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Welcome to the Boutis research laboratory web page. The groups' primary interest focuses on nuclear magnetic resonance applications in condensed matter systems and methodology development. The projects we pursue are driven by biological applications, and on occasion, allow students to work on improving methods (e.g. pulse sequences) and hardware for solid state and diffusion NMR. While the group focuses largely on studies of elastin, a protein giving the elastic fiber of vertebrate tissues resilience, we have also worked on problems relating to structural studies of silk, artificial silk scaffolds, and recently on cortical bone. These projects have attracted many undergraduate students, including minority students and students from underrepresented groups (through NIH SCORE and NSF LSAMP support). Examples of abstracts from some of our recent studies are provided below, highlighting the highly interdisciplinary nature of our work.

F. Gul-E-Noor, C. Singh, A. Papaioannou, N. Sinha and G. S. Boutis. "The Behavior of Water in Collagen and Hydroxyapatite Sites of Cortical Bone: Fracture, Mechanical Wear, and Load Bearing Studies" Journal of Physical Chemistry C 119 (37), 21528-21537, 2015 PDF

The mechanical properties of cortical bone, which is largely composed 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 time scales 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.

M. Silverstein, K. Bilici, SW. Morgan, Y. Wang, Y. Zhang, G S. Boutis, G.S (2015). "13C, 2H nmr studies of structural and dynamical modifications of glucose exposed porcine aortic elastin" Biophysical Journal, 108, 1758-1772, 2015 PDF

Elastin, the principal component of the elastic fiber of the extracellular matrix, imparts vertebrate tissues with remarkable resilience and longevity. The present work focuses on elucidating dynamical and structural modifications of porcine aortic elastin exposed to glucose by solid state NMR spectroscopy 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, while the measured correlation times of tumbling motion of water across the samples were similar. 13C magic angle spinning NMR methods were applied to investigate structural and dynamical modifications following glucose treatment. While 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 13C-1H internuclear vectors in the glucose treated sample are larger than in untreated samples pointing to a more rigid structure. The 13C cross polarization spectra reveal a notable increased alpha-helical character in the alanine motifs following glucose exposure. Results from molecular dynamics simulations are provided adding further insight into dynamical and structural changes of a short repeat [VGPVG]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. Most notably, the energetic contribution in the retractive force due to peptide-glucose interactions of the motif VPGVG increases and appears to be an important contribution to the observed stiffening in glucose treated elastin.

A. Papaioannou, M.Louis, B. Dhital, H.P. Ho, E. Chang, G.S. Boutis, "Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry" Biochimica et Biophysica Acta, Proteins and Proteomics, 1854(5):391-401, 2015 PDF

Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning 13C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the 13C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry are reported.

Ukpebor,O.T., A. Shah, E. Bazov, G.S. Boutis "Inverse Temperature Transition of Elastin Like Motifs in Major Ampullate Dragline Silk: MD Simulations of Short Peptides and NMR Studies of Water Dynamics" Soft Matter, 10 (5) 773-785, 2014. PDF

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 50C. 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 60C.

Downing, K., M. Billah, E. Raparia, A. Shah, M. Silverstein, A. Ahmad, G.S. Boutis "The role of mode of delivery on elastic fiber architecture and vaginal vault elasticity: a rodent model study" Journal of the Mechanical Behavior of Biomedical Materials, 29, 190-198, 2014.PDF

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 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 were 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.

Zelenova, Y., S. W. Morgan, G. S. Boutis, "Effects of experimental imperfections on a spin counting experiment" Solid State Nucl. Magn. Reson. 53, 27-37 2013. PDF

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, and phase errors 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.

C. Sun, O. Mitchell, J. Huang, G. S. Boutis. "NMR Studies of Localized Water and Protein Backbone Dynamics in Mechanically Strained Elastin" J. Phys. Chem. B, 115 (47), 13935-13942. 2011 PDF

We report on measurements of the dynamics of localized waters of hydration and the protein backbone of elastin, a remarkable resilient protein found in vertebrate tissues, as a function of the applied external strain. Using deuterium 2D T1-T2 NMR, we separate four reservoirs in the elastin-water system characterized by water with distinguishable mobilities. The measured correlation times corresponding to random tumbling of water localized to the protein is observed to decrease with increasing strain and is interpreted as an increase in its orientational entropy. The NMR T1and T1r relaxation times of the carbonyl and aliphatic carbons of the protein backbone are measured and indicate a reduction in the correlation time as the elastomer strain is increased. It is argued, and supported by MD simulation of a short model elastin peptide [VPGVG]3, that the observed changes in the backbone dynamics gives rise to the development of an entropic elastomeric force that is responsible for elastins' remarkable elasticity.

Ma, X., C. Sun, J. Huang, G.S. Boutis. "Thermal Hysteresis in the Backbone and Side Chain Dynamics of the Elastin Mimetic Peptide [VPGVG]3 Revealed by 2H NMR." J. Phys. Chem. B.116 (1),555564. 2012 PDF

We report on experimental measurements of the backbone and side chain dynamics of the elastin mimetic peptide [VPGVG]3 by 2H NMR echo spectroscopy and 2D T-T2 correlation relaxometry. The T1 and T2 relaxation times of the Gly alpha-deuterons and Val alpha-, beta- and gamma-deuterons of a hydrated sample reveal a thermal hysteresis when the temperature is raised from -10C to 45C and then subsequently cooled back to -10C. In addition, near 30C we observe a reduction in the slope of the T1(T) and T2(T) heating curves, indicating a structural change that appears to be correlated well to the known inverse temperature transition of this peptide. The thermal dependence of the correlation times of the Gly alpha-deuterons are well fit by an Arrhenius Law, from which we have measured Eact=(20.0 +/- 3.1) kJ/mol when the sample is heated, and Eact=(10.9 +/- 2.8) kJ/mol when cooled. Molecular dynamics simulations support the notion that the measured activation energy is determined largely by the extent of localized water, which is observed to decrease with increasing temperature from approximately 25C to 42C.