculation of a composite pulse for 2H broadband excitation by average Hamiltonian theory.

Quadrupolar echo NMR spectroscopy of solids often requires RF pulse excitation that covers spectral widths exceeding 100 kHz. In a recent work we found out that a four pulse, composite pulse COM-II ( [Formula: see text] ), provided robust broadband excitation for deuterium quadrupolar echo spectroscopy. Moreover, when combined with an eight step phase cycle, spectral distortions arising from finite pulse widths were greatly supressed. In this paper we report on a theoretical analysis COM-II with 8-step phase cycle by average Hamiltonian theory. This treatment is combined with the fictitious spin-1 operator formalism, and the mechanism of the 8-step phase cycling that minimizes the spectral distortions is discussed.

The Behavior of Water in Collagen and Hydroxyapatite Sites of Cortical Bone: Fracture, Mechanical Wear, and Load Bearing Studies.

The mechanical properties of cortical bone, which is largely comprised of collagen, hydroxyapatite, and water, are known to hinge on hydration. Recently, the characteristics of water in bone have drawn attention as potential markers of bone quality. We report on the dynamics, diffusion, population, and exchange of water in cortical bone by NMR relaxation and diffusion methodologies. Relaxation measurements over timescales ranging from 0.001 to 4.2 s reveal two distinguishable water environments. Systematic exposure to ethylenediaminetetraacetic acid or collagenase reveals one peak in our 2D relaxation map belonging to water present in the hydroxyapatite rich environment, and a second peak with shorter relaxation times arising from a collagen rich site. Diffusion-T2 measurements allowed for direct measurement of the diffusion coefficient of water in all observable reservoirs. Further, deuterium relaxation methods were applied to study cortical bone under an applied force, following mechanical wear or fracture. The tumbling correlation times of water reduce in all three cases, indicating that water dynamics may be used as a probe of bone quality. Lastly, changes in the relative populations and correlation times of water in bone under an applied force suggest that load bearing occurs largely in the collagen rich environment and is reversible.

13C, 2h NMR studies of structural and dynamical modifications of glucose-exposed porcine aortic elastin.

Elastin, the principal component of the elastic fiber of the extracellular matrix, imparts to vertebrate tissues remarkable resilience and longevity. This work focuses on elucidating dynamical and structural modifications of porcine aortic elastin exposed to glucose by solid-state NMR spectroscopic and relaxation methodologies. Results from macroscopic stress-strain tests are also presented and indicate that glucose-treated elastin is mechanically stiffer than the same tissue without glucose treatment. These measurements show a large hysteresis in the stress-strain behavior of glucose-treated elastin-a well-known signature of viscoelasticity. Two-dimensional relaxation NMR methods were used to investigate the correlation time, distribution, and population of water in these samples. Differences are observed between the relative populations of water, whereas the measured correlation times of tumbling motion of water across the samples were similar. (13)C magic-angle-spinning NMR methods were applied to investigate structural and dynamical modifications after glucose treatment. Although some overall structure is preserved, the process of glucose exposure results in more heterogeneous structures and slower mobility. The correlation times of tumbling motion of the (13)C-(1)H internuclear vectors in the glucose-treated sample are larger than in untreated samples, pointing to their more rigid structure. The (13)C cross-polarization spectra reveal a notably increased α-helical character in the alanine motifs after glucose exposure. Results from molecular dynamics simulations are provided that add further insight into dynamical and structural changes of a short repeat, [VPGVG]5, an alanine pentamer, desmosine, and isodesmosine sites with and without glucose. The simulations point to changes in the entropic and energetic contributions in the retractive forces of VPGVG and AAAAA motifs. The most notable change is the increase of the energetic contribution in the retractive force due to peptide-glucose interactions of the VPGVG motif, which may play an important role in the observed stiffening in glucose-treated elastin.

Revisiting NMR composite pulses for broadband (2)H excitation.

Quadrupolar echo NMR spectroscopy of static solids often requires RF excitation that covers spectral widths exceeding 100 kHz, which is difficult to obtain due to instrumental limitations. In this work we revisit four well-known composite pulses (COM-I, II, III and IV) for broadband excitation in deuterium quadrupolar echo spectroscopy. These composite pulses are combined with several phase cycling schemes that were previously shown to decrease finite pulse width distortions in deuterium solid-echo experiments performed with two single pulses. The simulations and experiments show that COM-II and IV composite pulses combined with an 8-step phase cycling aid in achieving broadband excitation with limited pulse width distortions.

Inverse temperature transition of elastin like motifs in major ampullate dragline silk: MD simulations of short peptides and NMR studies of water dynamics.

Using deuterium 2D T1 − T2 Inverse Laplace Transform (ILT) NMR, we have investigated the distribution, population, and dynamics of waters of hydration in major ampullate N. clavipes and A. aurantia silk as a function of temperature. In both samples studied, correlation times much larger than that of free water are measured, and in some cases, appear to increase with increasing temperature over the range of 5 to 60 °C (corresponding to reduced tumbling). In addition, the experimental data point to a reduction in the population of water localized in the silk with increasing temperature in the range of 20 to 50 °C. Molecular dynamics simulations were performed to probe the thermal characteristics of a variety of repeating motifs found in the two silk samples. The repeating motifs GLGSQ, GAAAAAAG, GPGGY, GPGQQ, GPSG, and GPSGPGS found in N. clavipes, GLGSQ, GYGSG, GPGSG, and GPGSQ found in A. aurantia silk were found to exhibit a thermal property observed in short elastin peptides known as the "inverse temperature transition". This is a well known characteristic exhibited by short peptides consisting of (VPGXG)n motifs (where X is any amino acid other than proline) found in elastin--a protein responsible for the elasticity of vertebrate tissues. In qualitative agreement with experimental measurements of water in the silks, all the peptides studied in simulation show evidence of an increase in sidechain contacts and peptide hydrogen bonds, concomitant with a decrease in radius of gyration and localized water as the temperature is raised from approximately 5 to 60 °C.

The role of mode of delivery on elastic fiber architecture and vaginal vault elasticity: a rodent model study.

We report on an experimental study of the role of mode of delivery and pregnancy on the architecture of vaginal elastic fibers and vaginal vault elasticity in female Sprague-Dawley rats. In primiparous rats submitted to spontaneous or Cesarean delivery and virgin rats submitted to simulated delivery, the tortuosity of elastic fibers (defined as the ratio of length to end-to-end distance) was observed to decrease when measured from two days to two weeks postpartum. In addition, the measured tortuosity of elastic fibers in multiparous rats was greater than that of virgin rats. The tortuosity of elastic fibers of all rats measured at two days postpartum was found to be similar to that of multiparous rats. At two weeks postpartum the measured tortuosity of vaginal elastic fibers was indistinguishable from virgin rats, regardless of the delivery method. Borrowing from the field of polymer physics, a model is suggested that connects elastic fiber tortuosity to the resulting tension under an applied stress; fibers having high tortuosity are expected to provide less structural support than more linear, low tortuosity fibers. To probe the macroscopic effects in elasticity due to architectural changes observed in elastic fibers, we have measured the stiffness of the vaginal vault in each cohort using a pressure-infusion system. The vaginal vault stiffness of all primiparous rats measured two weeks postpartum was greater than that measured two days postpartum. In addition, the vaginal vault of virgin rats was stiffer than that of multiparous rats. These observations confirmed that vaginal vault elastic fibers undergo significant remodeling due to pregnancy and parturition, and that the complex remodeling may be a significant contributor to tissue elasticity. Remarkably, regardless of the mode of delivery or simulated tissue trauma, elastic fiber tortuosity is observed to decrease from two days to two weeks postpartum indicating the onset of repair and recovery of tissue stiffness.

NMR Studies of Thermo-responsive Behavior of an Amphiphilic Poly(asparagine) Derivative in Water.

The thermo-responsive behavior of a unique biocompatible polymer, poly(N-substituted α/ß-asparagine) derivative (PAD), has been studied with several NMR methods. The 1H and 13C solution NMR measurements of the PAD in DMSO-d6 were used to investigate the isolated polymer and perform spectral assignments. By systematic addition of D2O we have tracked structural changes due to aggregation and observed contraction of hydrophilic side chains. Solution and cross polarization / magic angle spinning (CP/MAS) 13C NMR approaches were implemented to investigate the aggregates of the PAD aqueous solution during the liquid to gel transition as the temperature was increased. At temperatures near 20 °C, all of the peaks from the PAD were observed in the 13C CP/MAS and 13C solution NMR spectra, indicating the presence of polymer chain nodes. Increasing the temperature to 40 °C resulted in a partial disentanglement of the nodes due to thermal agitation and further heating resulted in little to no additional structural changes. Deuterium T1-T2 and T2-T2 two-dimensional relaxation spectroscopies using an inverse Laplace transform, were also implemented to monitor the water-PAD interaction during the phase transition. At temperatures near 20 °C the dynamical characteristics of water were manifested into one peak in the deuterium T1-T2 map. Increasing the temperature to 40 °C resulted in several distinguishable reservoirs of water with different dynamical characteristics. The observation of several reservoirs of water at the temperature of gel formation at 40 °C is consistent with a physical picture of a gel involving a network of interconnected polymer chains trapping a fluid. Further increase in temperature to 70 °C resulted in two non-exchanging water reservoirs probed by deuterium T2-T2 measurements.

Effects of experimental imperfections on a spin counting experiment.

Spin counting NMR is an experimental technique that allows a determination of the size and time evolution of networks of dipolar coupled nuclear spins. This work reports on an average Hamiltonian treatment of two spin counting sequences and compares the efficiency of the two cycles in the presence of flip errors, RF inhomogeneity, phase transients, phase errors, and offset interactions commonly present in NMR experiments. Simulations on small quantum systems performed using the two cycles reveal the effects of pulse imperfections on the resulting multiple quantum spectra, in qualitative agreement with the average Hamiltonian calculations. Experimental results on adamantane are presented, demonstrating differences in the two sequences in the presence of pulse errors.

On the inverse temperature transition and development of an entropic elastomeric force of the elastin mimetic peptide [LGGVG](3, 7).

We report on a molecular dynamics simulation based study of the thermal and mechanical properties of the elastin mimetic peptide [LGGVG](n) (n = 3, 7). Our findings indicate that this peptide undergoes an inverse temperature transition as the temperature is raised from ~20 °C to 42 °C. The thermal behavior is similar to what has been observed in other well studied short mimetic peptides of elastin. Both [LGGVG](n) (n = 3, 7) peptides exhibit an increase in the number of side chain contacts and peptide-peptide hydrogen bonds when the temperature is raised from ~20 °C to 42 °C. These observations are accompanied by a decrease in the number of proximal water molecules and number of peptide-water hydrogen bonds. This work also reports on a comparison of the thermal and mechanical properties of [LGGVG](3) and [VPGVG](3) and quantifies the interaction with surrounding waters of hydration under mechanically strained conditions. It is demonstrated, via a quasi-harmonic approach, that both model peptides exhibit a reduction in the population of low-frequency modes and an increase in population of high-frequency modes upon elongation. The shift in population of frequency modes causes the peptide entropy to decrease upon elongation and is responsible for the development of an entropic force that gives rise to elasticity. These observations are in disagreement with a previously published notion that model elastin peptides, such as [VPGVG](18), increase in entropy upon elongation.