^15N标记蛋白GAL4（62）的2D NMR实验

利用自编的脉冲程序,采用预饱和和自旋锁定对水峰进行双重抑制的方法,得到了＾１５Ｎ标记蛋白ＧＡＬ４（６２）的２Ｄ＾１Ｈ－＾１５ＮＨＳＱＣ、ＨＳＱＣ－ＮＯＥＳＹ、ＨＳＱＣ－ＴＯＣＳＹ谱,并对这几个谱在蛋白质＾１Ｈ谱的归属中所到的作用作了讨论。     

根际优势菌耐药菌株的获得及其15N标记

对两株具有反硝化活性的根际优势细菌进行了耐药性和^１５Ｎ标记;研究了该双标记菌株在土壤中的动态及其活性。结果表明,所用双标记方法是可行的,应用标记菌株可以追踪其在土壤中的消长。结果还表明,标记^1５Ｎ菌株的^１５Ｎ丰度与供试培养基中氮源的^１５Ｎ丰度相近;标记菌株的反硝化活性与出发菌株的相同。     

15N交叉标记有机与无机肥料氮的转化与残留

有机无机肥配施能够培肥土壤,改善土壤氮素供给,但目前有机无机肥配施主要集中在化肥氮的研究,忽略秸秆氮对化肥氮转化的影响。为了解秸秆还田对不同氮源转化和残留的影响,采用¹⁵N对尿素和水稻秸秆进行交叉标记,在两种不同肥力水稻土 (粘土矿物类型为1 ∶ 1型红黄泥和2 ∶ 1型紫潮泥) 进行水稻盆栽试验。设置对照(CK),单施尿素(¹⁵NU)、标记尿素与稻草配施(¹⁵NU-S) 和标记稻草与尿素配施(¹⁵NS-U)4个处理。结果表明,水稻吸收的氮素60%以上来自土壤氮,土壤氮素肥力相对较低的红黄泥较之紫潮泥对肥料氮的依赖更强;水稻生长期间微生物同化的尿素氮占标记底物的百分数红黄泥为1.8%-8.3%,紫潮泥为1.8%-19.2%;微生物同化的秸杆氮占标记底物的百分数红黄泥为1.7%-5.0%,紫潮泥为2.0%-6.2%。而粘土矿物固持的尿素氮占标记底物的百分数,红黄泥为0.3%-2.1%,紫潮泥为3.5%-18.7%;粘土矿物固持的秸杆氮红黄泥为0.2%-0.9%,紫潮泥为1.7%-5.0%。水稻成熟期尿素氮的残留率,红黄泥¹⁵NU处理、¹⁵NU+S分别为14.5%和17.0%,紫潮泥分别为16.9%和17.1%。秸秆氮的残留率分别为红黄泥38.8%、紫潮泥41.5%;有机无机肥配施提高了微生物同化化肥氮的能力,降低了粘土矿物晶格固持化肥氮的水平。有机无机配施提高了化肥氮利用率同时,提高了有机形态氮残留,降低了无机形态氮(矿质氮+固定态铵)的残留。     

微生物发酵合成L－缬氨酸－15N

采用含有稳定同位素^１５Ｎ－硫铵为主要氮源的专用发酵培养液配方和相应的工艺条件,在国内首次用微生物直接发酵研制成Ｌ－缬氨酸－^１５Ｎ高丰度精制产品。产品^１５Ｎ丰度９７．６８％,反比原料^１５Ｎ－硫铵丰度下降０．５３％,Ｌ－缬氨酸－^１５Ｎ产酸率最高达４％以上（未校正）。每克^1５Ｎ－硫铵可得到１克以上的Ｌ－缬氨酸－^１５Ｎ（分析值）。提取精制收率平均为８０－９０％（     

15N标记稻草中N、C在羊体内的转化和利用

应用¹⁵N标记稻草饲喂3只山羊,以探明羊对稻草N、C化合物的消化、吸收、排泄和转化规律.结果表明,已宰杀的2只羊消化、吸收、转化为羊机体的¹⁵N占试验日粮中¹⁵N富集总量的38.54和23.78%,平均为31.16%.3只羊从粪尿中排泄的¹⁵N各占饲料中¹⁵N的34.78、33.88和33.18%,平均为33.95±0.80%,已屠宰的2只羊对饲料¹⁵N总回收率为73.32和56.96%,损失率为26.68和43.04%.饲料¹⁵N的回收利用率与饲料中氨基酸的消化率(%)相吻合.1、2、3号羊对饲料碳水化合物的消化率分别为76.40、68.66和65.19%.其中饲喂2、3号羊的饲料中都含稻草50%左右,羊对碳水化合物的平均消化率为66.93%.     

15N同位素稀释法评估甘蔗的生物固氮量

为评估甘蔗生物固氮量,采用15N同位素稀释法,以木薯为参比植物,进行温室桶栽试验.结果表明:甘蔗全生育期植株固氮11.3514％ Ndfa,固氮量每桶0.9269 g.甘蔗根、茎、叶的固氮百分率和固氮量大小依序为叶＞茎＞根.叶的固氮百分率(13.2668％ Ndfa)略高于植株,但两者差异不显著.甘蔗植株全氮量中来自空气氮(生物固氮)、肥料氮和土壤氮的比例分别为11.3514％、7.6857％、80.9629％.甘蔗的氮肥利用率为58.7583％.甘蔗根、茎、叶各部位均有固氮现象,生产上可以用叶代替植株来评估甘蔗的生物固氮量.     

山东潍坊地下水硝酸盐污染现状及δ15N溯源

采用均匀布点选取了山东潍坊居民区、粮田和蔬菜种植区等区域地下水为研究对象,分析了地下水硝态氮含量及污染来源,结果表明:潍坊地区地下水硝态氮平均含量为28.1 mg/L ,按照国家地下水质量标准(GB/T 14848-93)属于Ⅲ类水;饮用水井硝态氮平均含量为23.3 mg/L, 最高值达到了150 mg/L,对国家饮用水标准(10 mg/L)超标率高达73.5%,严重超标率达50%;不同土地利用方式下地下水硝态氮含量不同,设施蔬菜种植区最高,其次是露地蔬菜种植区, 小麦-玉米种植区地下水硝态氮含量较低,但都超过了WHO饮用水中硝酸盐的最大允许含量50 mg/L的规定(折合为硝态氮11.3 mg/L);硝酸盐与水质离子之间的相关性以及水质分析相关的派珀图分析显示地下水硝酸盐污染与氮肥施用有关;根据硝酸盐δ¹⁵N的稳定同位素溯源分析,潍坊地区地下水硝酸盐41.5%来自于化肥,14.6%来自于生活污水,其他是来自化肥、生活污水和家畜粪尿的混合污染。综上,潍坊市地下水硝酸盐污染非常严重,已经对当地居民的身体健康造成了潜在的威胁;因此,亟需从源头控制做起,减少肥料的过量投入和生活污水的随意排放,以控制硝酸盐的继续污染及改善当地水环境。     

凋落叶多样性对杉木幼苗生长及吸收15N标记硫铵的影响

利用¹⁵N硫铵研究了凋落叶多样性对杉木幼苗生长及养分吸收的影响.结果表明,凋落叶多样性的增加有利于盆栽杉木幼苗的生长.杉木、火力楠、红栲和刺楸4种凋落叶混合处理后,杉木幼苗的生长量最大;杉木、火力楠、刺楸3种凋落叶混合处理后的杉木幼苗生长量次之,其它依次为杉木、火力楠、红栲3种凋落叶混合处理>杉木和刺楸凋落叶处理>杉木和红栲凋落叶处理>对照>杉木和火力楠2种凋落叶混合处理>杉木凋落叶处理.就杉木幼苗对硫铵氮的吸收率而言,不作任何处理的杉木幼苗吸收率最高,其次为杉木、火力楠、红栲和刺楸4种凋落叶混合处理,其它依次为杉木、火力楠、刺楸3种凋落叶混合处理和杉木、火力楠、红栲3种凋落叶混合处理>杉木和刺楸凋落叶处理>杉木和红栲凋落叶处理>杉木和火力楠2种凋落叶混合处理>杉木凋落叶处理.另外,用凋落叶处理后,土壤中硫铵氮的残留量比不作凋落叶处理的土壤多,硫铵氮的总回收量也比不作凋落叶处理的土壤大幅增加,而且凋落叶多样性的增加也会增加硫铵氮的残留量.     

15N示踪控释氮肥的氮肥利用率及去向研究

选用¹⁵N同位素标记的新型回收塑料包膜控释肥和大颗粒尿素,采用池栽试验研究夏玉米-冬小麦轮作体系中肥料氮的去向及利用率。结果表明,整个轮作体系中,控释肥处理（PCU）作物吸收的肥料氮为241.03 kg/hm,高于尿素处理（Urea)的211.02 kg/hm。控释肥处理施用的肥料氮主要残留在0~40 cm土层,而尿素处理则残留在0~60 cm土层,控释肥延缓了肥料氮向土壤深层迁移的趋势。在夏玉米和冬小麦轮作体系中,控释肥处理的氮肥利用率（32.86%,32.47%）高于尿素处理（28.23%,30.16%）。在冬小麦季,控释肥处理损失率相比尿素处理从36.07% 降至28.75%,而夏玉米季,控释肥处理损失率相比尿素处理从37.17%降至29.50%。玉米季控释肥处理与尿素处理差异不显著,但在冬小麦季控释肥处理的产量显著高于尿素处理。因此,在玉米和小麦整个生长季,新型回收塑料包膜控释肥的养分释放与作物养分需求吻合,既提高氮肥利用率,也降低了肥料氮的损失。     

栓皮栎叶片δ13C和δ15N的纬向趋势及其影响因子

以东亚广布种栓皮栎为例,通过对南北样带(26°-40° N)上7个群体的调查取样,并结合2个生活史阶段,探讨栓皮栎碳氮同位素比值随纬度环境的变异规律.结果表明: 在纬向梯度上,栓皮栎叶片δ¹³C和δ¹⁵N随纬度的增加分别呈现非线性的增加和下降趋势,且成年树叶片δ¹³C和δ¹⁵N均显著高于幼树;同时,树龄和纬度对叶片δ¹⁵N和δ¹³C均无显著交互作用,表明栓皮栎幼树和成年树纬向环境变化的响应较为一致.随机森林模型结果显示,栓皮栎叶片δ¹⁵N主要受土壤养分,如土壤有机质、磷和氮含量的影响,而叶片δ¹³C主要受水分因子,如空气相对湿度、降水量等的影响.     

NMR localization of the O-mycoloylation on PorH,a channel forming peptide from Corynebacterium glutamicum

PorH and PorA are two small peptides that, in complex, form a voltage-dependent ion channel in the outer membrane of Corynebacterium glutamicum. Specific post-translational modifications on PorA and PorH are required for the formation of a functional ion channel. The assignment of PorH proton NMR chemical shifts in DMSO, allowed identifying unambiguously the exact position of the PorH O-mycoloylation on Ser 56 side chain. This was further confirmed by site directed mutagenesis and mass spectrometry. Together with the previously published localization of PorA mycoloylation, this provides the complete primary structure characterization of this outer membrane porin.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

Experiments and strategies for the assignment of fully13 C/15N-labelled polypeptides by solid state NMR

High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully¹³ C/¹⁵N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue¹³ C-¹⁵N correlation experiments with¹³ C-¹³C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using ¹⁵N instead of¹³ C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the ¹⁵N and ¹³C resonances in human ubiquitin.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

Improved sensitivity in indirect monitoring of chemical shifts of proton-heteronuclear spin pairs (1H-13C and 1H-15N) in 3D and 4D NMR spectroscopy

In three-dimensional and four-dimensional experiments on doubly labelled proteins not only heteronuclear (¹³C or ¹⁵N) but also proton (¹H) frequencies are often indirectly monitored, rather than being directly observed. In this communication we show how in these experiments by overlaying ¹H and heteronuclear evolutions one can obtain decreased apparent relaxation rates of ¹H signals, yielding improved sensitivity. The new method applies to spin pairs like ¹H-¹⁵N, as in amide groups, or ¹H-¹³C, as in methine groups of alpha or aromatic systems.     

A 31p and 15N NMR study of the extraction of uranyl nitrate by Di-2-ethylhexyl phosphoric acid

Low temperature ³¹P and ¹⁵N NMR spectroscopy was used to investigate the species forming in the organic layer following the extraction of uranium from nitric acid solutions with di-2-ethylhexyl phosphoric acid. It was found that uranium is extracted from neutral solutions as the 1:2 complex UO₂A₂ regardless of what anion is present. For dilute nitric acid solutions, the uranium is extracted both as associated and mixed nitrato species. As the nitric acid concentration of the aqueous layer increases, the mixed nitrato complex, UO2(NO₃)A·HA, becomes predominant.     

15N resonance assignments of oxidized and reducedChromatium vinosum high-potential iron protein

The¹⁵N resonances in reduced and oxidizedChromatium vinosum high-potential iron protein have been assigned by use of¹H-¹H COSY spectra and¹H-¹⁵N HMQC, HMQC-COSY, and HMQC-NOESY spectra. Unambiguous assignment of 70 of 85 backbone¹⁵N resonances in the reduced protein and 62 of 85 resonances in the oxidized protein are made, as are 12 of 21 side-chain¹⁵N resonances.