Mechanistic Insights into the Role of Water in Backbone Dynamics of Native Collagen Protein by Natural Abundance ¹⁵N NMR Spectroscopy

Collagen being the most abundant animal protein is an integral component of muscle, connective tissues, cartilage, and bones, having function to provide strength, load bearing, and flexibility to organisms. The structural and functional role of water mediated hydrogen bonding network of collagen has been debated. We present here a solid–state nuclear magnetic resonance (ssNMR) spectroscopy method to observe water dependent backbone dynamics of collagen protein in its absolute native environment inside bone. The load bearing function of collagen is directly dependent on its backbone dynamics. The picoseconds backbone dynamics has been measured by relaxation rate measurement (R₁) of natural isotopic abundance ¹⁵N (∼0.37%) resonance. The sensitivity of natural abundance ¹⁵N ssNMR spectrum is enhanced by paramagnetic doping of copper-ethylenediaminetetraacetic acid (Cu (II) EDTA) inside bone matrix. Exclusive backbone resonances originating from proline/hydroxyproline (Pro/Hyp) and glycine (Gly) resonances are well resolved in ¹⁵N spectrum, giving access to relaxation rates from native collagen backbone. The water dependent site-resolved relaxation measurements have provided mechanistic insights into role of water mediated hydrogen bonding network in native collagen backbone dynamics. Our study has given new insights in the understanding of water dependent native collagen dynamics, functions, and stability

Comparison of a stochastic- and a deterministic-based action potential.

<p>The deterministic action potential (blue dashed line) reproduces the result of Hu et al; their action potential initiates at the AIS and spreads to the soma and apical dendrite. Aligned, peaked to peak, is a second action potential (solid red line) using stochastic Na-channels (both Nav 1.2 and Nav 1.6). Both action potentials are generated by the same somatic current-step of 1 nA. Inset y-axis goes from -55 mV to -48 mV (increments of 1 mV); inset x-axis goes from 4.8 ms to 5.2 ms (increments of.05 ms).</p

TTS relative frequency histogram and overlaid inverse Gaussian distribution with the same mean and variance.

<p>(A) is generated by a current-step of 0.67 nA, the mean TTS is 13.38 ms (vertical line) and the variance is 0.022 ms². (B) is generated by Poisson synaptic activation (λ = 55.8 events/ms), the mean TTS is 14.46 ms (vertical line) and the variance is 1.25 ms². One thousand simulations produce each of the histograms. Current and synaptic activations begin at TTS = 0. Notice the x-axis scale difference.</p

Inverse TTS approximates a linear function of excitation.

<p>Excitation is either (A) a point dendritic current-step or (B) a spatially dispersed, synaptic activation. Lines are best linear fits (see text). Each point is an average of 120 excitations from rest. The error bars (SEM) for the current-step are within the plot points. All points but the highest intensities always had spike initiation at the AIS. At the largest intensity on each curve, the spike originated in the dendrite 20 percent of the time.</p

Distributions for Λ and associated mutual information values.

<p>Distributions for Λ and associated mutual information values.</p

TTS relative frequency histogram and overlaid inverse Gaussian distribution with the same mean and variance.

<p>(A) is generated by a current-step of 0.67 nA, the mean TTS is 13.38 ms (vertical line) and the variance is 0.022 ms². (B) is generated by Poisson synaptic activation (λ = 55.8 events/ms), the mean TTS is 14.46 ms (vertical line) and the variance is 1.25 ms². One thousand simulations produce each of the histograms. Current and synaptic activations begin at TTS = 0. Notice the x-axis scale difference.</p

Synaptic shot-noise far exceeds Na-channel shot-noise.

<p>Random synaptic activation greatly increases the variation in TTS. (A) The only variation in TTS using a current-step is due to the stochastic nature of Na-channel activation. TTS variance increases as individual Na-channel conductance events get larger while keeping constant. By comparison in (B), the synaptic conductance events create much more variance. Note the y-axis scale differences. A current-step of 0.7 nA generates the data of (A). In (B), stochastic activation for each point is on average the same with a total conductance of 16.6 nS. Error bars are SEM. Lines are best linear fits (see text).</p

Predominant Role of Water in Native Collagen Assembly inside the Bone Matrix

Bone is one of the most intriguing biomaterials found in nature consisting of bundles of collagen helixes, hydroxyapatite, and water, forming an exceptionally tough, yet lightweight material. We present here an experimental tool to map water-dependent subtle changes in triple helical assembly of collagen protein in its absolute native environment. Collagen being the most abundant animal protein has been subject of several structural studies in last few decades, mostly on an extracted, overexpressed, and synthesized form of collagen protein. Our method is based on a ¹H detected solid-state nuclear magnetic resonance (ssNMR) experiment performed on native collagen protein inside intact bone matrix. Recent development in ¹H homonuclear decoupling sequences has made it possible to observe specific atomic resolution in a large complex system. The method consists of observing a natural-abundance two-dimensional (2D) ¹H/¹³C heteronuclear correlation (HETCOR) and¹H double quantum–single quantum (DQ-SQ) correlation ssNMR experiment. The 2D NMR experiment maps three-dimensional assembly of native collagen protein and shows that extracted form of collagen protein is significantly different from protein in the native state. The method also captures native collagen subtle changes (of the order of ∼1.0 Å) due to dehydration and H/D exchange, giving an experimental tool to map small changes. The method has the potential to be of wide applicability to other collagen containing biomaterials

Molecular Level Understanding of Biological Systems with High Motional Heterogeneity in Its Absolute Native State

Biological tissues possessing motional heterogeneity are difficult to comprehend at atomic level in their native state. Some of these tissues play crucial roles in support and movement of the body. Articular cartilage is one such tissue exemplified with highly mobile glycosaminoglycans (GAGS, correlation time ∼10^–9s) and relatively rigid collagen protein (correlation time ∼10^–6 s). Simultaneous study of both the components at atomic level is challenging and requires different sets of solid state nuclear magnetic resonance (ssNMR) experiments for mobile and rigid parts. Additionally, it is hard to get any quantitative information as one ¹³C one pulse experiment is time-consuming. We present here a ¹³C ssNMR experiment with complete molecular characterization of cartilage organic components in its native state. We achieved this by employing heteronuclear Overhauser enhancement with equilibration of magnetization by low power irradiation. Complete ¹³C resonances from GAGS and collagen were identified simultaneously with 2–6 times sensitivity enhancement. We are also providing a method to characterize GAGs (qualitatively and quantitatively) in native state thereby increasing its applicability in disease conditions where GAGS is degraded. This method is not only applicable to biological tissues such as cartilage but also to understand ultrastructural details of hydrogels and other motional heterogeneous biomaterials used in cartilage tissue engineering

One-Pot Tandem Hiyama Alkynylation/Cyclizations by Palladium(II) Acyclic Diaminocarbene (ADC) Complexes Yielding Biologically Relevant Benzofuran Scaffolds

A series of palladium acyclic diaminocarbene (ADC) complexes of the type <i>cis</i>-[(R¹NH)­(R²)­methylidene]­PdCl₂(CNR¹) [R¹ = 2,4,6-(CH₃)₃C₆H₂: R² = NC₅H₁₀ (<b>2</b>); NC₄H₈ (<b>3</b>); NC₄H₈O (<b>4</b>)] were used not only to perform the C_sp²–C_sp Hiyama coupling between aryl iodide and triethoxysilylalkynes but also to subsequently carry out the one-pot tandem Hiyama alkynylation/cyclization reaction between 2-iodophenol and triethoxysilylalkynes, giving a convenient time-efficient access to the biologically relevant benzofuran compounds. The palladium ADC complexes (<b>2</b>–<b>4</b>) were conveniently synthesized by the nucleophilic addition of secondary amines, namely, piperidine, pyrrolidine, and morpholine on the <i>cis</i>-{(2,4,6-(CH₃)₃C₆H₂)­NC}₂PdCl₂ in moderate yields (ca. 61–66%)