Whooping cough proteins evolving 'unusually' fast

逆转大脑衰老！Nature：年轻大脑中的神奇蛋白，可提高记忆力，让衰老大脑「重返青春」导读大脑老化是痴呆和神经退行性疾病的根源，给家庭和社会带来巨大负担。

前期对模式生物的系统性研究表明适当的外界干预能够逆转包括大脑在内的多种组织的生物学衰退。

例如，年轻血浆的输注可使老年大脑恢复活力并恢复记忆功能。

然而，大脑受到了脑屏障的保护，这在一定程度上可能会限制这些干预措施的获取，进而阻碍它们的功能效应。

脑脊液（Cerebrospinal fluid, CSF）与脑细胞密切相关，它携带信号，指导发育过程中神经元祖细胞的增殖和特异性。

然而，脑脊液蛋白组成会随着人类年龄的增长而变化，表现为炎症蛋白的增加和脑源性神经营养因子等生长因子的减少。

不过，脑脊液中的这些变化是否与年龄相关的认知能力下降有关尚不清楚。

2022 年 5 月 11 日，来自斯坦福大学医学院神经学与神经科学系的科研团队在国际顶级期刊 Nature 发表了题为 Young CSF restores oligodendrogenesis and memory in aged mice via Fgf17 的研究性文章，他们发现将年轻的脑脊液直接注入衰老的大脑可以明显改善记忆功能，其中少突胶质细胞对这种恢复最敏感，他们还进一步确定了 Fgf17 是在衰老的大脑中恢复少突胶质细胞功能的关键靶点。

图片来源：Nature主要研究内容幼龄小鼠脑脊液对少突胶质细胞的调控首先，该团队试图测试向老年小鼠注入年轻的脑脊液是否能改善海马与衰老相关的损伤，如学习和记忆。

他们对 20 个月大的老鼠进行了足部电击，然后小鼠被随机分为两组，分别注入人工脑脊液（aCSF）或幼鼠脑脊液（YM-CSF），在3 周后进行记忆测试。

结果发现，注射 YM-CSF 的小鼠对恐惧记忆保存得更好。

由于海马在与年龄相关的认知衰退中起着核心作用，并且与脑脊液非常接近，因此他们通过转录组测序来评估注入年轻脑脊液后对海马转录组的影响。

未折叠蛋白反应英语The Unfolded Protein Response.The unfolded protein response (UPR) is a cellular signaling pathway that is activated when the endoplasmic reticulum (ER) experiences a buildup of unfolded or misfolded proteins. This response is crucial for maintaining cellular homeostasis and preventing the accumulation of potentially harmful proteins. The UPR serves to restore ER function by enhancing protein folding capacity, reducing protein translation, and promoting the degradation of damaged proteins.The ER is a crucial organelle responsible for protein synthesis, folding, and trafficking. When the ER is unable to cope with the demand for protein folding, it triggers the UPR to address the imbalance. This imbalance can be caused by various factors such as changes in cellular metabolism, environmental stress, or mutations that affect protein folding.The UPR is initiated by three ER-resident transmembrane proteins: protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). Under normal conditions, these proteins are bound to the ER chaperone BiP/GRP78, which inhibits their activation. However, when unfolded proteins accumulate in the ER, BiP/GRP78 dissociates from these sensors, allowing them to initiate the UPR.PERK activation leads to the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which attenuates global protein synthesis. This reductionin protein synthesis reduces the load on the ER, allowingit to focus on folding the existing proteins. Additionally, phosphorylated eIF2α promotes the translation of specific mRNAs, including those encoding transcription factors such as ATF4, which induce the expression of genes involved in amino acid metabolism, oxidative stress resistance, and chaperone synthesis.IRE1α activation leads to its endonuclease activity, which splices the mRNA of the transcription factor XBP1. This splicing event converts XBP1 from an inactive form to an active form that can regulate the expression of genes involved in ER expansion, lipid metabolism, and protein degradation.ATF6 activation leads to its translocation to the Golgi apparatus, where it is cleaved to release its cytosolic domain. This cleaved ATF6 fragment then enters the nucleus and activates the expression of genes encoding chaperones, ER-associated degradation (ERAD) components, and other proteins that enhance ER function.Collectively, these UPR signaling branches aim to restore ER homeostasis by enhancing protein folding capacity, reducing protein synthesis, and promoting the degradation of damaged proteins. If the ER stress persists despite these adaptive responses, the UPR can also trigger apoptotic signaling, leading to cell death.The UPR plays a crucial role in maintaining cellularprotein homeostasis and preventing the accumulation of potentially harmful proteins. Its activation is a highly conserved mechanism across different cell types and organisms, indicating its importance in maintainingcellular function and survival.In summary, the unfolded protein response is a complex cellular signaling pathway that is activated in response to ER stress. It involves the activation of three ER-resident sensors, PERK, IRE1α, and ATF6, which trigger adaptive responses to restore ER homeostasis. These responses include enhancing protein folding capacity, reducing protein synthesis, and promoting the degradation of damaged proteins. The UPR is crucial for maintaining cellular protein homeostasis and preventing the accumulation of potentially harmful proteins.。

重组胶原蛋白免疫学
重组胶原蛋白是指利用基因工程技术将胶原蛋白的基因导入到
宿主细胞中，通过宿主细胞的表达和合成来获得胶原蛋白。

胶原蛋
白是一种重要的结构蛋白，它在人体中起着支撑和连接组织的作用。

重组胶原蛋白的免疫学研究主要涉及其在免疫调节、免疫应答和免
疫治疗等方面的应用。

从免疫学角度来看，重组胶原蛋白可以被视为一种外源抗原，
当其进入人体后，会引发免疫系统的应答。

这种免疫应答包括先天
免疫应答和获得性免疫应答。

先天免疫应答是指对抗原的非特异性
应答，包括炎症反应等；而获得性免疫应答则是指针对特定抗原的
免疫应答，包括T细胞和B细胞介导的免疫应答。

研究人员可以利用重组胶原蛋白来研究免疫系统对抗原的识别、处理和应答机制，从而深入了解免疫系统的功能和调节机制。

此外，重组胶原蛋白还可以作为免疫治疗的候选药物，用于治疗免疫相关
疾病，如风湿性关节炎、系统性红斑狼疮等。

研究人员可以通过调
节重组胶原蛋白的结构和功能，来影响免疫系统的应答，从而达到
治疗疾病的目的。

总的来说，重组胶原蛋白在免疫学领域具有重要的研究和应用前景，可以帮助人们更好地理解免疫系统的功能和调节机制，同时也为免疫相关疾病的治疗提供了新的思路和方法。

血清残余胆固醇水平对冠心病的影响及临床意义陈翠1，2，杨莉婷3，唐陶1，2，徐浩2，刘茂41.川北医学院附属医院遗传与产前诊断中心，四川南充637000；2.川北医学院检验医学院，四川南充637000；3.南充市中心医院检验科，四川南充637000；4.川北医学院附属医院心血管内科，四川南充637000【摘要】目的探讨血清残余胆固醇(RC)水平对冠心病的影响及其临床意义。

方法回顾性分析2019年6月至2020年6月因胸闷胸痛于川北医学院附属医院心内科住院行冠脉造影检查的230例患者的临床资料，根据冠脉造影结果分为冠心病组190例和非冠心病组40例(CON 组)，根据临床诊断标准又将冠心病患者分为稳定性心绞痛组(SAP 组)70例和急性冠脉综合征组(ACS 组)120例。

比较三组患者的一般资料和RC 水平，采用Spearman 秩相关分析RC 水平与Gensini 评分的相关性，采用多因素Logistic 回归分析影响冠心病发生的风险因素，绘制受试者工作特征曲线(ROC)分析RC 对冠心病发生的预测价值。

结果ACS 组、SAP 组和CON 组患者的性别、年龄、吸烟史、高血压史、LP(a)比较差异均无统计学意义(P >0.05)，但ACS 组和SAP 组患者的总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、载脂蛋白A (ApoA)、载脂蛋白B (ApoB)水平明显高于CON 组，而高密度脂蛋白胆固醇(HDL-C)水平明显低于CON 组，且ACS 组患者的TC 、LDL-C 、ApoB 水平明显高于SAP 组，差异均有统计学意义(P <0.05)；ACS 组和SAP 组患者的RC 水平分别为(0.98±0.37)mmol/L 、(0.86±0.23)mmol/L ，明显高于CON 组的(0.68±0.16)mmol/L ，且ACS 组的RC 水平明显高于SAP 组，差异均具有统计学意义(P <0.05)；经Spearman 秩相关分析结果显示，RC 水平与Gensini 评分呈正相关(P <0.05)；经多因素Logistic 回归分析结果显示，年龄、吸烟、RC 、LDL-C 、ApoA 为冠心病的独立危险因素(P <0.05)；经ROC 分析结果显示，血清RC 预测冠心病发生的曲线下面积(AUC)为0.755，灵敏性和特异性分别为53.20%和87.50%。

记忆力“治愈者”：注射“长寿”蛋白可增强猴子记忆力首个灵长目研究揭示了蛋白质klotho改善认知的功效，或预示着向着临床应用更近了一步。

一项研究发现，给老年猴子注射一种“长寿因子”蛋白能增强它们的认知功能。

该结果7月3日发表于《自然-衰老》（Nature Aging）[1]，或能用于开创神经退行性疾病的新疗法。

注射klotho后，老年恒河猴在记忆测试中的表现更佳。

来源：Arno Burgi/AFP via Getty klotho是一种体内自然产生的蛋白质，会随年龄增长而减少。

这是首次证明恢复klotho 的水平能提升灵长目的认知功能。

之前的小鼠研究显示，注射klotho能延长小鼠寿命，增强突触可塑性（synaptic plasticity）[2]，突触可塑性是指在名为突触的节点控制神经元之间通信的能力。

伦敦大学学院的英国痴呆症研究所的神经科专家Marc Busche说：“由于灵长类和人类在遗传学和生理学上具有相似性，这或许暗示了对人类认知障碍的治疗潜力。

”klotho蛋白以古希腊纺织生命线的命运三女神之一——克洛托（Clotho）的名字命名。

猴子记忆测试该研究给平均年龄22岁的老年恒河猴（Macaca mulatta）注射了单次的klotho，并在注射前后测试了这些猴子的认知能力。

研究团队的测试方法是通过一次行为学实验来测试它们的空间记忆：研究人员先将奖励食物放在多个小室中的一个里，然后将这些小室遮盖起来，再让猴子回忆奖赏食物的所在位置。

研究共同作者、加州大学旧金山分校的医生-研究员Dena Dubal将这个实验比作在停车场找车，或是在听到一串数字后的几分钟再回忆出来。

年龄越大，完成这类任务也越困难。

注射了klotho的猴子在这些实验中的表现显著提高，在注射前，他们发现正确小室的概率是45%，而注射后的概率为60%左右，这种改善能维持至少两周时间。

和之前的小鼠研究不同，这次只要相对低剂量的klotho就能起效。

一种本周氏蛋白测定的简易对照
谢晏如
【期刊名称】《现代检验医学杂志》
【年(卷),期】2002(017)004
【摘要】@@ 本周氏蛋白是一种免疫球蛋白的轻链或及聚合体,可大量出现在多发性骨髓瘤或轻链病患者的尿液中.
【总页数】1页(P2-2)
【作者】谢晏如
【作者单位】浙江省浦江县人民医院,浙江,浦江,322200
【正文语种】中文
【中图分类】R446.12+9
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3.一种鉴别蒲氏钩蝠蛾蛹及成虫雌雄的简易方法 [J], 喻浩;张剑霜;张古忍
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因版权原因，仅展示原文概要，查看原文内容请购买。

维持胚胎干细胞自我更新状态的分子信号途径
高远;宋后燕
【期刊名称】《细胞生物学杂志》
【年(卷),期】2005(27)4
【摘要】胚胎干细胞是从胚胎植入前期胚泡内细胞团分离的细胞,可以长久维持对称性自我更新的未分化状态。

多种胞内外细胞因子介导的信号途径参与这种状态的调节。

现对胚胎干细胞自我更新途径分子机制进行综述,并提出有待进一步阐明的相关问题。

【总页数】4页(P379-382)
【关键词】胚胎干细胞;自我更新;分子信号途径;信号途径;分子机制;状态;内细胞团;胚胎植入;细胞因子;对称性
【作者】高远;宋后燕
【作者单位】复旦大学分子医学教育部重点实验室复旦大学生物医学研究院干细胞与组织工程研究所
【正文语种】中文
【中图分类】Q813;B978
【相关文献】
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2.蜂王浆中的Royalactin具有维持胚胎干细胞多能性和自我更新的作用 [J], 钱家乐;胡福良
3.胚胎干细胞自我更新的信号转导途径和体外培养系统的研究进展 [J], 朱江;汪燕;刁永书
4.胚胎干细胞自我更新的信号转导途径和体外培养系统的研究进展 [J], 刘西茹;丘彦;徐红兵
因版权原因，仅展示原文概要，查看原文内容请购买。

波谱学杂志第26卷第4期2009年12月　Chinese Journal of M agnetic Resonance Vo l.26No.4　Dec.2009Article:1000-4556(2009)04-0437-20High-Resolution Solid-State NMR Spectroscopy ofMembrane Bound Proteins and Peptides Alignedin Hydrated LipidsFU R i-q iang(Center for Interdisciplinary M agnetic Res onance,National Hig h M agnetic Field Lab oratory,1800East Paul Dirac Drive,Tallahassee,Florida,32310,USA)A bstract:Solid-sta te nuclear mag netic r eso nance(N M R)o f alig ned samples has been rapidlyeme rged a s a successful and impor tant spect roscopic appro ach for hig h-resolution str uctural char acte rizatio n o f membrane-bo und pro teins and pe ptides in their“na tive-like”hydra ted lipid bilaye rs.Because the structure s,dynamics,and functions of membrane-bo und pro teins and peptides are highly asso ciated w ith he te rog eneo us native environments,proteins and pe ptides are prepared for so lid-state N M R measurements in the presence o f either bilay ers that are me-chanically alig ned on glass pla te s o r mag netically aligned bicelles.O rienta tion de pendent aniso-tropic spin nuclear interactio ns fro m these aligned pro teins and peptides can be o btained.These orienta tional restr aints can be assembled into hig h-resolution three-dimensional structur es.Driven by sig nificant advances in sample preparation pro tocols as well as N M R probes and o the r metho do lo gy developments in the pa st decade,the alig ned sample N M R appr oach has been w ell developed and become an effective w ay for structural characteriza tion of membrane-bound pro-teins and peptides.T his r eview intr oduces hig h resolution so lid-state N M R spectr osco py o f alig ned samples and summa rizes rece nt methodolog y develo pments in this arena.Key words:so lid-state N M R,membr ane-bound pro tein,o rienta tional co nst raint,hy drated lip-idsC LC number:O482.53 Document co de:AReceived date:3Aug.2009Biography:Fu Ri-qiang(1966-),m ale,major in Nuclear M agnetic Resonance Spectroscopy,T el:+1-8506445044, E-mail:rfu@.　＊Corresponding au th or.438波 谱 学 杂 志 第26卷　IntroductionThe characterizatio n of membrane bound proteins and peptides is very challenging in structural biology,in part because their structures,dy namics and functions are hig hly related to their biolo gical heterog eneous membrane environments[1-3].As an example, integral m em brane protein structures are severely underrepresented in the Pro tein Data Bank(PDB)(w w w.rcsb.o rg/pdb/),composing as little as about0.5%of protein structures deposited in the PDB,altho ug h they represent as m uch as o ne third of the pro teins from most geno mes[4].These membrane pro teins are bio logically very impo r-tant,carrying out transpo rt and signaling functions o n the surface of cells and organ-elles.M any of these proteins are highly dy namic and inv olve multiple confo rm atio nal and functional sta tes that are sensitive to the pro teins'environment.On the o ther hand, membranes allow fo r the establishment of electric,chemical and m echanical po tentials, w hich can be modulated and conve rted into othe r fo rms o f energy through the action of membrane pro teins.While fo r many amphipa thic ca tionic antimicrobial peptides,the ac-tual m ode of action directly inv olves interactions w ith cell membranes[5-8].Therefo re,it is fundam entally im po rtant to cha racterize the structures of membrane bound pro teins and peptides in their w ell hydrated native membrane enviro nm ent in o rder to understand their structure-functio n relationships that pe rfo rm vital functio ns at cell membranes.In past decades,many spectroscopic me thods,such as X-ray[9,10],Infrared spec-tro scopy[11,12],mag netic resonance spectro sco py[13,14],have been used to characterize the structures of membrane bound pro teins and peptides.Am ong these structural ap-proaches,solid-state nuclear magnetic resonance(NM R)[15]has the inherent ability to detect single atomic sites and hence e xhibits m any advantages for o btaining high-resolu-tion structures of proteins and peptides in their membrane bound states,since they often invo lve multiple confo rm ational and functional states w ith a sig nificant degree of lo cal and do main dy namics.Biolo gical solid-state NM R techniques[16-20]have been rapidly developed in the past few years for structural characterization o f mem brane bound proteins and peptides.Cur-rently,there are tw o complimentary NM R technologies that are primarily used to g ain structural insights:the magic ang le spinning(M AS)and the aligned sample appro ach. Fo r M AS NM R,uno riented samples are spun around the axis tilted at54.7°from the applied external m ag netic field,yielding“so lution-like”hig h-reso lution so lid-state NM R spectra w ithout requiring an isotropic m otio n on a nano second tim escales[21].For the a-lig ned sam ple appro ach,samples that have a unique alig nment with respect to a single axis,such as the m ag netic field axis and the bilaye r no rmal,are prepared so that aniso-tro pic high-reso lution solid-state NM R spectra can be obtained.These orientatio nal de-pendent aniso tropic spin interactions provide bo th mechanisms fo r dispersing the reso-nances in solid-sta te NM R spectra and for providing structural restraints to assem ble hig h-reso lution three-dimensional structures.In the past few years,the alig ned sam pleapproach has been mo re developed for membrane bound pro teins and peptides o riented in hy drated “native -like ”lipid bilay ers ,leading to the depo sit o f ove r ten membrane pro -tein structures in the PDB (i .e .,1M AG ,1PJD ,1EQ8,1M P6,1NYJ ,2H 95,1M ZT ,1PI7,2GOF ,2GOH ,1H 3O )[22-32].This review focuses on recent me thodology devel -o pments in the biological solid -state NM R of alig ned samples .1　Structural constraints in aligned samplesThe primary to ols used fo r the NM R structural determination of alig ned mem brane -bo und pro teins and peptides that have a unique orientation w ith respect to the mag netic field axis of the NM R spectro meter are the measurement of orientatio nal constraints ,derived fro m o bservatio ns of a v ariety o f aniso tropic nuclear spin interactions ,such as chemical shifts ,hetero -nuclear interactions ,and quadrupolar inte ractio ns .Fig .1(a )show s an α-helical seg ment .The enlarged insert illustrates a peptide plane ,the building blo ck of an ideal α-helical structure .Any given alignment o f the α-helical segm ent in the e xternal magnetic field B 0co rrespo nds to a specific o rientation of aniso tropic nuclear spin interactio ns w ith respect to B 0.Fo r instance ,the NH vecto r o f a peptide plane is tilted from B 0by the ang le θ,as indicated in Fig .1(b ).When all of the α-helical seg -m ents are random ly alig ned in B 0,as tho ug h the ang le θfo r the same peptide plane is unifo rmly distributed all o ver the sphere ,the resulting15N -1H dipolar spectrum has a ty pical “Pake -pattern ”lineshape .Ho wever ,w hen all of the α-helical seg ments have the same alig nment in B 0,the o bse rved15N -1H dipolar splitting Δνfro m the sam e peptide plane equals to the m agnitude ν∥of the N -H dipo lar interaction multiplied by the o rien -tation dependence :Δν=ν∥(3co s 2θ-1),(1)as show n in Fig .1(b ).Since the N -H bond in the peptide plane is cov alent ,the magni -tude ν∥is know n ,based on the N -H bond leng th .Therefo re ,the o rientation depend -ent 15N -1H dipola r splitting directly corresponds to the orientatio n of the N -H vector with respect to B 0.Similarly ,fo r a g iven α-helical seg ment ,the amide 15N chemical shift aniso tropy of a peptide plane can be defined by th ree Euler angles θ11,θ22,and θ33,as show n in Fig .1(c ).When the alig nments of all α-helical segm ents are rando mly alig ned w ith respect to B 0,the resulting 15N chemical shift spectrum has a typical chemical shift pow de r pat -tern .Ho wever ,w hen all of the α-helical seg ments have the same alignment in B 0,the observed 15N chemical shift σobs depends o n the three principal elements ,σ11,σ22,and σ33,of the amide 15N chemical shift tensor and their o rientations with re spect to B 0:σob s =σ11cos 2θ11+σ22co s 2θ22+σ33cos 2θ33,(2)as illustrated in Fig .1(c ).A gain ,the three principal elements σ11,σ22,and σ33can be in -dependently obtained .It is w o rth no ting that ,fo r the peptide planes in an ideal α-helix ,439　第4期　F U Ri -qiang :High -Resolution Solid -State NM R Spectr osco py of M embr ane Bound P ro teins and P eptides A ligned in Hydr ated L ipidsthe variatio ns of the amide 15N chemical shift tenso r are very minimal .Co nsequently ,the orientation of the amide 15N chemical shift tenso r w ith respect to B 0can be deter -mined from the orientation dependent 15N chemical shift reso nance of the aligned sam -ples .It is w orth noting that the obse rved anisotropic 15N chemical shift resonance fro m the alig ned sample is dispersed over the entire w idth of the amide 15N chem ical shift ani -sotropy (～200),rather than the iso tro pic chemical shift rang e (<10)in the M AS NM R spectra .Therefo re ,the data from the o rientation dependent nuclear spin interac -tions co nstrain the orientation of a specific m olecular site w ith respect to B 0.By obtai -ning numerous restraints ,all with respect to the sam e alig nment axis ,high -reso lution three -dim ensio nal structure s can be achieved .Fo r example ,the very first hig h resolu -tion three -dimensional structure of the g ramicidin A (gA )aligned in lipid bilayers (the PDB #1M AG )w as characterized by solid -state NM R using 120precise o rientational re -straints from numerous specifically labeled sites [22,23].Fig .1　NM R spectra of anisotropic spin interactions from α-helical segm ents under differen t alignmen ts .(a )A α-heli -cal segment .For an ideal α-helical structure ,the buildin g block is a peptide plane ,as illustrated in the enlarged insert ;(b )The orientation of the15N -1H vector w ith respect to the m agnetic field B 0,as defined by the angle θ,and the 15N -1H dipolar spectra at differen t alignment conditions ;(c )Orientation of the amide15N ch emical shift anisotropy w ith respect to B 0,as defined by three Euler angles θ11,θ22,and θ33,and the15N chemical shift spectra at differentalign men t conditions .Since the orientatio nal restraints result fro m aniso tropic nuclear spin interactions with respect to the same alig nment axis (e .g .,B 0),they are abso lute and independent with each other ,meaning that the er rors associated w ith individual restraints do not sum w hen multiple restraints are used for defining helical structures .Ho wever ,the o rien -taional restraints can no t be used to define the relative position between helices ,w hich can only be o btained by distance restraints ,ano ther type of structural constraints based on the spatial distances betw een tw o nuclei .Distances repre sent relative constraints in that they restrict the position of one mo lecule site relative to ano ther .Thus ,precise dis -tance restraints a re an impor tant com plement to orientational restraints fo r defining ter -tiary and quarte rnary pro tein structure [15].For instance ,g A mo nom eric structure in phospholipids bilayers is know n to have very hig h re solutio n through the extensive use 440波 谱 学 杂 志 第26卷　of o rientational restraints [22,23],but the dimme r interface can o nly be modeled in a lipid enviro nment throug h sym metric and absolute nature of the o rientational restraints .Fig .2(a )show s the positions o f the specifically labeled 13C -Val 1,15N -Ala 5gA in hy drated dimy ristoy lphosphatidy lcho line (DMPC )bilay ers and the 13C cross polarized MAS (CP -M AS )spectrum .Fo r these labels ,the distance betw een the intramo nom er13C and 15N sites is 0.82nm ,based on the hig h resolutio n gA m onomeric structure ,too lo ng to y ield any detectable dipolar co upling .On the o ther hand ,the intermo nome r 13C and 15N sitesacro ss the dimmer interface appear to be much closer .Fig .2(b )show s that the sig nal intensities at δC 171.0and 172.6,the tw o resonances from the peptide13C label ,are clearly dephased by the simultaneous frequency and amplitude m odulatio n (SFAM )[33]irradiatio ns on the 15N channel ,as indicated by ho rizontal dashed lines .When co nsider -ing the fast mo tio n around the channel ax is ,the dephasing ra te yields a motionally aver -aged distance of 0.43±0.01nm betw een these 13C and 15N sites acro ss the monome r -monomer junction ,w hich provides a direct evidence of the m onomer -m onomer g eo metry in the g A channel structure [34].A sy nergic use o f the aligned sam ple approach and the MAS measurements has been w idely used as a pow erful tool to cha racterize helical bund -ling and channel gating mechanism [27,35-39].Fig .2　(a )Position s of th e specifically labeled 13C -Val 1and 15N -Ala 5gramicidin A (gA )in hyd rated dimyristoylph os -ph atidylcholine (DM PC )bilayers and the corresponding13C CPM AS NM R spectrum .(b )A set of 13C CPM AS spectra at different dephasing times w ith (righ t )and w ithou t (left )the simultaneou s frequency and amp l itude modulation (S FAM )irradiation on the 15N ch annel .T he spectra w ere recorded at 315K (above th e phase transition temperature of DM PC ).T he peak at δC 174results from the carb onyl group of the lipids and is not affected by dephasing ,w hile the tw o res onances at δC 172.6and 171.0are deph ased by the S FAM irradiations on th e 15N spin .441　第4期　F U Ri -qiang :High -Resolution Solid -State NM R Spectr osco py of M embr ane Bound P ro teins and P eptides A ligned in Hydr ated L ipids442波 谱 学 杂 志 第26卷　2　Solid-state NMR of align ed samplesSince the earliest demo nstration[40]that high-re solutio n structural constraints could be o btained by solid-state NM R o f pro teins and peptides in hydrated but anisotropic en-vironm ents,the biolog ical solid-state NM R of aligned sample s has been rapidly emer-g ing as a successful and important technique fo r structural and dynamic characterization of membrane bo und pro teins and peptides in“native-like”hy drated lam ellar phase lipid enviro nments[14,26,29,30,41-46];this is driven by sig nificant advances in sam ple prepara-tions as well as N M R probe and methodo logy developments.2.1　Sample alignmentsMechanical and m ag netic alig nm ents are the tw o prim ary approaches fo r the uni-fo rm alig nment of mem brane bound proteins and peptides,bo th of w hich use a lipid bi-laye r enviro nment above the phase transition tem perature.Fig.3(a)outlines the sche-m atics fo r aligned sample preparation in a hydrated lipid environment.Once the labeled pro tein/peptide is incorporated into lipids,the lipo some is spread onto thin g lass slides. S uch slides are then stacked in a glass tube,hy drated and sealed.Several thousand hy-drated lipid bilayers can thus be alig ned mechanically betw een a pair o f g lass surfaces.A stack o f～50such slides alig ned with the bilayer no rm al pa rallel to B0makes fo r a high sensitivity sam ple using5～10mg of pro tein.Fig.3(b)show s the31P spectrum of an alig ned protein in hy drated DPM C bilay ers contained in a g lass tube.The31P sig nals re-sult from the head g roup of the lipids.The narrow resonance peak at the left side indi-cates that the majority of the lipids are w ell aligned betw een glass slides,w hile the w eak resonance at the right side implies that a small portion o f the lipids is still uno riented, w hich could be par tly from tho se at the edge of the glass slides.Recently,anodic aluminum oxide(AAO)substrates w ith flow-throug h nanopo res have been used to provide m acro sco pically aligned peptide-co ntaining lipid bilayers[47]. The mo st striking advantage with these flow-through lipid nano tube arrays is that high hy dration levels as w ell as pH and desirable ion and/o r drug concentrations co uld be eas-ily maintained and modified in a series of expe riments w ith the same sam ple w itho ut lo s-ing the lipid alig nm ent o r bilay ers fro m the nanopo res,avoiding any uncertainty during the sample preparation at those various conditions.The mag netic alig nment takes advantag e of bicelles,discoidal lipid ag gregates that are edge stabilized by sho rt chain lipids.So lutions o f these bicelles can be prepared in such a w ay that they are unifo rmly alig ned with the bilay er no rmal perpendicular or par-allel to B0,w itho ut the aid of g lass slides.In recent years,magnetically aligned bicelles have also become an im po rtant bilayer preparatio n fo r bio logical solid-state NM R of alig ned sam ples[48-51].Since any dispersio n o f m olecular o rientations w ith respect to B0will broaden the observed resonance s,much effo rt has gone into the improvement of sample alig nment. This includes:i)choices o f lipids;ii)improved protoco ls fo r aligned sam ple prepara-tions ;and iii )the availability of higher mag netic fields .Early on in the development of solid -sta te NM R structural biology ,mo st of the alig ned samples w ere prepared with DM PC .No w aday s ,a range o f diffe rent lipids and mix tures o f lipids have been used ,in -cluding saturated and unsaturated fatty acids as w ell as charged and zwitterionic lipids .Altho ug h so lid state NM R bilayer sam ples are far less co mplex than nativ e mem branes (e .g .,typically m ore than 100lipids of even a bacterial membrane ),they represents themost native -like membrane environm ents in many aspects [1-3](e .g .,by providing a het -erog eneous environment ,dielectric gradient from the lipid interfacial regio n to the low dielectric lipid interior ,and so on ).Sam ple preparatio n pro to cols have been g reatly im -proved over years with different types of lipids and lipids mix ture and will co ntinue to be improving .It has been sho w n very recently that high reso lution MAS spectra of m em -brane pro teins can be achieved with the use o f viral lipids [38,52].Furthermo re ,it is also evident that hig h B 0improves sample alignment [53,54],in part because it enlarges the an -isotropy of the diam ag netic o r paramag netic susceptibility [55-57],w hich either aids uni -fo rm alig nment of lipid bilayers o r minimizes the defects of lipid bilaye rs due to their flu -idity nature.Fig .3　(a )S chematics of sample p reparation for an al igned p rotein /pep tide in a hydrated lipid environment .(b )31P NM R spectrum of a p rotein sam ple oriented in hydrated DM PC bilayers .2.2　NMR probe developmentT raditionally ,solid -state NM R probes used a m ultiply turned solenoid coil as a sample coil ,tuned to different frequencies simultaneously (kno w n as sing le -coil double -resonances circuits fo r H -X probes )[58,59].H ow ev er ,this ty pe of probe desig ns is no longer efficient fo r biological so lid -state NM R ,especially at hig h fields .Biolog ical sam -ples are ty pically hy drated so that the heating associated w ith the electric fields genera -ted during high pow er 1H decoupling is sig nificant ,par ticularly at hig h fields ,resulting in sample dehydratio n or even destruction of the samples [60].The electric fields are pro -duced by the hig h voltage across the sam ple coil .A lthough the use of a balanced circuit 443　第4期　F U Ri -qiang :High -Resolution Solid -State NM R Spectr osco py of M embr ane Bound P ro teins and P eptides A ligned in Hydr ated L ipids444波 谱 学 杂 志 第26卷　moves the g rounding point fro m o ne end of the so lenoid coil to the center of the solenoid leading to a bette r B1homo geneity[61,62],it does not reduce the v oltage across the sole-noid,thus it has limited effects to prevent sample heating.Several clever desig ns have been propo sed to reduce sample heating by moving the large electric field aw ay from the sample.In the“scro ll”coil desig n[63],the electric field of the scroll is largely confined between concentric turns of foil,so it g reatly reduces sample heating.The scro ll's low inductance tends to reduce the1H drive vo ltag e,yet it presents a challenge[64]to the ef-ficient tuning and m atching of a low frequency channel such as15N.A slo tted Faraday shield betw een the coil and the sample can also reduce the electric fields[65].The fundamental problem invo lves using the same sample coil for both the hig h fre-quency(1H)and m uch lowe r frequency(e.g.,15N).It is very hard to have the same sample coil w orking efficiently at tw o very diffe rent frequencies.Fo r instance,sensitivi-ty at the low frequency larg ely depends on having relatively m ore turns.H owever,more turns lead to a large inductance fo r hig he r frequency thus leading to hig h voltages across the sample coil,w hich require the use o f hig h-voltage capacito rs in the circuitry and re-sult in a large electric field fo r sample heating.In addition,a1H frequency trap is re-quired for the lo w frequency channel,w hich furthe r reduces sensitivity.For aligned samples,due to the presence of g lass tube,glass slides and a significant po rtion of lipids and hy dration,the pro tein sample s being investig ated are limited,resulting in low sensi-tivity.Therefo re,it is desirable to have a bigg er sample coil so that m ore samples can be av ailable for detectio n in orde r to im pro ve the sensitivity.Again,a la rg e sample coil po-ses significant problem s asso ciated w ith the w aveleng th effects.Recently,the cro ssed-coil design[66]has been very successful in term s of reducing e-lectric fields and improving sensitivity on both hig h and low frequency channels.A par-ticularly efficient design for large vo lume,hig h field,static applications is know n as low electric field coil,o r“low-E”coil[67].The low-E coil assembly co nsists of a low-frequen-cy coil(e.g.,15N)nested w ithin a1H lo op gap resonato r(LGR).The15N coil and1H LG R are oriented in such a w ay that they produce B1fields in o rthog onal directio ns. Therefo re,no additio n trap is needed in the circuitries to isolate the tw o channels.For each channel,a sing le-coil sing le reso nance circuitry is established,so that each channel can be independently optimized.The1H LG R's inherently low electric field is further re-duced by Fa raday screening by the inner coil.I t has been demo nstrated that this desig n dramatically decreases sam ple heating fo r hydrated biological sam ples[67].2.3　Polarization inversion spin exchange at the magic angle(PISEMA)In aligned samples that have unique o rientation with respect to B0,the data from o-rientation-dependent nuclear spin interactions within a peptide plane permit the charac-terization of this peptide plane orientatio n w ith respect to the alignment axis,as long as the relativ e orientatio n of tho se spin interactions in the m olecular frame becom es know n.In a peptide plane,the15N chemical shift tenso r has been w ell characterized in-　Fig .4　Relative orientation of the 15N chem ical shift tensor and the 15N -1H dipolar in teraction in a peptide planedependently with respect to the 15N -1Hdipo lar interaction ,as show n in Fig .4.Therefo re ,traditional sepa rated -local -field(SLF )ex periments [68]can be used to cor -relate the o rienta tion -dependent ,aniso -tro pic 15N -1H dipolar coupling w ith 15Nchemical shift from a peptide plane ,thusto define the orientatio n of this peptideplane w ith respect to the alig nment axis .By obtaining the orientations o f all peptideplanes with respect to the sample alig n -m ent axis ,three -dimensional backbone conformation and to polo gy o f the alig ned samples can be achieved [69].Ho wever ,spec -tral resolution is the key to the success of using such S LF experiments.Fig .5　(a )Pu lse sequence for PIS EM A experim en ts ;(b )Tw o -dimensional PISEAM spectrum of 15N 1,3,5,7-labeled gramicidin A oriented in hydrated DM PC bilayers (at apep tide ∶lipid molar ratio of 1∶8)recorded on a 400M H z NM R spectrometer at 40℃.The first and mo st sig nificant ex peri -ment to im pro ve the S LF spectral resolu -tion w as polarizatio n inve rsion spin ex -chang e a t the m ag ic angle (PIS EM A )[70],as show n in Fig .5(a ).After cross polari -zatio n ,the pro to n magnetization is flippedto the magic ang le by a 35°pulse ,alongw hich the polarizatio n inversio n spin ex -chang e is established by applying a fre -quency switched Lee -Goldburg (FS LG )homo nuclear decoupling sequence [71]to theproton channel in sy nchronizatio n with180°phase alternation o f the spin -lockingfield applied to the 15N channel .The spinexchange takes place w hen the effective field in the proton channel and the applied15N spin -locking field fulfill the H artman -H ahn co nditio n .Fo r the traditional SLFexperiments ,the 15N sig nals a re evolved inthe x y plane during the indirect t 1dimension and thus affected no t o nly by the dipolar coupling s w ith directly bonded amide pro to ns but also by the dipolar inte ractio ns with other remote pro to ns .The latter leads to additio nal line broadening in the dipolar di -m ensio n .While fo r the PISEMA experim ents ,the dipolar flip -flo p te rm of the directly bo nded 15N -1H dipo lar co upling s are evo lved in the ro ta ting frame during the spin ex -change period w hile the couplings to o ther rem ote pro tons are effectively truncated [72].445　第4期　F U Ri -qiang :High -Resolution Solid -State NM R Spectr osco py of M embr ane Bound P ro teins and P eptides A ligned in Hydr ated L ipidsCo nsequently ,the spectral reso lution in the dipo lar dimension is improved dram atically ,as demonstrated in Fig .5(b ).Furtherm ore ,the 15N -1H dipolar coupling s are scaled by a large facto r (0.82)in the PIS EM A experim ents ,com pared to the traditional SLF exper -iments [71,73-75](0.57at mo st depending on hom onuclear deco upling sequences used in the indirect t 1dimensio n ).With the g reatly improved resolution ,the PISEMA ex peri -m ents have become a powe rful tool to obtain o rientational constraints fro m uniform ly la -beled pro teins and peptides .Since the building block of an ideal α-helical structure is a sing le peptide plane ,the PISEM A resonance patterns of a tilted helix ,as illustrated in Fig .6(a ),can be calculat -ed by sim ply rotating the peptide plane about its helical axis ,w hile calculating the 1H -15N dipolar coupling and 15N chemical shift observables .As show n in Fig .6(b ),the PISEM A resonances o f ideal α-helix clearly fo rm characte ristic w heels ,the so -called PI -SA w heels (polarity index slant ang le )[76,77],w hose size and po sitio n uniquely define the helical tilt with respect to B 0and the bilayer no rmal without reso nance assignments .The ro tation ρabo ut the helical axis h does no t affect the PISA w heel ,but does change the resonance positions of individual peptide planes on the w heels.Fig .6　(a )Definitions of helix tilt τand rotation ρaround the hel ical axis h in the laboratory frame .(b )Simu lated “w h eels ”rep resenting PISE M A resonance pattern of an ideal α-helix at differen t tilt angles τusing the 15N chemical shift tensors 'principal elements (σ11=31,σ22=54,σ33=202)and the relative orientation of th e dipolar and chemical shift tensors ,given by θ=17°,as sh own in Fig .4.For each tilt angle ,th ere are tw o symmetric w heels (b lack and g rey )mirrored along the zero frequency in the dipolar dimen sion .2.4　Other PISEMA type experimentsThe pe rfo rm ance o f the PISEM A ex periments largely depends o n the decoupling ef -ficiency of the FSLG o ff -resonance irradiatio n ,w hich is very sensitive to the setting of 1H carrier frequency .In the PISEM A spectra ,the 1H offse t effects include [78],1)the bro adening and thus attenuation of the dipolar oscillatio n peaks in the dipolar dimen -sion ;2)increase of unwanted artifacts at zero -frequency of the dipolar dimension ,and3)e xperimental 1H -15N dipolar coupling s la rg er than w hat they should be .S uch effects greatly co mpromise the reso lution of the PISEMA spectra ,especially at hig h fields ,446波 谱 学 杂 志 第26卷　。