13C magnetic resonance evidence for anisotropic molecular motion in collagen fibrils

Relaxation times and integrated intensities have been obtained from dipolar decoupled ¹³C magnetic resonance spectra of reconstituted fibrils of chick calvaria collagen enriched at the glycine C^a and C′ positions. The data obtained are consistent with a model in which collagen molecules reorient about the long axis of the helix with a rotational diffusion constant (R₁) of ~10⁷ s^?1, a value similar to that expected for the helix in solution. Data obtained from natural abundance ¹³C spectra of native (crosslinked) calf achilles tendon and rat tail tendon provide evidence of rapid anisotropic reorientation for at least 75% of the carbons in these tissues. Hence, our preliminary data indicate that, in these materials, the intermolecular interactions in the fibrilar collagen lattice can accommodate rapid reorientation at a majority of carbon sites.     

A 13C nuclear magnetic resonance study of the interaction of ligands with arginine residues in dihydrofolate reductase.

¹³C nmr spectra of $\text{Streptococcus faecium}$ dihydrofolate reductase containing [¹³C-guanidino] arginine and ligand complexes with the labeled enzyme are reported. The spectrum of the native enzyme shows 5 well-resolved resonances (the enzyme contains 8 Arg) with a chemical shift range of 1.2 ppm. Addition of ligands to the enzyme produces distinct changes in the enzyme spectrum, and demonstrates that ¹³C nmr of labeled protein can be used in studies of protein-ligand interactions. An example of the use of ¹³C-depleted material is also presented.     

Multinuclear magnetic resonance studies of collagen molecular structure and dynamics

We have measured the percentages of cis and trans Gly-Pro and X-Hyp peptide bonds in thermally unfolded type I collagen. ¹³C-nmr solution spectra show that 16% of the Gly-Pro and 8% of the X-Hyp bonds are cis in unfolded chick calvaria collagen. These results support the hypothesis that cis–trans isomerization is that rate-limiting step in the propagation of the collagen triple helix. We have used multinuclear solid-state nmr to study the molecular dynamics of the collagen backbone in tendon, demineralized bone, and intact bone as a function of temperature, hydration, and pH. These studies show that collagen backbone motions are characterized by a broad distribution of correlation times, τ, covering the range from 10^?4 to 10^?9 s. In the case of nonmineralized collagen, the root-mean-square fluctuations in azimuthal angle, γ_rms, range from ca. 10° when τ ～ 10^?9 s to ca. 30° when τ < 10^?4 s; in the case of bone collagen, γ_rms values are about half as large as those found in nonmineralized collagen. Backbone motions are negligible at temperatures below ?25°C. This is also the case at 22°C when demineralized bone collagen is lyophilized. In contrast, flexibility of hydrated demineralized bone collagen greatly increases as pH is lowered from 7 to 2. The more limited flexibility observed at neutral pH is a consequence of the intermolecular interactions that contribute to fibril organization and strength. However, the fibrils retain significant flexibility at physiological pH, enabling them to distribute stress and dissipate mechanical energy.     

Inhibition by collagen chains of lysyl hydroxylase of chick embryo and of WI-38 human lung fibroblasts.

Lysyl hydroxylase from chick embryos was strongly inhibited by heat-denatured collagens from various vertebrate sources, and by separated a chains and β components of rat tail tendon collagen. The kinetics exhibited with this enzyme when heat-denatured calf or rabbit skin collagens was used showed a mixed type of inhibition. On the other hand, a preparation of homologous heat-denatured 4,5-³H-L-lysine-labeled collagen, in itself an extremely poor substrate for the hydroxylase, showed non-competitive inhibition with a K_i of about 8–9 μM. Finally, the lysyl hydroxylase preparations from WI-38 fetal human lung fibroblasts and from transformed WI-38 cells (WI-38 VA 13) were also inhibited by heat-denatured collagens or, where tested, by separated collagen chains.     

Influence of neutral salts on the hydrothermal stability of acid-soluble collagen

The thermal stability of acid-soluble collagens was studied by circular dichroism (CD) spectroscopy. Adult bovine dermal collagen (BDC), rat-tail tendon collagen (RTC), and calf skin collagen (CSC) were compared. Despite some variability in amino acid composition and apparent molecular weight, the CD spectra for helical and unordered collagen structures were essentially the same for all the sources. The melting of these collagens occurs as a two-stage process characterized by a pretransition (T _p) followed by complete denaturation (T _d). The characteristic temperatures vary with the source of the collagen; for mature collagens (BDC, RTC) T _p = 30°C and T _d = 36deg;C, and for CSC T _p = 34°C and T _d = 40°C. Neutral salts, NaCl or KCl, at low concentrations (0.02–0.2 M) appear to bind to the collagens and shift the thermal transitions of these collagens to lower temperatures.     

Mobility of elastin chains as determined by 13C nuclear magnetic resonance

Relaxation times and integrated intensities of ¹³C have been obtained from nuclear magnetic resonance spectra of elastin in unstretched calf ligamentum nuchae and indicate that about 80% of the backbone carbonyl carbons have short rotational correlation times, τ_R ～ 40 nanoseconds. τ_R is reduced by only a factor of two when the ligament is in contact with 2 m-KCNS, a strong denaturant. By contrast, the highly ordered chains of collagen in insoluble calf achilles tendon give no spectrum until denatured in 2 m-KCNS, when t_R decreases by many orders of magnitude. These results show that elastin is composed largely of highly mobile chains under physiological conditions, suggesting that configurational entropy has an important role in its elastic properties.     

Cross-polarization NMR of N-15 labeled soybeans

Cross-polarization ¹⁵N nmr spectra of ¹⁵N-labeled soybean seeds, pods, and leaves have been obtained at 9.12 MHz both with and without high-speed sample rotation at the magic angle. Spectral resolution is sufficient to permit a determination of the relative concentrations of amide and amine nitrogens, as well as of a few specific amino acid residues of proteins in the solid, intact samples. Utilization by soybean of nitrogen from labeled fertilizer in the presence of dinitrogen fixation can be determined from these spectra. A double-cross polarization ¹³C nmr spectrum of a spinning, ¹⁵N-labeled seed has been obtained in which resonances are observed only from these carbons directly bonded to nitrogens. This technique leads to a qualitative estimate of amino-acid composition of the protein which is complementary to that obtained directly from the ¹⁵N nmr spectrum.     

液相色谱/质谱联用法分析不同年龄鼠皮肤中I型、III型胶原蛋白相对含量

哺乳动物皮肤真皮中胶原蛋白含量约为70%,主要为是I型、III型胶原蛋白,本实验利用稀酸溶解和酶法提取了大鼠皮肤中的总胶原蛋白,将胶原蛋白粗提品在60℃变性后用胰蛋白酶进行降解,液相色谱/质谱联用法分析了两种胶原蛋白的特征多肽,利用特征多肽比较了不同生长期大鼠皮肤中I型和III型胶原蛋白相对含量。结果表明,大鼠皮肤中的III型胶原蛋白的相对含量随生长期延长逐渐降低,而I型胶原蛋白的相对含量逐渐升高,8周后两种胶原蛋白的比例趋于稳定。本实验结果表明使用高效液相色谱/质谱联用法分析组织中的胶原蛋白类型及其动态变化具有可行性,为更好的临床应用提供了实验基础。     

Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen

3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5–18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro⁷⁰⁷ and newly identified sites at Pro⁷¹⁶ and Pro⁷¹⁹, at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.     

NMR study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR chemical shift contour plots

The metastable state silk I structures of Bombyx mori silk fibroin in the solid state were studied on the basis of ¹⁵N- and ¹³C-nmr chemical shifts of Ala, Ser, and Gly residues. The ¹⁵N cross-polarization magic angle spinning (CP/MAS) nmr spectra of the precipitated fraction after chymotrypsin hydrolysis of B. mori silk fibroin with the silk I and silk II forms were measured to determine the ¹⁵N chemical shifts of Gly, Ala, and Ser residues. For comparison, ¹⁵N CP/MAS nmr chemical shifts of Ala were measured for [¹⁵N] Ala Philosamia cynthia ricini silk fibroin with antiparallel β-sheet and α-helix forms. The ¹³C CP/MAS nmr chemical shifts of Ala, Ser, and Gly residues of B. mori silk fibroin with the silk I and silk II forms, as well as ¹³C CP/MAS nmr chemical shifts of Ala residue of P. c. ricini silk fibroin with β-sheet and α-helix forms, are used for the examination of the silk I structure. Both silk I and α-helix peaks are shifted to a lower field than silk II (β-sheet) for the Cα carbons of the Ala residues, while both Cβ carbon peaks are shifted to higher field. However, the silk I peak of the ¹⁵N nucleus of the Ala residue is shifted to lower field than the silk II peak, but the α-helix peak is shifted to high field. Thus, the difference in the structure between the silk I and α-helix is reflected in a different manner between the ¹³C and ¹⁵N chemical shifts. The Cα and Cβ chemical shift contour plots for Ala and Ser residues, and the Cα plot for the Gly residue, were prepared from the Protein Data Bank data obtained for 12 proteins and used for discussing the silk I structure quantitatively from the conformation-dependent chemical shifts. The plots reported by Le and Oldfield for ¹⁵N chemical shifts were also used for the purpose. All these chemical shift data support Fossey's model (Ala: ϕ = −80°, φ = 150°, Gly: ϕ = −150°, φ = 80°) and do not support Lotz and Keith's model (Ala: ϕ = −104.6°, φ = 112.2°, Gly: ϕ = 79.8°, φ = 49.7° or Ala: ϕ = −124.5°, φ = 88.2°, Gly: ϕ = −49.8°, φ = −76.1°) as the silk I structure. © 1997 John Wiley & Sons, Inc.     

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Magic-angle-spinning solid-state ¹³C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-¹³C,¹⁵N labeled samples. To overcome this problem, we present a method for assigning ¹³C chemical shifts and secondary structures from unresolved two-dimensional ¹³C–¹³C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts and cross-peak intensities. The experimental ¹³C–¹³C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C^α, C^β, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved ¹³C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.     

Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation.

Types I and III collagens were solubilized from fetal human skin by limited digestion with pepsin and precipitated by dialysis against 0.02 M Na₂HPO₄. Heat denaturation of the collagens in 2 M guanidine-HCl, pH 7.5, resulted in the precipitation of the contaminant pepsin which could be removed by centrifugation. Renaturation of the denatured collagens by dialysis against deionized water at 22° for 2 hours selectively precipitated the type III collagen fibrils. Type I collagen remained in solution. The simplicity and high recovery (77%) make this a suitable approach for the rapid estimation of type III collagen in small tissue samples.     

13C/1H high power double magnetic resonance investigation of collagen backbone motion in fibrils and in solution

${}^{13}\text{C}{}^1\text{H}$ high power double magnetic resonance spectroscopy was used to investigate the mobility of the collagen peptide backbone. [1-¹³C]- and [2-¹³C]-glycine-labeled collagen samples (with >50% enrichment in ¹³C) were prepared via chick calvaria culture. ¹³C n.m.r.² spectra of labeled reconstituted collagen fibrils, of labeled helical collagen in solution, and of unlabeled bovine Achilles tendon collagen were obtained with scalar decoupling and with dipolar decoupling of protons. Proton-enhanced spectra were also obtained using cross-polarization techniques. n.m.r. parameters (linewidths, lineshapes, T₁ values, nuclear Overhauser enhancements, and cross polarization enhancements) were measured for the labeled samples and for collagen in natural abundance. Comparison of ¹³C n.m.r. parameters for bovine Achilles tendon fibrils and for reconstituted chick calvaria collagen fibrils established that chick calvaria collagen is a good model for the molecular dynamics of collagen in vivo.Spin-lattice relaxation times and nuclear Overhauser enhancements for [1-¹³C]- and [2-¹³C]glycine-labeled collagen indicated that R₁ ~2 × 10⁷s^?1 in solution, where R₁ is the diffusion constant for reorientation about the long axis of the molecule. A substantially smaller value for R₁ (2.6 × 10⁶s^?1) was calculated for an axially symmetric ellipsoid of revolution having dimensions appropriate to the collagen helix. The discrepancy between the rigid ellipsoid and n.m.r. values of R₁ suggests that the collagen molecule undergoes torsional reorientation, as well as rod-like reorientation, about its long axis.The T₁ and NOE values measured in the glycine-labeled fibrils show that rapid axial motion (R₁ ~ 10⁷s^?1) persists in the fibrillar state. In the collagen fibril the full width of the glycyl carbonyl powder pattern is 103 p.p.m. This value is substantially smaller than the rigid lattice value, 144 p.p.m., which provides further evidence for motion in the fibril. The observed powder pattern is axially asymmetric, which shows that certain azimuthal orientations are energetically preferred in the fibril. Taken together, the n.m.r. data provide strong evidence that rapid reorientation of the helix backbone occurs in the fibrils. This result shows that formation of a fibrillar structure does not require the existence of a unique set of intermolecular interactions at the helical surfaces.     

Human aorta collagens: evidence for three distinct species

Three different molecular species of collagen and a soluble form of elastin were obtained by digestion of human aortas with pepsin. Two of the three collagens contain $\text{1}\text{2}$ cystine, present in interchain disulfide crosslinkages, and appear to represent type IV collagen previously described in basement membranes and type III collagen, recently found in fetal skin. The third collagen species is type I, the molecule found in a wide variety of connective tissues including skin, bone, tendon and ligaments.     

Isolation and biochemical characterization of collagens from seaweed pipefish, Syngnathus schlegeli

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the seaweed pipefish (Syngnathus schlegeli) and partially characterized. The amount of collagens isolated in the subsequent treatments was 5.5% of ASC and 33.2% PSC on the basis of lyophilized pipefish body weight, respectively. According to the electrophoretic pattern and CM-cellulose column chromatogram, the collagens might be classified as type I collagens, containing α1 and α2 chain. The imino acid content of collagen from pipefish was lower than those of mammalian collagens as also were the denaturation temperatures (Td) of collagens were 34.8°C and 35.1°C, respectively. This study shows that there is a possibility to use pipefish collagen as the alternative source of collagen from industrial purposes and subsequently it may evaluate the economical value of the seaweed pipefish.     

The X-Pro peptide bond as an nmr probe for conformational studies of flexible linear peptides

The equilibrium between the cis and trans forms of X-Pro peptide bonds can readily be measured in the ¹³C nmr spectra. In the present paper we investigate how observation of this equilibrium could be used as an nmr probe for conformational studies of flexible polypeptide chains. The experiments include studies by ¹³C nmr of a series of linear oligopeptides containing different X-L -Pro peptide bonds, with X = Gly, L -Ala, L -Leu, L -Phe, D -Ala, D -Leu, and D -Phe. Overall the study confirms that X-Pro peptide bonds can generally be useful as ¹³C nmr probes reporting the formation of nonrandom conformation in flexible polypeptide chains. It was found that the cis–trans equilibrium of X-Pro is greatly affected by the side chain of X and the configuration of the α-carbon atom of X. On the basis of these observations some general rules are suggested for a practical applications of the X-Pro nmr probes in conformational studies of polypeptide chains.     

A 13C nmr and esr study on the structure of the different forms of the cobalt-bleomycin A2 complex

¹³C nmr and esr spectra have been recorded from oxygenated cobalt-bleomycin A₂. It appears that (a) in oxygenated cobalt-bleomycin all the cobalt is in the Co³⁺ oxidation state, (b) the ¹³C nmr data indicate a structure of the Co-bleomycin complex in which the nitrogens of the pyrimidine, the imidazole, and the secondary and primary amine of the diaminopropanoic amide are involved as planar equatorial ligands, and (c) free bleomycin forms a “dimer” with Co-bleomycin.     

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.     

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {¹H}-¹³C solid-state NMR spectroscopy. We interpret ¹³C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a ¹³C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen.