RESUMO
Silk fibroin is stored in the silk glands of Bombyx mori silkworms as a condensed aqueous solution called liquid silk. It is converted into silk fibers at the silkworm's spinnerets under mechanical forces including shear stress and pressure. However, the detailed mechanism of the structural transition of liquid silk to silk fibers under pressure is not well understood. Magic angle spinning (MAS) in solid-state nuclear magnetic resonance (NMR) can exert pressure on liquid samples in a quantitative manner. In this study, solid-state NMR was used to quantitatively analyze the impact of pressure on the structural transition of liquid silk. A combination of 13C DD-MAS and CP-MAS NMR measurements enabled the conformation and dynamics of the crystalline region of the silk fibroin (both before (Silk Ip) and after (Silk IIp) the structural transition) to be detected in real time with atomic resolution. Spectral analyses proposed that the pressure-induced structural transition from Silk Ip to Silk IIp proceeds by a two-step autocatalytic reaction mechanism. The first reaction step is a nucleation step in which Silk Ip transforms to single lamellar Silk IIp, and the second is a growth step in which the single lamellar Silk IIp acts as a catalyst that reacts with Silk Ip molecules to further form Silk IIp molecules, resulting in stacked lamellar Silk IIp. Furthermore, the rate constant in the second step shows a significant pressure dependence, with an increase in pressure accelerating the formation of large stacked lamellar Silk IIp.
Assuntos
Bombyx , Fibroínas , Animais , Seda/química , Bombyx/química , Fibroínas/química , Espectroscopia de Ressonância Magnética/métodos , Conformação ProteicaRESUMO
Skin plays an important role in protecting the human body from the environment, dehydration, and infection. Burns, wounds, and disease cause the skin to lose its role, but tissue-engineered skin substitutes offer the opportunity to restore skin loss. Silk fibroin from Bombyx mori (SF) has proven to be an excellent wound dressing material. In this study, we aim to develop an excellent wound dressing material by introducing three-residue sequence Arg-Gly-Asp (RGD), which is the most well-known adhesion site of fibronectin, in the films of SF and the model peptide. Its usefulness as a wound dressing material was evaluated both in vitro and in vivo. First, we showed that the flexible structures of the RGD sequence are still maintained in SF with a rigid antiparallel ß-sheet structure using NMR in association with excellent wound dressings of SF containing RGD. Then, in in vitro experiments, two types of normal cells derived from human skin, normal human neonatal epidermal keratinocytes and normal human neonatal dermal fibroblasts, were used to evaluate the cell adhesion. On the other hand, in in vivo experiments, the study was conducted using a rat model of a whole skin layer defect wound. The results showed that the high-functionalized SF developed here has the potential to play a significant role in the field of wound dressings.
Assuntos
Bombyx , Fibroínas , Animais , Ratos , Humanos , Fibroínas/química , Bombyx/química , Cicatrização , Oligopeptídeos/química , Peptídeos/química , Bandagens , Seda/químicaRESUMO
The conformational transition of [3-13C]Ser- and [3-13C]Tyr-Antheraea yamamai silk fibroin before spinning induced by stretching was investigated with 13C CP/MAS NMR spectroscopy. The α-helix content of the silk fibroin before stretching was found to be 31.6% based on the Ala and Ser peaks. With increasing stretching ratio, the α-helix and the random coil Ala Cß peaks decreased gradually, while the ß-sheet peak was observed at a stretching ratio of ×5 and increased rapidly upon further stretching. For Ser residue, the α-helix peak decreased monotonically with increasing stretching ratio, but the random coil peak increased slightly till the stretching ratio of ×5 and then decreased. A small ß-sheet peak was observed before stretching and then increased rapidly starting from the stretching ratio of ×7. In contrast, a gradual decrease of random coil peak and an increase of ß-sheet peak were observed for the Tyr residue. The results of this investigation may be helpful for further studies of fiber formation mechanism in A. yamamai and in the future design of artificial silk materials.
Assuntos
Bombyx , Fibroínas , Mariposas , Animais , Fibroínas/química , Bombyx/química , Conformação Proteica , Seda/química , Espectroscopia de Ressonância Magnética/métodosRESUMO
Spider dragline silk is a biopolymer with excellent mechanical properties. The development of recombinant spider silk protein (RSP)-based materials with these properties is desirable. Formic acid (FA) is a spinning solvent for regenerated Bombyx mori silk fiber with excellent mechanical properties. To use FA as a spinning solvent for RSP with the sequence of major ampullate spider silk protein from Araneus diadematus, we determined the conformation of RSP in FA using solution NMR to determine the role of FA as a spinning solvent. We assigned 1H, 13C, and 15N chemical shifts to 32-residue repetitive sequences, including polyAla and Gly-rich regions of RSP. Chemical shift evaluation revealed that RSP is in mainly random coil conformation with partially type II ß-turn structure in the Gly-Pro-Gly-X motifs of the Gly-rich region in FA, which was confirmed by the 15N NOE data. In addition, formylation at the Ser OH groups occurred in FA. Furthermore, we evaluated the conformation of the as-cast film of RSP dissolved in FA using solid-state NMR and found that ß-sheet structure was predominantly formed.
Assuntos
Formiatos/química , Proteínas de Insetos/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas Recombinantes/química , Seda/química , Animais , Bombyx , Conformação ProteicaRESUMO
Spider dragline silk has unique characteristics of strength and extensibility, including supercontraction. When we use it as a biomaterial or material for textiles, it is important to suppress the effect of water on the fiber by as much as possible in order to maintain dimensional stability. In order to produce spider silk with a highly hydrophobic character, based on the sequence of ADF-3 silk, we produced recombinant silk (RSSP(VLI)) where all QQ sequences were replaced by VL, while single Q was replaced by I. The artificial RSSP(VLI) fiber was prepared using formic acid as the spinning solvent and methanol as the coagulant solvent. The dimensional stability and water absorption experiments of the fiber were performed for eight kinds of silk fiber. RSSP(VLI) fiber showed high dimensional stability, which is suitable for textiles. A remarkable decrease in the motion of the fiber in water was made evident by 13C solid-state NMR. This study using 13C solid-state NMR is the first trial to put spider silk to practical use and provide information regarding the molecular design of new recombinant spider silk materials with high dimensional stability in water, allowing recombinant spider silk proteins to be used in next-generation biomaterials and materials for textiles.
Assuntos
Seda , Água , Seda/química , Água/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas Recombinantes/química , Materiais Biocompatíveis/química , Proteínas de ArtrópodesRESUMO
Silk fibroin (SF) from Bombyx mori has superior properties as both a textile and a biomaterial, and has been used to functionalize the surfaces of various medical inorganic materials including titanium (Ti). In this study, we endowed SF with reversible binding ability to Ti by embedding a titanium binding motif (minTBP-1 and RKLPDA). Artificial SF proteins were first created by conjugating gene cassettes for SF motif (AGSGAG) and minTBP-1 motif with different ratios, which have been shown to bind reversibly to Ti surfaces in quartz crystal microbalance analyses. Based on these results, the functionalized SF (TiBP-SF) containing the designed peptide [TS[(AGSGAG)3 AS]2 RKLPDAS]8 was prepared from the cocoon of transgenic B. mori, which accelerates the ossific differentiation of MC3T3-E1 cells when coated on titanium substrates. Thus, TiBP-SF presents an alternative for endowing the surfaces of titanium materials with osseointegration functionality, which would allow the exploration of potential applications in the medical field.
Assuntos
Diferenciação Celular , Materiais Revestidos Biocompatíveis/química , Fibroínas/química , Osteogênese , Titânio/química , Motivos de Aminoácidos , Animais , Bombyx , Linhagem Celular , Fibroínas/genética , CamundongosRESUMO
Recently, considerable attention has been paid to Bombyx mori silk fibroin by a range of scientists from polymer chemists to biomaterial researchers because it has excellent physical properties, such as strength, toughness, and biocompatibility. These appealing physical properties originate from the silk fibroin structure, and therefore, structural determinations of silk fibroin before (silk I) and after (silk II) spinning are a key to make wider applications of silk. There are discrepancies about the silk I structural model, i.e., one is type II ß-turn structure determined using many solid-state and solution NMR spectroscopies together with selectively stable isotope-labeled model peptides, but another is α-helix or partially α-helix structure speculated using IR and Raman methods. In this review, firstly, the process that led to type II ß-turn structure by the authors was introduced in detail. Then the problems in speculating silk I structure by IR and Raman methods were pointed out together with the problem in the assignment of the amide I band in the spectra. It has been emphasized that the conformational analyses of proteins and peptides from IR and Raman studies are not straightforward and should be very careful when the proteins contain ß-turn structure using many experimental data by Vass et al. In conclusion, the author emphasized here that silk I structure should be type II ß-turn, not α-helix.
Assuntos
Fibroínas/química , Fibroínas/metabolismo , Seda/química , Animais , Bombyx/química , Proteínas de Insetos/química , Espectroscopia de Ressonância Magnética/métodos , Peptídeos/química , Conformação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Seda/metabolismo , Espectrofotometria Infravermelho/métodos , Análise Espectral Raman/métodosRESUMO
Recently, Bombyx mori silk fibroin (SF) has been shown to be a suitable material for vascular prostheses for small arteries. In this study, we developed a softer SF graft by coating water-dispersed biodegradable polyurethane (PU) based on polycaprolactone and an SF composite sponge on the knitted SF vascular graft. Three kinds of 13C solid-state nuclear magnetic resonance (NMR), namely carbon-13 (13C) cross-polarization/magic angle spinning (MAS), 13C dipolar decoupled MAS, and 13C refocused insensitive nuclei enhanced by polarization transfer (r-INEPT) NMR, were used to characterize the PU-SF coating sponge. Especially the 13C r-INEPT NMR spectrum of water-dispersed biodegradable PU showed that both main components of the non-crystalline domain of PU and amorphous domain of SF were highly mobile in the hydrated state. Then, the small-diameter SF artificial vascular grafts coated with this sponge were evaluated through implantation experiments with rats. The implanted PU-SF-coated SF grafts showed a high patency rate. It was confirmed that the inside of the SF grafts was covered with vascular endothelial cells 4 weeks after implantation. These results showed that the water-dispersed biodegradable PU-SF-coated SF graft created in this study could be a strong candidate for small-diameter artificial vascular graft.
Assuntos
Prótese Vascular , Fibroínas/química , Poliuretanos/química , Seda/química , Animais , Bombyx/química , Células Endoteliais/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Teste de Materiais , Poliuretanos/farmacologia , Ratos , Seda/farmacologia , Alicerces Teciduais/química , Água/químicaRESUMO
Bombyx mori silk fibroin (SF) fibers with excellent mechanical properties have attracted widespread attention as new biomaterials. However, the structural details are still not conclusive. Here, we propose a lamellar structure for the crystalline domain of the SF fiber based on structural analyses of the Ala Cß peaks in the 13C cross-polarization/magic angle spinning NMR spectra of (Ala-Gly)m (m = 9, 12, 15, and 25) and 13C selectively labeled (Ala-Gly)15 model peptides. Namely, three Ala Cß peaks with relative intensities of 1:2:1 obtained by deconvolution were assigned to two kinds of ß-sheet and a ß-turn, which are interpreted as a lamellar structure formed by repetitive folding using ß-turns every eighth amino acid, for which the basic structure is (Ala-Gly)4 in an antipolar arrangement. The dynamics and intermolecular arrangement were further studied using 13C solid-state spin-lattice relaxation time observations and the rotational echo double resonance experiments, respectively.
Assuntos
Bombyx , Fibroínas , Alanina , Animais , Glicina , Espectroscopia de Ressonância Magnética , SedaRESUMO
This review will introduce very recent studies using solid-state nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulation on the structure and dynamics of spider dragline silks conducted by the author's research group. Spider dragline silks possess extraordinary mechanical properties by combining high tensile strength with outstanding elongation before breaking, and therefore continue to attract attention of researchers in biology, biochemistry, biophysics, analytical chemistry, polymer technology, textile technology, and tissue engineering. However, the inherently non-crystalline structure means that X-ray diffraction and electron diffraction methods provide only limited information because it is difficult to study the molecular structure of the amorphous region. The most detailed picture of the structure and dynamics of the silks in the solid state experimentally have come from solid-state NMR measurements coupled with stable isotope labeling of the silks and the related silk peptides. In addition, combination of solid-state NMR and MD simulation was very powerful analytical tools to understand the local conformation and dynamics of the spider dragline silk in atomic resolution. In this review, the author will emphasize how solid-state NMR and MD simulation have contributed to a better understanding of the structure and dynamics in the spider dragline silks.
Assuntos
Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Seda/química , Seda/metabolismo , AnimaisRESUMO
Packing structures of polyalanine regions, which are considered to be the reason for the extremely high strength of spider dragline silks, were studied using a series of sequential peptides: (Glu)4GlyGlyLeuGlyGlyGlnGlyAlaGly(Ala)nGlyGlyAlaGlyGlnGlyGlyTyrGlyGly(Glu)4 (n = 3-8) using 13C solid-state NMR spectroscopy. The conformations of (Ala)n in the freeze-dried peptides changed gradually with increasing n from random coils to α-helices with partial antiparallel ß-sheet (AP-ß) structures. Conversely, all the insolubilized peptides, n = 6-8 after low-pH treatment and n = 4-8 after formic acid/methanol treatment, formed AP-ß structures with significant amounts of staggered packing arrangements. These results are different from previously obtained results for pure alanine oligopeptides, that is, AP-ß (Ala)n formed rectangular packing for less than n = 6 but staggered packings for n ≥ 7. The 13C-labeled peptides were also used to confirm the staggered packing arrangements from NMR dynamics. Furthermore, a MD simulation supported the observed results.
Assuntos
Isótopos de Carbono/química , Peptídeos/química , Seda/química , Aranhas/química , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Conformação Proteica em Folha betaRESUMO
To the best of our knowledge, there are currently no reports on primary uterine ependymoma. We operated on a 38-year-old woman suspected with uterine fibroids; surgical findings led to the diagnosis of primary anaplastic ependymoma of the uterus. Most extraneural ependymomas arising from the ovary and perigenital peritoneum occur in women of reproductive age and express estrogen and progesterone receptors. The etiology of such tumors is unknown, and a treatment strategy has not been established yet. Therefore, it is essential to accumulate cases for identifying mechanisms underlying the pathogenesis and disease progression to facilitate diagnosis and development of an effective treatment.
Assuntos
Ependimoma/patologia , Neoplasias Uterinas/patologia , Feminino , Humanos , Útero/patologiaRESUMO
The beta (ß)-sheet structures of oligopeptides and polypeptides can be formed in anti-parallel (AP) and parallel (P) forms, which is an important feature to understand such structures. In principle, P- and AP-ß-sheet structures can be identified by the presence (AP) or absence (P) of inter-strand 1 HNH /1 HNH correlations on a diagonal in the corresponding 2D 1 H double quantum (DQ)/1 H single quantum (SQ) spectrum due to the different inter-strand 1 HNH /1 HNH distances between the two arrangements. However, the 1 HNH /1 HNH peaks overlap with the 1 HNH3+ /1 HNH3+ peaks, which always give cross-peaks regardless of the ß-sheet arrangement. The 1 HNH3+ /1 HNH3+ peaks disturb the observation of the presence/absence of 1 HNH /1 HNH correlations and the assignment of 1 HNH and 1 HNH3+ is not always available. Here, 3D 14 N/1 H DQ/1 H SQ correlation solid-state NMR experiments at fast magic angle spinning (70â kHz) are introduced to distinguish AP- and P-ß-sheet structures. The 14 N dimension allows the distinction of 1 HNH /1 HNH peaks from 1 HNH3+ /1 HNH3+ peaks with clear assignments of 1 HNH and 1 HNH3+ . In addition, the high natural abundance of 1 H and 14 N enables 3D 14 N/1 H DQ/1 H SQ experiments of oligo-alanines (Ala3-6 ) in four hours without isotope labelling.
RESUMO
The conformational analysis of spider dragline silks is difficult because of the amorphous character of the silks. In this article, the fractions of several conformations were determined for three 47-mer peptides, (Glu)4(Ala)6GlyGly12Ala13Gly14GlnGlyGlyTyrGlyGlyLeuGlySerGlnGly25Ala26Gly27-ArgGlyGlyLeuGlyGlyGlnGly35Ala36Gly37(Ala)6(Glu)4, with three underlined 13C-labeled blocks using a 13C CP/MAS NMR method. The conformations of the 13C-labeled sites change significantly depending on the location of the labeled blocks when treated with trifluoroacetic acid, low pH, and freeze-drying. The conformations of Ala36 and Gly37 residues are strongly influenced by the specific conformation of the (Ala)6 sequence at the C-terminal side, but those of other residues, Ala13 and Gly14, and Ala 26 and Gly27, are basically not influenced by the conformations of (Ala)6. Through hydration of the ß-sheet peptide, sharp peaks with random coil could be observed depending on the position of the residue, and this result could be interpreted via the change in the Ramachandran map obtained from the molecular dynamics simulation.
Assuntos
Fibroínas/química , Proteínas de Insetos/química , Espectroscopia de Ressonância Magnética/métodos , Fragmentos de Peptídeos/química , Seda/química , Aranhas/química , Ácido Trifluoracético/química , Animais , Isótopos de Carbono , Liofilização , Concentração de Íons de Hidrogênio , Conformação Molecular , Simulação de Dinâmica MolecularRESUMO
The effects of water on the structure and dynamics of natural and regenerated silk fibroin (SF) samples were studied using 13C solid-state nuclear magnetic resonance (NMR) spectroscopy. We prepared different types of SF materials, sponges, and fibers with different preparation methods and compared their NMR spectra in the dry and hydrated states. Three kinds of 13C NMR techniques, r-INEPT, CP/MAS, and DD/MAS, coupled with 13C isotope labeling of Ser, Tyr, and Ala residues were used. In the hydrated sponges, several conformations, that is, Silk I* and two kinds of ß-sheets, A and B, random coil, and highly mobile hydrated random coil were observed, and the fractions were determined. The fractions were remarkably different among the three sponges but with only small differences among the regenerated and native fibers. The increase in the fraction of ß-sheet B might be one of the structural factors for preparing stronger regenerated SF fiber.
Assuntos
Bombyx/química , Fibroínas/química , Ressonância Magnética Nuclear Biomolecular , Água/química , Alanina/química , Animais , Isótopos de Carbono/química , Estrutura Secundária de Proteína , Serina/químicaRESUMO
Samia cynthia ricini is one of the wild silkworms and its silk fibroin (SF) consists of alternatively repeating poly-l-alanine (PLA) sequences as crystalline domain and glycine-rich sequences as noncrystalline domain; the structure is similar to those of spider silk and other wild silkworm silks. In this paper, we proposed a new staggered model for the packing arrangement of the PLA sequence through the use of the Cambridge Serial Total Energy Package program and a comparison of the observed and calculated chemical shifts of the PLA sequence with the Gauge Including Projector Augmented Wave method. The new model was supported by the interatomic distance information from the cross peaks of Ala Cß dipolar-assisted rotational resonance (DARR) spectrum of the PLA sequences in S. c. ricini SF fiber. In addition, three 13C NMR peaks observed in the ß-sheet region were assigned to the carbons with different environments in the same model, but not assigned to different ß-sheet structures.
Assuntos
Fibroínas/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Bombyx/química , Bombyx/fisiologia , Isótopos de Carbono , Cristalização , Cristalografia , Fibroínas/isolamento & purificação , Espectroscopia de Ressonância Magnética , Conformação Proteica em Folha betaRESUMO
Polyalanine (polyA) sequences are well known as the simplest sequence that naturally forms anti-parallel ß-sheets and constitute a key element in the structure of spider and wild silkworm silk fibers. We have carried out a systematic analysis of the packing of anti-parallel ß-sheets for (Ala)n, n = 5, 6, 7 and 12, using primarily 13C solid-state NMR and MD simulation. HFIP and TFA are frequently used as the dope solvents for recombinant silks, and polyA was solidified from both HFIP and TFA solutions by drying. An analysis of Ala Cß peaks in the 13C CP/MAS NMR spectra indicated that polyA from HFIP was mainly rectangular but polyA from TFA was mainly staggered. The transition from the rectangular to the staggered arrangement in (Ala)6 was observed for the first time from the change in the Ala Cß peak through heat treatment at 200 °C for 4 h. The removal of the bound water was confirmed by thermal analysis. This transition could be reproduced by MD simulation of (Ala)6 molecules at 200 °C after removal of the bound water molecules. In this way, the origin of the stability of the different packing arrangements of polyA was clarified.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Peptídeos/química , Solventes/química , Varredura Diferencial de Calorimetria , Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , TermogravimetriaRESUMO
Samia cynthia ricini (S. c. ricini) is one of the wild silkworms. Their silk fibroins have been paid attention as potentially valuable biomedical materials as well as Bombyx mori silk fibroins, but detailed information on the packing arrangement of the fibers is still not currently well understood at a molecular level. In this study, 34 mer model peptides, GGAGGGYGGDGG(A)12GGAGDGYGAG with different 13C labeled positions have been synthesized as a typical sequence of the primary structure of S. c. ricini silk fibroins made up of tandemly repeated sequences of polyalanine as the crystalline region and glycin-rich sequences as the non-crystalline region. The heterogeneous structure was obtained from the determination of the fraction of several conformations depending on the position of the Ala residue by 13C cross polarization/magic angle spinning NMR. The packing arrangement was studied by 13C dipolar assisted rotational resonance NMR and packing in a staggered arrangement rather than a rectangular arrangement of this peptide with an anti-parallel ß-sheet structure was clarified, which is in good agreement with our previous report on the packing arrangement of (Ala)7 with an anti-parallel ß-sheet structure.
Assuntos
Bombyx/metabolismo , Fibroínas/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Isótopos de Carbono/química , Marcação por Isótopo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/síntese química , Estrutura Secundária de Proteína , Seda/químicaRESUMO
In order to develop small-diameter vascular grafts, it is necessary to evaluate endothelialization, especially, in the center part at early stage. For implantation of vascular grafts of 1 cm in length to abdominal aortae of rat, endothelial cells can be formed easily by stretching anastomosis. We evaluated the endothelialization in the center part of vascular grafts by implanting vascular grafts using transgenic (TG) silk fibroin (SF) of 3 cm in length. Vascular grafts were prepared 1.5 mm in diameter and 1 and 3 cm in length using wild type (WT) SF and TG SF by braiding structure, respectively. The grafts were removed after 2 weeks or 3 months and evaluated pathologically. Endothelialization was not confirmed totally after 3 months of implantation. However, endothelialization in the center part of grafts was significantly higher in TG SF than in WT SF. No significant difference was found regarding tissue infiltration and internal diameter. The TG SF revealed migration of the endothelial cells into the center part of the vessels at the early stage. Also, tissue infiltration and remodeling is expected using SF. The 3 cm length vascular grafts can be evaluated as a new experimental system.
Assuntos
Aorta Abdominal/cirurgia , Bioprótese , Prótese Vascular , Endotélio Vascular/ultraestrutura , Doenças Vasculares/cirurgia , Enxerto Vascular/métodos , Animais , Aorta Abdominal/ultraestrutura , Modelos Animais de Doenças , Feminino , Microscopia Eletrônica de Varredura , Ratos , Ratos Sprague-Dawley , Doenças Vasculares/patologiaRESUMO
(14)N/(14)N correlations are vital for structural studies of solid samples, especially those in which (15)N isotopic enrichment is challenging, time-consuming and expensive. Although (14)N nuclei have high isotopic abundance (99.6%), there are inherent difficulties in observing (14)N/(14)N correlations due to limited resolution and sensitivity related to: (i) low (14)N gyromagnetic ratio (γ), (ii) large (14)N quadrupolar couplings, (iii) integer (14)N spin quantum number (I = 1), and (iv) very weak (14)N-(14)N dipolar couplings. Previously, we demonstrated a proton-detected 3D (14)N/(14)N/(1)H correlation experiment at fast magic angle spinning (MAS) on l-histidine·HCl·H2O utilizing a through-bond (J) and residual dipolar-splitting (RDS) based heteronuclear multiple quantum correlation (J-HMQC) sequence mediated through (1)H/(1)H radio-frequency driven recoupling (RFDR). As an extension of our previous work, in this study we show the utility of dipolar-based HMQC (D-HMQC) in combination with (1)H/(1)H RFDR mixing to obtain sensitivity enhanced (14)N/(14)N correlations in more complex biological solids such as a glycyl-l-alanine (Gly-l-Ala) dipeptide, and parallel (P) and antiparallel (AP) ß-strand alanine tripeptides (P-(Ala)3 and AP-(Ala)3, respectively). These systems highlight the mandatory necessity of 3D (14)N/(14)N/(1)H measurements to get (14)N/(14)N correlations when the amide proton resonances are overlapped. Moreover, the application of long selective (14)N pulses, instead of short hard ones, is shown to improve the sensitivity. Globally, we demonstrate that replacing J-scalar with dipolar interaction and hard- with selective-(14)N pulses allows gaining a factor of ca. 360 in experimental time. On the basis of intermolecular NH/NH distances and (14)N quadrupolar tensor orientations, (14)N/(14)N correlations are effectively utilized to make a clear distinction between the parallel and antiparallel arrangements of the ß-strands in (Ala)3 through the observation of inter-ß-sheet correlations.