Biosynthesis and characterization of [15N]actinomycin D and conformational analysis by nitrogen-15 nuclear magnetic resonance

We describe the production and characterization of actinomycin D labeled with 15N at all twelve nitrogen positions. Cultures of Streptomyces parvulus were incubated in the presence of racemic [15N]glutamic acid and, following an initial delay, labeled antibiotic was produced. Evidence is presented that the D enantiomorph of glutamic acid was ultimately used for actinomycin biosynthesis. The 15N NMR spectrum at 10.14 and 20.47 MHz of the labeled drug in CDCl3 is presented. All nitrogens except the phenoxazone chromophore nitrogen are inverted when spectra are obtained under broad-band proton irradiation conditions. All 15N resonances have been assigned, and the proton-nitrogen one-bond coupling constants were determined in CDCl3 to be 92.5 +/- 0.3 Hz for the valine and threonine amide protons by both 1H and 15N NMR. 15N NMR spectra were also obtained in dimethyl sulfoxide, methanol, and water in order to probe solvent interactions with the peptide nitrogens and carbonyl groups. Large downfield shifts (greater than 5 ppm) were seen for the Pro, sarcosine, and methylvaline resonances when the solvent was changed from dimethyl sulfoxide to water. Smaller downfield shifts were observed for the Val and Thr peaks. These results are discussed in terms of a model for the solution conformation of the actinomycin pentapeptide rings based on different hydrogen-bonding interactions in the monomer in organic solvents and the dimer which is formed in water.     

Nitrate Inhibition of Legume Nodule Growth and Activity : II. Short Term Studies with High Nitrate Supply

Soybean plants (Glycine max [L.] Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR⁺ rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR⁻ rhizobia appeared to be too low to explain the inhibition of nitrogenase. Carbohydrate composition was similar in control nodules and nodules receiving 15 millimolar nitrate suggesting that the inhibition of nitrogenase by nitrate was not related to the availability of carbohydrate.

Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N₂ fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR⁺ and one NR⁻. The high nitrate treatment did not affect the allantoate/allantoin ratio or the proportion of amino N or ureide N in bacteroids (4%) and cytosol (96%).

     

Automated evaluation of chemical shift perturbation spectra: New approaches to quantitative analysis of receptor-ligand interaction NMR spectra

This paper presents new methods designed for quantitative analysis of chemical shift perturbation NMR spectra. The methods automatically trace the displacements of cross peaks between a perturbed test spectrum and the reference spectrum (or among a series of titration spectra), and measure the changes of chemical shifts, heights, and widths of the altered peaks. The methods are primary aimed at the (1)H-(15)N HSQC spectra of relatively small proteins (<15 kDa) assuming fast exchange between free and ligand-bound states on the chemical shift time scale, or for comparing spectra of free and fully bound states in the slow exchange situation. Using the (1)H-(15)N HSQC spectra from a titration experiment of the 74-residue Pex13p SH3 domain with a Pex14p peptide ligand (14 residues, K (d)= approximately 40 microM), we demonstrate the scope and limits of our automatic peak tracing (APET) algorithm for efficient scoring of high-throughput SAR by NMR type HSQC spectra, and progressive peak tracing (PROPET) algorithm for detailed analysis of ligand titration spectra. Simulated spectra with low signal-to-noise ratios (S/N ranged from 20 to 1) were used to demonstrate the reliability and reproducibility of the results when dealing with poor quality spectra. These algorithms have been implemented in a new software module, FELIX-Autoscreen, for streamlined processing, analysis and visualization of SAR by NMR and other high-throughput receptor/ligand interaction experiments.     

Analysis of flash-induced FTIR difference spectra of the S-state cycle in the photosynthetic water-oxidizing complex by uniform 15N and 13C isotope labeling

Protein bands in flash-induced Fourier transform infrared (FTIR) difference spectra of the S-state cycle of photosynthetic water oxidation were analyzed by uniform (15)N and (13)C isotopic labeling of photosystem II (PS II). The difference spectra upon first- to fourth-flash illumination were obtained with hydrated (for the 1800-1200 cm(-)(1) region) or deuterated (for the 3500-3100 cm(-)(1) region) films of unlabeled, (15)N-labeled, and (13)C-labeled PS II core complexes from Thermosynechococcus elongatus. Shifts of band frequencies upon (15)N and (13)C labeling provided the assignments of major peaks in the regions of 3450-3250 and 1700-1630 cm(-)(1) to the NH stretches and amide I modes of polypeptide backbones, respectively, and the assignments of some of the peaks in the 1600-1500 cm(-)(1) region to the amide II modes of backbones. Other prominent peaks in the latter region and most of the peaks in the 1450-1300 cm(-)(1) region exhibited large downshifts upon (13)C labeling but were unchanged by (15)N labeling, and hence assigned to the asymmetric and symmetric COO(-) stretching vibrations, respectively, of carboxylate groups in Glu, Asp, or the C-terminus. Peak positions corresponded well with each other among the first- to fourth-flash spectra, and most of the bands in the first- and/or second-flash spectra appeared with opposite signs of intensity in the third- and/or fourth-flash spectra. This observation indicates that the protein movements in the S(1)-->S(2) and/or S(2)-->S(3) transitions are mostly reversed in the S(3)-->S(0) and/or S(0)-->S(1) transitions, representing a catalytic role of the protein moieties of the water-oxidizing complex. Drastic structural changes in carboxylate groups over the S-state cycle suggest that the Asp and/or Glu side chains play important roles in the reaction mechanism of photosynthetic water oxidation.     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form

All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H. Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue )]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763-1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe.2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe.2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

短期内^15N标记的铵盐和硝酸盐在青藏高原高寒草甸中的运移规律

运用^15N稳定性同位素示踪技术,对高寒草甸植物和土壤微生物固持沉降氮的能力及沉降氮在小嵩草（Kobresia pygaea)草甸中的运移规律进行了研究。施肥2周后,NO3^-－^15N和NH4^ －^15N的总恢复率分别为73．5％和78％。无论是NO3^-－^15N,还是NH4^ －^15N植物所固持的^15N总是比土壤有机质或者是土壤微生物固持的多。4周后,70．6％的NO3^-－^15N和57．4％的NH4^ －^15N被固持在土壤和植物中。其中,土壤微生物所固持。在施肥6周和8周后,NO3^-－^15N的总恢复率分别为58．4％和67％,而NH4^ －^15N的总恢复率分别为43．1％和49％。植物和土壤微生物所固持的NO3^-－^15N比NH4^ －^15N多。在整个实验期间,植物固持的NO3^-N较多,而且比土壤微生物固持了较多^15N。由于无机氮的含量一直很低,无机氮库所固持的^15N一般不超过1％。上述结果意味着短期内植物在高寒草甸中对沉降氮的去向起着决定作用。     

Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

We introduce a Python-based program that utilizes the large database of ¹³C and ¹⁵N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D ¹³C–¹³C, ¹⁵N–¹³C, or 3D ¹⁵N–¹³C–¹³C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D ¹³C–¹³C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the Cα and Cβ chemical shifts, the highest-ranked PLUQ assignments were 40–60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO–Cα–Cβ or N–Cα–Cβ), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.     

Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation.

Cultured cells of a Rhizobium phaseoli wild-type strain (CE2) possess b-type and c-type cytochromes and two terminal oxidases: cytochromes o and aa3. Cytochrome aa3 was partially expressed when CE2 cells were grown on minimal medium, during symbiosis, and in well-aerated liquid cultures in a complex medium (PY2). Two cytochrome mutants of R. phaseoli were obtained and characterized. A Tn5-mob-induced mutant, CFN4201, expressed diminished amounts of b-type and c-type cytochromes, showed an enhanced expression of cytochrome oxidases, and had reduced levels of N,N,N',N'-tetramethyl-p-phenylenediamine, succinate, and NADH oxidase activities. Nodules formed by this strain had no N2 fixation activity. The other mutant, CFN4205, which was isolated by nitrosoguanidine mutagenesis, had reduced levels of cytochrome o and higher succinate oxidase activity but similar NADH and N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activities when compared with the wild-type strain. Strain CFN4205 expressed a fourfold-higher cytochrome aa3 content when cultured on minimal and complex media and had twofold-higher cytochrome aa3 levels during symbiosis when compared with the wild-type strain. Nodules formed by strain CFN4205 fixed 33% more N2 than did nodules formed by the wild-type strain, as judged by the total nitrogen content found in plants nodulated by these strains. Finally, low-temperature photodissociation spectra of whole cells from strains CE2 and CFN4205 reveal cytochromes o and aa3. Both cytochromes react with O2 at -180 degrees C to give a light-insensitive compound. These experiments identify cytochromes o and aa3 as functional terminal oxidases in R. phaseoli.     

Effects of heating at neutral and acid pH on the structure of beta-lactoglobulin A revealed by differential scanning calorimetry and circular dichroism spectroscopy

The structural change of beta-lactoglobulin A (betaLG A) on heating was measured at pH 3.0 and 7.5 with UV absorption difference spectra, differential scanning calorimetry (DSC), and circular dichroism (CD). At pH 3.0, betaLG A showed a reversible structural change by heating at 80 degrees C, while an irreversible change was observed and molecular aggregates of betaLG were formed by heating at 95 degrees C. DSC analysis of betaLG A gave endothermic peaks at 75 degrees C and 90 degrees C at pH 7.5, and 90 degrees C at pH 3.0. At pH 7.5, betaLG A modified with N-ethylmaleimide (NEM-betaLG A) gave two endothermic peaks: at 72 degrees C and 90 degrees C. CD spectra of betaLG A heated at various temperatures and pHs were measured and the spectra at pH 3.0 and 7.5 were not changed by heating to 95 degrees C and 80 degrees C, respectively. Unheated NEM-betaLG A gave a spectrum similar to that of heated betaLG A, suggesting that the secondary structure was changed by NEM treatment.     

Probable site of allantoin formation in nodulating soybean plants

Nodulated soybean plants contain high concentration of allantoin in all parts. Excision of nodules from the roots brought about a marked decrease in allantoin. To examine the function of nodules in allantoin production, nodulated and nodule-detached soybeans were fed with ¹⁵NH₃ for 1 week. High abundance of ¹⁵N was found in the amino acid-N fraction of both plants. In the root and stem of the nodulated plants, ca 80% of the nitrogen in this fraction was derived from the NH₃ added in the medium. Excess ¹⁵N was detected also in allantoin-N fraction, but the ¹⁵N content was very low in contrast to that in amino acid-N fraction. The site involved in the allantoin formation and the possible significance of its synthesis are discussed in relation to symbiotic nitrogen fixation.     

尖叶拟船叶藓的77K荧光光谱及对强光照的短期适应

报道了东亚特有濒危植物尖叶拟船叶藓(Dolichomitriopsis diversiformis)在不同光质的光照诱导下的低温77K荧光光谱及状态转移的初步研究结果,实验中,尖叶拟船叶藓在77K下出现了3条发射带,分别是F680、F685、F720nm,并没有出现存在于大部分高等植物中的F695nm和F740nm两个峰．经过PSⅡ光诱导后、在77K下出现了F680nm,这个峰在77K下出现是首次报道,而以前的研究认为只在4K下才出现这一条光谱带,这一结果表明尖叶拟船叶藓叶绿体的两个光系统结构与其他高等植物存在着差异。在自然光下,PSⅡ与PSⅠ的总能量比是2.04,经过15min的PSⅡ光(670nm)诱导后,PSⅡ与PSⅠ的总能量比变成了1．28(状态2),当用15min的PSⅠ光(716nm)照射后,PSⅡ与PSⅠ的总能量比从2．04变成了3．4l(状态1)。在自然光下,由尖叶拟船叶藓的光系统的外部LHCⅡ所吸收的激发能是整个光系统激发能的21．19％．这说明尖叶拟船叶藓对光的短期调节能力是21．19％．尖叶拟船叶藓的光系统的外部LHCⅡ有51．7％位于PSⅡ中,48．3％在PSⅠ中.     

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as (15)N. By utilising hydroponic media that contain (15)N inorganic salts as the sole nitrogen source, near to 100% (15)N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled (14)N- and (15)N-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of (14)N/(15)N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the (14)N and (15)N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct (14)N and (15)N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2).     

Solid-state 15N NMR analysis of highly 15N-enriched plant materials

Solid-state ¹⁵N NMR spectroscopy was used to determine the chemical nature of nitrogen in ¹⁵N-enriched material from the roots and stems of wheat (Triticum aesitivum), field pea (Pisum sativum) and kikuyu grass (Pennisetum clandestinum) and from the roots, stems and leaves of a eucalyptus species (Eucalyptus globulus). Nitrogen-15 cross polarization (CP) spectra of the materials were all very similar, with 64–75% of total signal assigned to amide N. Spin counting analysis indicated that 37–80% of potential signal was accounted for in the CP spectra, and that NMR observability using the CP technique (N _obs -CP) was higher for stems and leaves than for roots, and higher for wheat and eucalyptus than for peas and kikuyu. The ¹⁵N direct polarization (DP) spectra contained higher proportions of signal assigned to amine (up to 22%) and nitrate (up to 17%), and less assigned to amide N (50–72%) than the corresponding CP spectra. Spin counting analysis indicated that 68–93% of potential signal was accounted for in the DP spectra, confirming the DP technique to be more quantitatively reliable than CP.     

Novel approach for peptide quantitation and sequencing based on 15N and 13C metabolic labeling

Here we describe a method for protein identification and quantification using stable isotopes via in vivo metabolic labeling of the hyperthermophilic crenarchaeon Sulfolobus solfataricus. Stable isotope labeling for quantitative proteomics is becoming increasingly popular; however, its usefulness in protein identification has not been fully exploited. We use both 15N and 13C labeling to create three different versions of the same peptide, corresponding to the unlabeled, 15N and 13C labeled versions. The peptide then appears as three different peaks in a TOF-MS scan and three corresponding sets of MS/MS spectra are obtained. With this information, the elemental carbon and nitrogen compositions for each peptide and each fragment can be calculated. When this is used as a constraint in database searching and/or de novo sequencing, the confidence of a match is increased (for an example intact peptide from 34 choices to 1). This makes the method a useful proteomic tool for both sequenced and unsequenced organisms. Furthermore, it allows for accurate protein quantitation (standard deviations over >4 peptides per protein were within 10%) of three phenotypes in one MS experiment. Abundances for each peptide are calculated by determining the relative areas of each of the three peaks in the TOF-MS spectrum.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

较长时间内~(15)N标记的氨态氮和硝态氮在小嵩草草甸中的运移规律(英文)

运用15N稳定性同位素技术,对15N标记的硝酸盐和铵盐在输入小嵩草(KobresiapygaeaC.B.Clarke)草甸11~13个月后的运移规律进行了研究。在经历11~13个月后,进入无机氮库中的15N很少,一般不超过所输入氮素的1%,而较多的15N为土壤有机质、土壤微生物和植物所固持。NO3--15N和NH4 -15N在小嵩草草甸中的运移规律差异很大。在11、12和13个月后,NO3--15N的总恢复率分别为92.83%、92.64%和79.96%;而NH4 -15N的则分别为49.6%、63.33%和66.22%。两者的差异在土壤有机质、土壤微生物和植物等库之间的分配中更加明显。输入NO3--15N时在11、12个月后植物所固持的15N最多,而土壤微生物和土壤有机质所固持的15N比较接近,而在13个月后,土壤有机质和植物所固持的15N接近,而土壤微生物所固持的15N下降许多;当输入NH4 -15N,土壤有机质所固持的15N比植物和土壤微生物所固持的都多,而且植物所固持的15N比较稳定,而土壤微生物所固持的15N则有较大变化。这表明在较长的时间内嵩草草甸对NO3-和NH4 的固持能力是不一样的。     

Root Nodule Enzymes of Ammonia Assimilation in Alfalfa (Medicago sativa L.) : DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN

Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO₃⁻ at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO₃⁻-N per hectare and harvested plants treated with 40 kilograms NO₃⁻-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N₂-fixing capacity were accompanied by similar changes in levels of nodule soluble protein.     

In vivo labeling with stable isotopes as a tool for the identification of unidentified peaks in the metabolome analysis of Corynebacterium glutamicum by GC/MS

Of the hyphenated techniques used for metabolic profiling of cell and tissue extracts, GC/MS is in some ways advantageous as it allows the simultaneous fingerprinting of chemically very different metabolites, and the electron impact mass spectra recorded in many cases lead to unambiguous identification of the compounds. However, prior to chromatography, the hydrophilic substances of the cell extracts have to be converted to vaporizable derivatives, the mass spectra of which often are not known or not listed in the available spectral libraries, even if they are derived from simple biochemicals. Thus, numerous chromatographic peaks remain as yet unidentified. Attempts to identify these peaks afford the acquisition of more data on these compounds. The value of in vivo labeling of metabolites with (13)C and (15)N for this purpose is described.     

Advances towards resonance assignments for uniformly—13C, 15N enriched bacteriorhodopsin at 18.8 T in purple membranes

Solid state NMR spectra from uniformly (13)C, (15)N enriched bacteriorhodospin (bR) purified from H. salinarium were acquired at 18.8 T using magic angle spinning methods. Isolated resonances of 2D (13)C-(13)C spectra exhibited 0.50-0.55 ppm line-widths. Several amino acid types could be assigned, and at least 12 out of 15 Ile peaks could be resolved clearly and identified based on their characteristic chemical shifts and connectivities. This study confirms that high resolution solid state NMR spectra can be obtained for a 248 amino acid uniformly labeled membrane protein in its native membrane environment and indicates that site-specific assignments are likely to be feasible with heteronuclear multidimensional spectra.     

Rotational-echo Double-resonance in Complex Biopolymers: a Study of Nephila Clavipes Dragline Silk

Rotational-Echo Double-Resonance (REDOR) NMR on strategically 13C and 15N labeled samples is used to study the conformation of the LGXQ (X = S, G, or N) motif in the major ampullate gland dragline silk from the spider Nephila clavipes. A method is described for calculating REDOR dephasing curves suitable for background subtractions, using probability distributions of nitrogen atoms surrounding a given carbon site, which are developed from coordinates in the Brookhaven Protein Data Bank. The validity of the method is established by comparison to dephasings observed from natural abundance 13C peaks for G and A. Straightforward fitting of universal REDOR dephasing curves to the background corrected peaks of interest provide results which are not self-consistent, and a more sophisticated analysis is developed which better accounts for 15N labels which have scrambled from the intended positions. While there is likely some heterogeneity in the structures formed by the LGXQ sequences, the data indicate that they all form compact turn-like structures.