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Affinity capturing for targeting proteins into micro and nanostructures

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TRENDSAffinity capturing for targeting proteins into micro and nanostructuresChangjiang You&Maniraj Bhagawati&Andreas Brecht&Jacob PiehlerPublished online:20January2009#Springer-Verlag2009Abstract Protein immobilization into micro and nano-scaled patterns opens exciting possibilities in fundamental and applied research.Developing efficient capturing tech-niques while preserving the structural and functional integrity of the proteins on surfaces is a key challenge for surface scientists.In this paper,current techniques for site-specific protein immobilization into engineered surface architectures are reviewed.Fundamental principles for functional protein immobilization on solid supports are discussed and popular affinity-based recognition pairs and their application for capturing proteins into nano and microstructures are presented.Keywords Protein immobilization.Microstructure. Nanostructure.Patterning.Nitrilotriacetic acid. BiotinylationIntroductionProtein immobilization on solid supports is rapidly gaining importance for analytical applications such as functional proteomics[1],diagnostics,and drug screening[2].These applications require multiplexed immobilization of proteins into arrays in a functional manner.Moreover,the extraor-dinary broad spectrum of protein functions and their highly specific recognition of cognate ligands make them very attractive functional elements for constructing biotechno-logical sensors and actuators.For such applications,even more sophisticated targeting of proteins into microscopic and nanoscopic structures is required in order to obtain efficient integration of protein building blocks into func-tional assemblies.Immobilization of proteins is frequently achieved by direct adsorption or by chemical reactions with solid supports,and patterns are readily obtained by photolithography,microcontact printing,piezodispensing, or through the use of scanning probe microscopy-based techniques[3–5].These applications are successful if either very robust proteins such as antibodies are involved,or if only a minor fraction of the protein needs to be active for further functional analysis.For many proteins and applica-tions,however,functional attachment of proteins to solid supports is a key challenge because of the metastable nature of proteins and their wide-ranging physicochemical prop-erties.Depending on the sensitivity of the detection scheme and the size of the structures,activity of10–90%of the immobilized protein is desired.Two main issues have turned out to be of key importance for successful functional protein immobilization:1.because protein interaction with the bare solid supportfrequently causes denaturation and loss of function, protein-compatible layers are required for shielding the surface;and2.site-specific attachment of the protein to the surface isrequired in order to ensure homogeneous functionality with the intact protein activity.For assembling proteins into micro and nanostructures,the protein coupling approach must be compatible with the respective patterning technique.This review will focus on surface engineering approaches,which aim to site-specificallyAnal Bioanal Chem(2009)393:1563–1570DOI10.1007/s00216-008-2595-6Dedicated to Prof.Günter Gauglitz on the occasion his65th birthday.C.You:M.Bhagawati:J.Piehler(*)Department of Biophysics,University of Osnabrück,Barbarastr.11,49076Osnabrück,Germanye-mail:piehler@uos.deA.BrechtCovalys Biosciences AG,Witterswil,Switzerlandcapture proteins to structured surfaces rendered biocom-patible by appropriate surface modification.Moreover, promising techniques employed to pattern protein-capturing surfaces into micro and nano-scaled structures will also be introduced.In order to make surface biocompatible,molecular layers of bio-inert,hydrophilic polymers have been successfully employed.Next to dextran hydrogels,dense layers of poly (ethylene glycol)(PEG)have been proven efficient for shielding surfaces against proteins[6].While relatively long PEGs with a molecular mass of2000–5000g mol−1 yield the most biocompatible surfaces,very dense layers of short oligo(ethylene glycol)chains with3–6monomer units as obtained by self-assembly of alkyl thiolates monolayers are also capable of very efficiently shielding of protein interactions with the surface[7].Non-covalent assembly of lipids and lipid-like molecules into mono and bilayers has also been very successfully used for rendering surfaces biocompatible[8].Lipid-based surface architectures,how-ever,are significantly more fragile than covalently attached layers and are particularly sensitive to exposure to air. Protein-repellent surfaces are not suitable for protein immobilization by physisorption and,therefore,require further functionalization with chemical or biochemical anchoring groups for protein capturing.Covalent coupling of proteins to surfaces through reactive groups on amino acid side-chains is frequently used for tethering protein to surfaces,and numerous procedures have been described. Such chemical reactions are not very selective and for most proteins yield highly heterogeneous attachment.Moreover, these reactions can involve residues important for function and thus affect the activity of the protein.Therefore,protein capturing techniques are desired,which site-specifically tether proteins to surfaces.Monoclonal antibodies have been used very successfully for protein immobilization because these very robust proteins can be efficiently physisorbed by surfaces and they sometimes bind proteins very stably.This approach,however,requires a suitable monoclonal antibody which recognizes the protein of interest without affecting its function.Moreover,antibodies are large proteins and not ideally suited for high-resolution structured surface fabrication processes.A more generic strategy for site-specific tethering involves fusion of a protein or peptide tag to the protein of interest,which is used as a recognition unit for capturing to surfaces.This approach originates from affinity chromatography and several recognition pairs are already available[9].One critical issue for protein immobilization,however,is that stable attachment of the protein to the surface is highly important for most analytical applications,while affinity chromatography requires reversible binding.Thus,only recognition pairs with high binding stability have found successful application in surface engineering.Moreover,efficient recognition elements are mostly proteins,most of which can only be handled under physiological conditions, whereas surface fabrication processes involving patterning techniques frequently require robust molecules.For these reasons,efficient recognition units for capturing proteins on surfaces are scarce.Currently most approaches rely on either the interaction between biotin and streptavidin or related proteins,or on the complexation of immobilized transition metal ions by oligohistidine tags.These concep-tionally different approaches for protein immobilization and their application towards functional protein patterning in micro and nanometre dimensions will be described in detail in the following text.Biotin-mediated protein immobilizationCapturing proteins on surfaces by interaction of co-factor biotin with the proteins avidin/streptavidin and their derivatives is a classic approach in solid-phase assays first described by Bayer and Wilchek[10].Biotin rapidly binds to avidin/streptavidin in4:1stoichiometry in a quasi-irreversible,non-covalent interaction.Because avidin is rather positively charged,the neutral variants streptavidin or neutravidin are used for surface functionalization in order to minimize non-specific interactions.The binding stoichiometry of the biotin/streptavidin complex enables sandwich binding architectures on surfaces,where biotiny-lated surfaces are reacted with streptavidin,and the remaining biotin binding sites are used to capture biotiny-lated proteins(Fig.1c).The stable chemical property of biotin makes it easy to go through relatively harsh fabrication conditions during the build up of a micro or nano-structure,while the high affinity of biotin/streptavidin interaction provides steady protein binding on the surface. For using this versatile interaction,biotin has to be attached to the protein of interest.Chemical biotinylation of proteins with standard reagents such as amine-reactive N-hydroxy-succinimide esters in most cases is a statistical process yielding heterogeneously and multiple labelled species.In contrast,enzymatic biotinylation elegantly enables site-specific attachment of biotin and biotin derivatives.For this purpose,the biotin ligase BirA from E.coli has been very successfully employed;it is capable of selectively ligating biotin to a15amino acid peptide tag(AviTag or AP-tag, Fig.1d)[11].Novel enzymatic biotinylation techniques employ phosphopantetheinyl transfer of biotinylated Coen-zyme A derivatives to peptidyl or acyl carrier proteins[12] or even short recombinant peptides[13](Fig.1e).These reactions are highly specific and can be efficiently carried out without prior purification of the protein of interest, enabling capture of proteins from crude lysates.Also, fusion with an intein tag and subsequent reaction with a1564 C.You et al.biotinylated cysteine has been successfully employed for site-specific protein biotinylation for immobilization on surfaces [14].Surface fabrication requires immobilization of streptavi-din,which can be directly attached to the surface by physisorption or chemisorption.Alternatively,surfaces can be functionalized with biotin,which is a small,stable organic molecule highly compatible with surface manufac-turing processes.Chemical linkage of biotin to surfaces through its carboxyl group is straightforward,and numer-ous biotin derivatives for surface modification including biotin-functionalized OEG-thiols and lipids are commer-cially available,yielding well-defined surface assemblies [15,16].Direct adsorption of neutravidin on to surfaces and its subsequent micropatterning by deep UV irradiation has been demonstrated to be an efficient technique for creating laterally structured surfaces for the immobilization of biotinylated proteins [17].Patterning of streptavidin and derivatives directly adsorbed or coupled to surfaces by photolithography has been successfully employed for functional surface patterning;lateral resolution down to submicron dimensions has been achieved [18].However,more elegant and defined approaches are based on surface biofunctionalization by chemical reaction with a suitable biotin derivative followed by self-assembly of the strepta-vidin layer.Different chemical approaches employing this method for functional surface patterning have been devel-oped.Microcontact printing (Fig.2a)of biotin on to different activated polymer surfaces has been described [19].Various photochemical approaches have been developed for pattern-ing biotinylation of surfaces.Chemical biotinylation of a PEG polymer brush after photo-deprotection of caged surface amines by UV-irradiation through a mask yielded patterns of streptavidin on a highly biocompatible surface [20].Functional streptavidin patterns on polystyrene and nitrocellulose surfaces were successfully obtained by use of a photoactivable derivative of biotin [21].More versatile surface patterning was achieved by direct,spatially con-trolled photochemical biotinylation by using a thiol-ene reaction [22].This approach can,in principle,be used for spatio-temporally controlled protein immobilization on the stage of a confocal microscope.An inverse patterning approach was chosen by Majima and coworkers,who synthesized a nitroveratryl-based,cleavable biotin-farnesyl conjugate which was incorporated into phospholipid membranes.Thus,biotin could be removed from these membranes by UV irradiation [23],which can also be used for in situ protein patterning.Similar capabilities for controlling both the position and the concentration of protein immobilized on a surface in situ was recently described by Costantino and coworkers [24].For this approach,which they termed laser-assisted protein adsorp-tion by photobleaching (LAPAP),biotin-4-fluorescein was adsorbed by a BSA-coated glass substrate,via the genera-tion of free radicals after fluorescein photobleaching,using a visible argon or diode laser,and used as a scaffold to bind proteins.For lateral organization of proteins in nanoscale dimen-sions,classic irradiation-based approaches are limited because of the diffraction-limited resolution of UV light.Templates with nm-sized structures such as bacterial S-layer or arrays of nanoparticles allow nanostructured surface patterning down to the molecular level [25].However,these approaches lack the flexibility to freely define thegeometryFig.1Immobilization through biotin/streptavidin interaction.(a )Structure of tetramericstreptavidin (ribbon model)with four biotin molecules (spacefill-ing model).(b )Structure of biotin.(c )Immobilization of a biotinylated protein in a sand-wich format.(d –f )Site-specific biotinylation through enzymatic reactions:(d )biotin ligase BirA-mediated biotinylation of the AP-tag;(e )phosphopantheteinyl-transferase (PPTase)-mediated transfer of phosphopantheteinyl-biotin to acyl or peptidyl carrier proteins or other peptide tags;(f )self-biotinylation of SNAP-tagged proteins using benzylgua-nin (BG)-biotin conjugatesAffinity capturing for targeting proteins into micro and nanostructures 1565of the patterns,because the templates rely on self assembly.In contrast,lithographic techniques based on modifications with the tip of an atomic-force microscope have proven capable of functionalizing surfaces in nanometre dimensions.Nanopatterned streptavidin has been obtained by removing the protein-repellent PEG coating on a silicon wafer by the AFM tip,thus creating sites for physisorption of streptavidin [26].A more general technique for nanostructured surface modification by the AFM tip developed by Mirkin and coworkers is dip pen nanolithography (DPN)[27](Fig.2c).This technique has been implemented for the functionaliza-tion of gold and glass-type surfaces with biotin [28–30].These structures were visualized by AFM and fluorescence microscopy after binding of streptavidin.A very elegant,optical approach for nanopatterning was implemented by Leggett and co-workers,who used the scanning near-field optical microscope (SNOM)to generate nanometre scale patterns of reactive aldehyde groups on a protein repellent OEG terminated SAM.The aldehyde patterns were subse-quently converted into a streptavidin nano-pattern by covalent reaction of amines on the protein with the surface aldehydes [31].Similar nano-scaled resolution ofbiotin(b)(I)(II)(III)(VI)(I)(II)(III)(VI)(I)(II)(III)(VI)(I)(II)(III)(VI)Fig.2Selected techniques for surface functionalization in micrometre and nanometre dimensions.(a )Microcontact printing.After inking a PDMS-stamp with a suitable ink such as an alkyl thiol (I ),a mono-molecular layer is transferred on to a planar substrate such as a gold surface only by the protruding regions of the stamp (II ,III ).By back-filling with a passivating compound,the remaining surface is protected against non-specific binding.(b )Surface patterning by UV-deprotection on surfaces.A chemically modified surface with functional groups caged by a photolabile protection group is illuminated through a mask (II )thus selectively uncaging the functional groups in the illuminated areas (III ).Subsequently,uncaged regions are functionalized by chemical reaction (VI ).(c )Dip-pen nanolithography.An AFM tip is inked with reactive molecules (I ),which are subsequently transferred on to the surface into nanometre-sized structures (II ,III ).The remaining regions are then protected by back-filling with a passivating compound (IV ).(d )Electron beam-induced chemical nanolithography.A surface coated with a suitable surface layer (e.g.a 4′-nitro-1,1′-biphenyl-4-thiol,SAM)is reduced and cross-linked by electron beam lithography (I ,II ).The non-exposed thiol is subsequently exchanged for a passivating compound,and the amine groups within the chemically modified region can be further functionalized1566 C.You et al.patterning was also achieved by using a combination of nanoimprint lithography(NIL)and molecular assembly patterning by lift-off(MAPL)[32].Immobilized transition metal ionsAlthough the biotin-streptavidin interaction enables highly stable tethering of proteins to surfaces,reversible affinity capturing not only provides oriented and homogeneous attachment but also the possibility of detaching the protein and thus repeated use of the same surface.For this purpose, complexation of histidine residues with immobilized tran-sition metal ions offers powerful solutions.Originally developed for affinity chromatography of histidine-rich proteins,this approach has gained tremendous importance for purification of recombinant proteins fused to a tag of6–10histidine residues[33].Transition metal ions such as Zn (II),Cu(II),Ni(II),or Co(II)are immobilized by chelating agents such an iminodiacetic acid(IDA)or nitrilotriacetic acid(NTA,Fig.3a),thus maintaining2or3coordination sites free for histidines.This interaction can be very efficiently disrupted by competing coordinators such as imidazole,which selectively and rapidly remove the protein from the surface under mild conditions(Fig.3c).For protein immobilization on surfaces,NTA has been favoured over IDA because it coordinates transition metal ions more stably.One key advantage of immobilized metal ions for capturing proteins on surfaces is the chemical nature of this recognition unit,enabling versatile and efficient surface chemistries.In a visionary paper published by Whitesides and coworkers in1996[34],they devised a mixed self-assembled monolayer of alkyl thiols terminated with a short OEG and an NTA moiety.Meanwhile a variety of procedures for NTA-functionalization of glass-type surfaces and gold surfaces[35,36]have been described.As an alternative approach,non-covalent self-assembly of NTA-functionalized lipids on solid supports(solid-supported lipid mono and bilayers)has been successfully employed for capturing His-tagged proteins onto surfaces[37].Akey Fig.3Adapters for protein immobilization.(a)Reversible loading ofNi(II)ions on to an immobilized NTA moiety.(b)Structure of the MCHtris-NTA with complexed Ni(II)ions.(c)Reversible binding of a his-tagged protein to Ni(II)-loaded tris-NTA upon addition of imidazole(blue).(d)A tris-NTA/biotin conjugate for reversible tethering ofproteins to streptavidin-functionalized surfaces(e).(f)Molecular print-boards as a generic platform for protein immobilization.Multivalentinteractions of bis-adamantyl(BA)conjugates(BA-biotin and BA-NTA)with immobilizedβ-cyclodextrin(blue)is employed forreversible functionalization of surfaces for protein capturingAffinity capturing for targeting proteins into micro and nanostructures1567problem,which was soon identified for many of these approaches,was that His-tagged proteins rather rapidly leaked from Ni-NTA surfaces.This problem was recently overcome by synthesis of multivalent chelator head(MCH) groups,supramolecular entities with2–4NTA moieties incorporated onto branched and cyclic scaffolds[38,39]. These MCH,which recognize oligohistidine-tags by mul-tivalent interactions(Figs.3b,c),were shown to bind His-tagged proteins with sub-nanomolar binding affinities[38]. Bottom-up chemistries for efficient attachment of these MCH to glass type surfaces via a protein-repellent PEG polymer brush have been reported[40],and biocompatible MCH-SAM on gold have been described[36,41].Using conjugates of MCH with biotin,streptavidin-functionalized supports can also be used for capturing His-tagged proteins, lending this technique an even greater versatility[42].Numerous approaches for Ni-NTA-based protein pat-terning by microcontact printing of reactive alkyl thiols followed by chemical coupling of NTA[43]and direct microcontact printing,or by backfilling of NTA derivatives, have been described[28,36,44,45].Next to multivalent NTA,also mono-NTA in high surface densities yields stable protein immobilization by multivalent binding on surfaces.Thus,the binding affinity can be varied by the density of NTA moieties on the surface.This feature was exploited for differential targeting of proteins into array elements with different NTA densities,which were obtained by combining microcontact printing with piezo-dispensing [44].After selective elution of proteins from arrays elements with a low density of NTA moieties by imidazole, these array elements were selectively refilled with a different protein[45].Thus,sequential assembly of binary protein microstructures was achieved.Like the patterning of biotin-functionalized surfaces,photochemical approaches have been used successfully to pattern NTA surfaces.One noticeable advantage for photochemistry-based patterning methods is that the patterning can be done on dimensions as large as a centimetre simultaneously[22].In one work[20],photosensitive silanes containing nitroveratryl(Nvoc)-caged amine groups and protein-repellent tetraethylene glycol units were used for modification of silica surfaces (Fig.2b).After masked irradiation by UV-irradiation and coupling with trisNTA,protein micropatterns were success-fully generated.Recently,we have established a generic method for photochemical micropatterning of NTA-func-tionalized glass-type surfaces by highly selective photo-destruction of the NTA moieties(unpublished results).Several approaches have demonstrated nanoscaled protein assembly by immobilized transition metal ions. The reversible nature of the His-tag/Ni-NTA interaction was exploited for a novel nanopatterning technique termed native protein nanolithography,which is based on selective removal and exchange of NTA-bound proteins by the tip of an atomic force microscope(AFM) [46].This was achieved by use of a novel AFM mode, which enabled removal of the protein without affecting the integrity of the NTA surface.Thus,functional immobili-zation of different cell-surface receptors into nanometre-sized structures was achieved.Moreover,nanostructured NTA was achieved by nanoimprint lithography[47],by functionalization of nanopatterned streptavidin with a biotin-MCH conjugate[28],and by electron beam-mediated chemical nanolithography[48](Fig.2d).Very elegantly,AFM based metal ion loading of immobilized NTA was also used for nanopatterning surfaces with proteins[49].DPN has also been used to spatially pattern NTA lipid into nanostructures.Selective adsorption of functionalized proteins based on streptavidin and His-tag were realized on these biomimetic membrane patterns with a resolution of200nm[30].The reversibility of the interaction between oligohistine and immobilized transition metal ions has the advantage that once the template is patterned,it can be regenerated and repeatedly loaded with fresh proteins.For some cases, however,stable attachment of proteins is desired,and several attempts to stabilize these NTA-Ni complexesTable1Patterning techniques used in protein affinity capturingPatterning technique Resolution Other characteristics Microcontact printing1–16μm(NTA hydrogel)[61],(biotin patterning)[19]Photolithography5–100μm(biotin surface)[22]Patterning areas can becentimetres in width Deep UV photolithography400nm(193nm UV,biotin patterning)[18]Scanning near-field photolithography150nm(biotin patterning)[31]Nanoimprint lithography75nm(biotinylated BSA surface)[32],700nm(NTAmonolayer)[47]e-Beam chemical nanolithography1μm,potentially10nm[48]Currently limited to goldsubstratesNative protein nanolithography:DPN, nanoshaving/nanografting 50nm(bisNTA surface)[46],200nm(biotin-lipid andNTA-lipid patterning)[30]Patterning areas limited by AFMscanning area1568 C.You et al.have been made,for example affinity mediated chemical crosslinking[50].PerspectivesIn the past ten years,tremendous progress has been made in surface engineering for protein immobilization,and power-ful patterning techniques for the nanoscale have been devised.Table1is a list of the different protein-patterning techniques summarized in this review.For self-assembly of functional protein micro and nanostructures,multiplexed protein capturing will be critically important.Molecular printboard approaches[42,51,52](Fig.3f)and multi-plexed DPN[23,30]have already paved the way towards multiplexing protein capturing on micro and nanoscale based on the recognition pairs described above.However, for real applications,more orthogonal recognition pairs will be required for protein capturing,and potential candidates have to be thoroughly explored.Several novel techniques are currently being explored for site-specific covalent protein immobilization into microarrays including highly specific biochemical reactions such as native chemical ligation[53],and Staudinger ligation[54].“Click chemis-try”utilizing azide alkyne Huisgen cycloaddition has found usage in protein immobilization[55].Self-immobilizing fusion proteins have been developed,employing enzymes which covalently bind to surface-coupled substrate ana-logues[56,57].Although some of these techniques—e.g. microcontact printing[58]—have already been used for lateral patterning,tailored surface modification is still missing.Also non-covalent recognition of peptide tags remains attractive,and promising solutions may be offered by oligonucleotide aptamers selected for high-affinity binding of proteins and peptides[59].Aptamers have already been employed for protein immobilization into microarrays[60],and are likely to replace antibodies in many fields.In this promising field,surface chemistries and patterning techniques still need to be devised. 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人类丝氨酸消旋酶的同源模建及其与多肽类抑制剂的分子对接_赵勇山

人类丝氨酸消旋酶的同源模建及其与多肽类抑制剂的分子对接_赵勇山

人类丝氨酸消旋酶的同源模建及其与多肽类抑制剂的分子对接赵勇山郑清川*张红星楚慧郢孙家钟(吉林大学理论化学研究所,理论化学计算国家重点实验室,长春130023)摘要：利用同源模建和分子动力学模拟方法构建了人类丝氨酸消旋酶(hSR)的三维结构,并利用profile -3D 和procheck 方法评估了模型的可靠性.在此基础上用分子对接程序(affinity)将多肽类抑制剂A 和B 分别与hSR 进行对接,获得了其复合物结构的理论模型.通过配体与受体之间相互作用能和结构分析给出了此类抑制剂与hSR 的具体结合方式,明确了hSR 与此类抑制剂结合时起重要作用的氨基酸残基,为基于人类丝氨酸消旋酶三维结构的药物设计提供重要的参考信息.关键词：人类丝氨酸消旋酶;分子动力学模拟;分子对接;同源模建中图分类号：O641Homology Modeling of Human Serine Racemase and Its Molecular Docking with Peptide InhibitorsZHAO Yong -ShanZHENG Qing -Chuan *ZHANG Hong -XingCHU Hui -YingSUN Chia -Chung(State Key Laboratory of Theoretical and Computational Chemistry,Institute of Theoretical Chemistry,Jilin University,Changchun 130023,P.R.China )Abstract ：The three dimensional structure of human serine racemase (hSR)was modeled and refined using homology modeling and molecular dynamics simulation.This model was assessed by profile -3D and procheck,which confirmed that the refined model was plex structures of peptide inhibitors with hSR were obtained and investigated through ligand -receptor docking studies by a molecular docking program,affinity.The binding pattern predicted by the affinity module revealed that important residues interacted with the peptide inhibitors and the module provided further refinement of the hSR/inhibitor binding interaction that may be used as a basis for new structure -based design efforts.Key Words ：Human serine racemase;Molecular dynamics simulation;Molecular docking;Homologymodeling[Article]物理化学学报(Wuli Huaxue Xuebao )Acta Phys.-Chim.Sin .,2009,25(3)：417-422Received:November 4,2008;Revised:December 11,2008;Published on Web:January 13,2009.*Corresponding author.Email:zhengqch@;Tel:+86431-88498962.国家自然科学基金(20573042)、国家科技支撑计划重点项目(2006BAE03B01)和高等学校博士点学科专项基金(20070183046)资助鬁Editorial office of Acta Physico -Chimica Sinica一直以来,人们普遍认为D 型氨基酸只存在于细菌和无脊椎动物体内[1,2],但是研究发现[3,4],依赖磷酸吡哆醛的人类丝氨酸消旋酶(hSR)在体内可催化丝氨酸从L 型转变为D 型,从而调节D 型丝氨酸在组织中的表达水平.近年来研究也证实,D 型氨基酸在高等有机体的脑部和其他一些组织,特别是在中枢神经系统中高度表达,并有着非常重要的作用[5-8],如D 型丝氨酸在哺乳动物的大脑中具有非常高的丰度,而且作为内源性配体,能够结合于N -甲基-D -天门冬氨酸(NMDA)受体的“甘氨酸”位点,从而调节该受体的活性[9-11].实验上证明NMDA 受体在神经的兴奋传递、神经元和海马区的突触的可塑性、视网膜的发育、学习与记忆等方面发挥着非常重要的作用,NMDA 受体的过度激活会导致神经细胞的死亡和引起脑缺血后的脑部损伤以及其他神经病理学方面的疾病[12].因此调节D 型丝氨酸在组织中的March 417Acta Phys.-Chim.Sin.,2009Vol.25表达水平对研究神经方面的疾病具有非常重要的意义[13-15].到目前为止,已有大量关于hSR的实验研究报道[12-15],表明hSR含有一个磷酸吡哆醛(PLP)结构域,并且结构保守区残基Lys50与PLP共价结合,在催化过程中把L型丝氨酸转换成D型丝氨酸.2006年,Dixon等人[16]利用高通量虚拟筛选和实验等手段获得了hSR的多肽类抑制剂,其研究结果表明本文所研究的多肽类抑制剂A和B(图1)能够很好地抑制hSR的活性并使其活性降低75%以上.但是, hSR三维结构详细信息的缺失,阻碍了对该酶的功能、催化机理等的进一步研究.同源模建和分子动力学模拟的方法能很好地应用于生物大分子的理论模拟,并指导和解释实验现象[17,18],本文采用这两种方法构建了hSR的三维结构模型,并通过分子对接和量子化学计算方法考察了多肽类抑制剂A和B与hSR的结合模式,以期为基于人类丝氨酸消旋酶的三维结构药物设计提供重要的参考信息.1计算方法1.1模型搭建以hSR的一级序列(来源于Swiss-Prot and TrEM BL数据库,编号:EC5.1.1.18)为探针,在NCBI数据库中利用protein blast程序搜索Brookheaven蛋白晶体数据库,得到模板蛋白裂殖酵母菌丝体的丝氨酸消旋酶的三维结构,收录号为1V71.整个模型的构建分为以下四个步骤.(1)利用clusterW程序进行序列比对,确定模板与序列之间的残基匹配.(2)模型的建立:根据序列比对的结果,采用modeler自动模建程序搭建hSR的三维结构并进行侧链异构化,对所得模型在pH为8.0条件下进行加氢、补建等化学修饰,然后把模板1V71中的辅酶磷酸吡哆醛的坐标赋到模建所得结构的相应位置,得到hSR的初始结构模型.(3)模型优化:将整个体系加入0.5nm TIP3P 水分子,借助Insight II程序包[15]中Discover3模块,首先采用最陡下降法(steepest descents,SD)进行500步的能量最小化,再采用共轭梯度法(conjugate gradient,CG)优化至能量梯度小于4.18×10-2kJ·mol-1·nm-1,能量优化采用分级方法,即先对溶剂水和蛋白质的氢原子进行能量优化,进而优化蛋白质的环区侧链、环区主链、结构保守区侧链和结构保守区主链,直至全部放开优化.优化后的hSR模型在没有任何约束条件下进行500ps分子动力学优化,所用力场为CVFF(consistent valence force field)力场.(4)模型的合理性评估:用profile-3D[19]和procheck[20]检查优化后模型的键长、键角及二面角的合理性,并计算了hSR与1V71主链原子的均方根偏差(RMSD).1.2分子对接hSR的活性位点的预测用binding-site程序、CASTp[21]程序以及CAVER[22]程序来确定.hSR与多肽类抑制剂的分子对接采用affinity程序[15].Affinity 是一套自动对接配体(客体)到受体(主体)的程序,对给定的由配体分子和受体分子组成的体系,它能自动地依据配体/受体复合物的能量得到最佳的配体和受体结合结构.分子对接时采用Monte Carlo极小化方法与模拟退火的方法,每次优化1000步,并考虑溶剂化效应,计算过程中采用cell multiple模型计算非键相互作用.配体和受体间的能量匹配用半经验的自由能计算方法来评价.1.3量子化学计算多肽类抑制剂A和B分子的几何构型见图1,它们都含有一个3-苯基丙乙酸部分和一个组氨酸部分.构型在6-31G(d,p)水平下采用B3LYP[23,24]方法优化,溶剂化模型采用CPCM模型[25](介电常数ε= 80).此外,在分子对接的基础上,截取抑制剂0.3nm 范围内的氨基酸残基获得截断hSR-抑制剂复合物模型.在B3LYP/6-31G(d,p)水平下分别计算抑制剂、氨基酸残基以及截断复合物的单点能,从而获得抑制剂与hSR之间的相互作用能.所有量子化学计算由Gaussian03程序包[26]来完成的.2结果与讨论2.1hSR三维结构的同源模建由自动模建程序modeler构建的初始模型,经图1多肽类抑制剂A和B的结构示意图Fig.1Structures of peptide inhibitors A and B418No.3赵勇山等：人类丝氨酸消旋酶的同源模建及其与多肽类抑制剂的分子对接过能量最小化和分子动力学模拟后,得到了hSR三维结构的合理模型,如图2所示.hSR的三维结构中含有14个α螺旋,10个β折叠和11个β转角. hSR与1V71主链原子的均方根偏差为0.054nm.在分子动力学的模拟过程中,如图3所示,体系的势能在50ps之后能量基本保持不变,蛋白质的RMSD 值也在最后达到了平衡.同时应用profile-3D对模建结构进行氨基酸残基的兼容性评测,其结构兼容性得分为130.13,远远高于临界得分60.89.从图4可以看出,绝大多数残基得分都是正值,均位于合理可信的位置,只有2个处于loop区的Tyr225和Ser296处于不合理范围之内,但它们远离活性位点,对后续研究没有不良影响.另外,还应用procheck 程序检查了hSR主链构型中的Φ-Ψ二面角的分布,图5给出的是Ramachandran plot检测图,图中处于合理区域的氨基酸比例大于99.6%,以上检测结果都表明,模建所得的hSR三维结构是合理可信的.2.2分子对接在同源模建所得hSR三维结构基础上,利用binding-site程序搜寻得到了hSR的2个可能的活图5人类消旋酶模建结构的Ramachandran图Fig.5Main-chain dihedral angles(Ramachandran plot)of the modeled human serine racemase A,B,and L represent the residues in most favoured regions;a,b,l, and p represent the residues in additional allowed regions;~a,~b,~l, and~p represent the residues in generously allowed regions.图2人类丝氨酸消旋酶三维结构的带状显示图Fig.2Ribbon representation of human serineracemaseDots model depicts pyridoxal phosphate(PLP).图3体系的势能曲线(a)以及蛋白质的RMSD曲线(b) Fig.3Potential energy of the system(a)and the root mean square deviation(RMSD)of the protein(b)图4人类丝氨酸消旋酶模建结构的profile-3D图Fig.4Profile-3D plot of the modeled human serineracemaseScore S means the compatibility of an amino acid sequence with aknown3D protein structure.419Acta Phys.-Chim.Sin.,2009Vol.25性位点,位点1由Lys50,Ser77,Gly79,His81,Pro147,Asn148,Gln149,Gly179,Gly180,Lys235,Ser236,Ser237,Gly239等残基和辅酶PLP 组成,而位点2由Ser78,Val99,Val100,Pro101,Cys122,Pro124,Gly127,Ser128等残基组成,根据消旋酶的催化机制以及实验上的相关报道[27],位点1中包含了催化残基Lys50、辅酶PLP 以及保守残基Gly179,Gly180等,因此选择位点1作为本文研究对象.我们还应用CASTp 和CAVER 位点预测程序验证所选位点的合理性.从图2中可以看出,hSR 的活性位点1位于蛋白质表面较为狭长的凹槽,并位于辅酶磷酸吡哆醛的上方.为了明确hSR 与多肽类抑制剂的相互作用情况,借助affinity 程序分别进行了hSR 与多肽类抑制剂A 和B 的分子对接研究,计算所得复合物的总能量(total energy),hSR 与配体的最低相互作用能(ΔE ),其主要作用的关键氨基酸残基分别列于表1中.hSR 与多肽类抑制剂A 的对接结果表明,二者的结合方式具有2种不同的空间取向.取向(1)在10个对接结果中为优势构象(图6(a)),其最低相互作用能为-347.86kJ ·mol -1.从图6(a)可以看出,抑制剂A 的咪唑环伸向活性位点的深处,其末端的羰基O 与E130的羧基O 形成水合氢键,K235的侧链氨基N 与3-苯基丙乙酸部分的氨基H 形成氢键,从而使3-苯基丙乙酸部分和引入的乙丁基恰好位于活性部位的空腔中.取向(2)与(1)的结合方式存在明显差异,选取其中相互作用能最低(-325.96kJ ·mol -1)的复合物构象来进行分析(图6(b)).抑制剂A 的3-苯基丙乙酸部分伸向活性位点的深处,3-苯基丙乙酸基团上的O 分别与S78和K235形成水合氢键,增强了复合物的稳定性.但是由于配体末端羰基O 与NH 分别与R129胍基和S78的羟基形成氢键,使配体引入的乙丁基暴露在hSR 的表面,从而减弱了多肽类抑制剂A 与hSR 之间的相互作用.从表1可以看到,借助affinity 程序计算得到的取向(1)的体系总能量和分子间相互作用能都比取向(2)的低,也就是说,取向(1)为最佳的作用模式.为了进一步验证抑制剂A 在hSR 中的结合方式,根据配体的作用机理,抽取对接结果中的抑制剂的几何结构,利用B3LYP 方法,在6-31G(d ,p )水平下,用截断复合物表1抑制剂A 和B 与hSR 对接的结果Table 1Results of docking runs for inhibitors A and B with hSRa)N is the total number of docking results,b)the lowest interaction energy of protein with the inhibitor obtained by docking results,including electrostatic and van der Waals interactions,c)the residues that form hydrogen bonds with the ligand,d)the interaction energy between the proteinand inhibitors calculated at B3LYP/6-31G(d ,p )level,e)the total energy of the complex from the docking results.energy in kJ ·mol -1Inhibitor Cluster N a ΔE b Contacting residue cEnergy d Total energy e A 16-347.86K235,E130-35.65-5.28×10424-325.96S78,R129,K235-15.05-5.25×104B15-462.79H76,N148,E130-68.95-5.22×10425-398.23D232,N148,H76,K235-52.96-5.21×104图6抑制剂A 与人类丝氨酸消旋酶活性位点的对接结果Fig.6Docking results of inhibitor A with hSR(a)cluster 1,(b)cluster 2420No.3赵勇山等：人类丝氨酸消旋酶的同源模建及其与多肽类抑制剂的分子对接模型计算了抑制剂A与蛋白质的相互作用能,从表1可以明显地看出,取向(1)的相互作用能明显的低于取向(2),从而进一步说明抑制剂A是以取向(1)的方式与hSR结合的.此外,还利用Ludi程序来评判所得复合物结构的合理性,取向(1)和(2)的打分分别为371和323,可以看出(1)的对接方式的打分远远高于(2),也进一步证明了抑制剂A是以(1)的方式与hSR结合的.对于抑制剂B,从得到的复合物的构象来看,在hSR活性位点中的结合情况也主要有两种方式,并且二者出现几率是一样的,对接结果见表1与图7.同样地,分别选取其中能量最低的构象进行讨论,以明确抑制剂B和hSR的具体作用方式.在方式(1)的最低相互作用能(-462.79kJ·mol-1)复合物构象中(图7(a)),抑制剂B的3-苯基丙乙酸部分伸向活性位点的深处,氨基酸残基E130与抑制剂B形成两个氢键,一个由E130的O与抑制剂的末端的NH形成,另一个是E130的O通过水分子与抑制剂B中间肽键上的O形成的水合氢键;残基N148也与抑制剂形成了两个氢键,即N148上的侧链O和氨基H分别通过水分子与抑制剂B末端的羰基O形成水合氢键.这些氢键对稳定空间体积较大的苯甲基有很大贡献.此外,咪唑环上的氮原子与H76的氨基H之间还有一个氢键存在.在方式(2)的最低相互作用能(-398.23kJ·mol-1)的复合物构象中,抑制剂B与hSR之间形成了四个氢键(图7(b)),抑制剂B 的咪唑环伸向活性位点的深处,但由于苯甲基所在的末端链太长,不能完全包埋在活性部位的空腔中而暴露于hSR的表面,导致了hSR和抑制剂B之间的相互作用减弱.为了进一步证明抑制剂B在hSR中结合的优势几何构型,同样的,利用B3LYP 方法,在6-31G(d,p)水平下,计算了方式(1)和(2)的相互作用能.结果如表1所示,方式(1)比(2)的相互作用能低,也就是说抑制剂是以方式(1)的结合方式与hSR结合的.方式(1)和(2)的Ludi打分分别为504和488.以上分析结果都表明,抑制剂B也是以(1)的方式结合在hSR中.3结论通过比较分析hSR与多肽类抑制剂A和B的对接结果,得到了hSR与抑制剂分子的最佳作用模式的详细理论信息.从二者之间的相互作用能、氢键的形成以及Ludi打分的数据来看,在多肽类抑制剂的R位点上引入体积较大的苯甲基(抑制剂B)更适合作为hSR的抑制剂.而且在hSR与多肽类抑制剂的对接结果中发现,人类丝氨酸消旋酶的Asn148, His76,Glu130,Lys235等残基在与抑制剂的作用过程中起到非常重要的作用.References1Corrigan,J.J.Science,1969,164:1422Corrigan,J.J.;Srinivasan,N.G.Biochemistry,1966,5:11853Panizzutti,R.;De Miranda,J.;Ribeiro,C.S.;Engelender,S.;Wolosker,H.Proc.Natl.Acad.Sci.U.S.A.,2001,98:52944De Miranda,J.;Santoro,A.;Engelender,S.;Wolosker,H.Gene, 2000,256:1835Nagata,Y.;Konno,R.;Yasumura,Y.;Akini,T.Biochem.J.,1989,图7抑制剂B与人类丝氨酸消旋酶活性位点的对接结果Fig.7Docking results of inhibitor B with hSR(a)cluster1,(b)cluster 2421Acta Phys.-Chim.Sin.,2009Vol.25257:2916Hashimoto,A.;Nishikawa,T.;Hayashi,T.;Fujii,N.;Harada,K.;Oka,T.;Takahashi,K.FEBS 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分子对接affinity评分

分子对接affinity评分

分子对接affinity评分一、引言分子对接是药物研发过程中的重要环节，通过计算机模拟预测分子间的相互作用，可以评估药物与靶点间的亲和力（affinity）。

affinity 评分是评价药物候选化合物结合靶点的强度和稳定性的重要指标。

本文将探讨分子对接affinity评分的概念、应用和方法。

二、分子对接affinity评分的概念affinity评分是指药物分子与靶点蛋白结合的能力或强度的度量。

它可以通过计算药物分子与靶点蛋白之间的相互作用能量来获得。

affinity评分越高，表示药物分子与靶点蛋白结合越紧密，具有更强的亲和力。

三、分子对接affinity评分的应用1. 药物研发：affinity评分可以帮助药物研发人员预测候选化合物与靶点之间的亲和力，从而筛选出具有潜力的药物候选化合物。

2. 药物优化：通过分析affinity评分，研发人员可以了解药物分子与靶点蛋白结合的机制，指导药物结构的优化设计，提高药物的亲和力和选择性。

3. 副作用预测：affinity评分还可以用于预测药物与非靶点蛋白的结合情况，从而评估药物的副作用风险。

四、分子对接affinity评分的方法1. 分子对接软件：常用的分子对接软件包括AutoDock、Glide、DOCK等，它们可以根据药物分子和靶点蛋白的结构进行计算和模拟，得到affinity评分。

2. 动力学模拟：通过分子动力学模拟方法，可以模拟药物分子与靶点蛋白结合的动态过程，进一步优化affinity评分的准确性。

3. 机器学习方法：近年来，机器学习方法在affinity评分中的应用越来越多。

通过建立模型，利用已知的药物-靶点配对数据进行训练，可以预测未知药物-靶点的affinity评分。

五、结论分子对接affinity评分是药物研发过程中的重要工具，可以帮助研发人员预测药物候选化合物与靶点之间的亲和力，指导药物结构的优化设计，并预测药物的副作用风险。

通过合理选择分子对接软件、动力学模拟和机器学习方法，可以提高affinity评分的准确性和可靠性，促进药物研发的进展。

合成生物学中不依赖限制性内切酶的分子克隆策略

合成生物学中不依赖限制性内切酶的分子克隆策略

合成生物学中不依赖限制性内切酶的分子克隆策略不依赖限制性内切酶的分子克隆策略是一种生物学的技术，用于将特定的DNA片段复制成多个复制物，以便进行进一步分析和研究。

它可以帮助遗传学家获取大量特定的DNA序列，并可以通过特定的实验室方法对这些序列进行功能分析。

一、概述不依赖限制性内切酶的分子克隆策略主要是通过外源保护化合物（TAP）来实现无限制酶性克隆技术（TFC）。

TAP通过专门表达的融合蛋白分泌到培养基中，形成一个不接受基因粒范围的抗胞内抗原的壳。

TAP可以把DNA片段放置在其中的交叉起始位点，然后可以使用TFC把DNA片段克隆到另一个载体上，最终得到重组基因。

另一个重要的技术是以有害突变为基础的PCR方法，即突变依赖PCR （MAPS），它是一种不需要保护性蛋白酶就可以进行无限制酶性复制的高通量克隆方式。

MAPS神经可以检测特定的基因突变区域，从而克隆出对应的特定DNA片段。

二、TAP和MAPS的原理理解TAP技术是不依赖限制性内切酶的一种分子克隆策略，其效率要高于传统的限制性内切酶的分子克隆策略。

其原理是：通过表达一种特殊的保护化合物，形成一个不可受阻基因粒范围的抗胞内抗原的壳，这个抗原壳可以将DNA片段放置在其中的交叉起始位点，然后可以利用TFC把DNA克隆到载体上。

MAPS方法是一种基于毒性突变的PCR技术，它可以检测特定的基因突变区域，从而克隆出特定的DNA片段，而不需要任何保护性酶。

三、TAP和MAPS的实验流程在TAP技术中，需要研究者完成以下几个步骤：（1）设计一对序列特异性引物，可以通过in silico方法预测是否存在交叉起始位点，以确定可克隆的最佳DNA片段；（2）在受体上，添加足够的融合蛋白，以保护邻接的交叉起始位点；（3）在源上提供足够多的DNA片段，通过TFC把这些DNA片段复制到受体上；（4）培养新生成的菌株，检测复制基因效率。

MAPS方法的实验流程如下：（1）把特定的DNA片段转录制备好，由此形成一组cDNA；（2）利用突变依赖的PCR技术，根据特定的cDNA的突变，选择相应的引物搭配；（3）复制特定的DNA片段；（4）把复制出的DNA片段克隆到载体上；（5）检测复制的DNA的效率。

NIJ防弹标准[英]

NIJ防弹标准[英]

ABOUT THE LAW ENFORCEMENT AND CORRECTIONSSTANDARDS AND TESTING PROGRAMThe Standards and Testing Program is sponsored by the Office of Science and Technology of the National Institute of Justice (NIJ), Office of Justice Programs, U.S. Department of Justice. The program responds to the mandate of the Homeland Security Act of 2002, which directed the Office of Science and Technology to establish and maintain performance standards in accordance with the National Technology Transfer and Advancement Act of 1995 (Public Law 104–113) to test and evaluate law enforcement technologies that may be used by Federal, State, and local law enforcement agencies. The Homeland Security Act of 2002 also directed the Office of Science and Technology to establish and maintain a program to certify, validate, and mark or otherwise recognize law enforcement technology products that conform to the standards mentioned above.The Standards and Testing Program is a basic and applied research effort that determines the technological needs of justice system agencies, sets minimum performance standards for specific devices, tests commercially available equipment against those standards, and disseminates the standards and the test results to criminal justice agencies nationally and internationally.The Office of Law Enforcement Standards (OLES) at the National Institute of Standards and Technology develops voluntary national performance standards for compliance testing to ensure that individual items of equipment are suitable for use by criminal justice agencies. The standards are based upon laboratory testing and evaluation of representative samples of each item of equipment to determine the key attributes, develop test methods, and establish minimum performance requirements for each essential attribute. In addition to the technical standards, OLES also produces technical reports and user guidelines that explain in nontechnical terms the capabilities of available equipment.The National Law Enforcement and Corrections Technology Center (NLECTC), operated by a grantee, coordinates a national compliance testing program conducted by independent laboratories. The standards developed by OLES serve as performance benchmarks against which commercial equipment is measured.Publications are available at no charge through NLECTC. Some documents are also available online at https:///justnet.html. To request a document or additional information, call 800–248–2742 or 301–519–5060, or write:National Law Enforcement and Corrections Technology Center2277 Research Boulevard, Mailstop 8J20850MDRockville,asknlectc@E-mail:World Wide Web address: This page intentionally left blank.U.S. Department of JusticeOffice of Justice ProgramsNational Institute of JusticeBallistic Resistance of Body ArmorNIJ Standard–0101.06Supersedes:NIJ 2005 Interim Requirements, Ballistic Resistance of Body Armor (August 2005) Also supersedes:NIJ Standard–0101.04 Rev. A, Ballistic Resistance of Personal Body Armor (June 2001) Coordination by:Office of Law Enforcement StandardsNational Institute of Standards and TechnologyGaithersburg, MD 20899–8102Prepared for:National Institute of JusticeOffice of Science and TechnologyWashington, DC 20531July 2008NCJ 223054N ATIONAL I NSTITUTE OF J USTICEDavid HagyDirectorJohn MorganDeputy Director for Science and TechnologyMarc CaplanChief, Operational Technologies DivisionDebra StoePhysical Scientist, Operational Technologies DivisionThe technical effort to develop this standard was conducted underInteragency Agreement 2003–IJ–R–029.This standard was formulated by the Office of Law Enforcement Standards (OLES) of the National Institute of Standards and Technology (NIST). The participants in the research andrevision of this standard were:Kirk Rice, Program Manager, Weapons and Protective SystemsMichael A. Riley, Program Manager, Weapons and Protective Systems, Testing and EvaluationAmanda Forster, Materials Research EngineerThe preparation of this standard was sponsored by the National Institute of Justice.F OREWORDThis document, NIJ Standard–0101.06, “Ballistic Resistance of Body Armor,” is a minimum performance standard developed in collaboration with the Office of Law Enforcement Standards (OLES) of the National Institute of Standards and Technology (NIST). It is produced as part of the Standards and Testing Program of the National Institute of Justice (NIJ), Office of Justice Programs, U.S. Department of Justice. This standard is a technical document that specifies the minimum performance requirements that equipment must meet to satisfy the requirements of criminal justice agencies and the methods that shall be used to test this performance. This standard is used by the NIJ Voluntary Compliance Testing Program (CTP) to determine which body armor models meet the minimum performance requirements for inclusion on the NIJ Compliant Products List. In addition, manufacturers, criminal justice agencies, and others may use the tests described in this standard to determine whether a particular armor design meets their own requirements. Users are strongly encouraged to have this testing conducted in accordance with the NIJ CTP. Procurement officials may also refer to this standard in their purchasing documents and require that equipment offered for purchase meet or exceed these requirements.This document is a testing and performance standard and provides precise and detailed test methods. Additional requirements, processes, and procedures for CTP participants are detailed in the NIJ Voluntary CTP Administrative Procedures Manual. Those seeking guidance concerning the selection and application of body armor should refer to the most recent revision of the Selection and Application Guide to Personal Body Armor, NIJ Guide 100, which is published as a separate document and explains in nontechnical language how to select equipment that provides the level of performance required by a purchasing agency.Publication of this revision of the standard does not invalidate or renderunsuitable any body armor models previously determined by NIJ to becompliant to either the NIJ 2005 Interim Requirements or the NIJ Standard–0101.04 Rev. A Requirements. While it may not necessary to remove theseexisting armors from service, agencies are advised to always require theirprocurements to meet or exceed the most recent and up-to-date version ofthis standard.Personal body armor that is independently tested to this standard bymanufacturers, purchasers, or other parties will not be considered as NIJ compliant unless the body armor is submitted and tested through the NIJ CTP and found incompliance with this standard.When manufacturers seek NIJ compliance of their armor to this standard and thearmor contains unique materials or forms of construction that may not have beenanticipated when this standard was drafted, NIJ may modify the test methods of the standard to take those features into account. If NIJ determines that the model meets the requirements of the standard, modified test methods, and the NIJ CTP, NIJ will include the armor on the NIJ Compliant Products List.NIJ standards are subjected to continued research, development, testing, review, and revision. This standard and its successors will be re-evaluated and modified as necessary. Because of ongoing advancements in ballistic-resistant materials, changes to armor designs and manufacturing techniques, and improvements in testing methods, NIJ reserves the right to withhold NIJ compliance status for body armor that meets the current requirements but is deemed by NIJ to pose a risk to officer safety. NIJ also reserves the right to alter or modify existing test methods and/or requirements to address perceived weaknesses in varying designs of body armor submitted for inclusion on the NIJ Compliant Products List.Technical comments and recommended revisions are welcome. Please send all written comments and suggestions to the Deputy Director, Office of Science and Technology, National Institute of Justice, Office of Justice Programs, U.S. Department of Justice, 810 Seventh Street, NW, Washington, DC 20531. Before citing this or any other NIJ standard in a contract document, users should verify that the most recent edition of the standard is used. The most recent edition will be available on the Justice Technology Information Network (JUSTNET) at , or write to the Director, Office of Law Enforcement Standards, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8102, Gaithersburg, MD 20899.John Morgan, Deputy Director for Science andTechnology, National Institute of JusticeC ONTENTS Foreword (v)Commonly Used Symbols And Abbreviations (xi)1. Purpose And Scope (1)2. NIJ Body Armor Classification (3)2.1 Type IIA (9 mm; .40 S&W) (3)2.2 Type II (9 mm; .357 Magnum) (3)2.3 Type IIIA (.357 SIG; .44 Magnum) (3)2.4 Type III (Rifles) (4)2.5 Type IV (Armor Piercing Rifle) (4)2.6 Special Type (4)3. Definitions (5)4. Sample Requirements and Laboratory Configuration (11)4.1 Test Samples (11)4.1.1 Flexible Vests and Jackets (11)4.1.2 Hard Armors and Plate Inserts (12)4.1.3 Accessory Ballistic Panels (17)4.1.4 Workmanship (17)4.1.5 Labeling (17)4.1.6 Armor Carriers With Removable Ballistic Panels (20)4.1.7 Armors With Built-In Inserts or Trauma Packs (20)4.2 Laboratory Configuration and Test Equipment (20)4.2.1 Range Configuration (20)4.2.2 Test Rounds and Barrels (22)4.2.3 Velocity Measurement Equipment (22)4.2.4 Armor Submersion Equipment (23)4.2.5 Armor Backing Material (23)5. Flexible Armor Conditioning Protocol (27)5.1 Purpose and Scope (27)5.2 Pretest Parameters (27)5.2.1 Storage of Armors (27)5.2.2 Pretest Calibrations (27)5.2.3 Test Conditions (27)5.3 Laboratory Configuration and Test Equipment (28)5.3.1 General Parameters (28)5.3.2 Controls (29)5.3.3 Test Interruption (29)5.3.4 Procedure (30)6. Hard Armor Conditioning Protocol (33)6.1 Purpose and Scope (33)6.2 Pretest Parameters (33)6.2.1 Storage of Armors (33)6.2.2 Pretest Calibrations (33)6.2.3 Test Conditions (33)6.3 Laboratory Configuration and Test Equipment (35)6.3.1 General Parameters (35)6.3.2 Controls (35)6.3.3 Test Interruption (35)6.3.4 Conditioning Procedure (36)7. Ballistic Test Methods (39)7.1 Purpose and Scope (39)7.2 Test Order (39)7.3 Workmanship Examination (39)7.3.1 Armor Carriers and Ballistic Panel Covers (39)7.3.2 Ballistic Panels (39)7.3.3 Label Examination (39)7.4 Sampling (40)7.5 Sample Acclimation (40)7.5.1 Inserts (40)7.6 Fair Hit Requirements for All Ballistic Tests (40)7.6.1 Minimum Shot-to-Edge Distance (40)7.6.2 Minimum Shot-to-Shot Distance (40)7.7 Backing Material Preparation and Sample Mounting for All Ballistic Tests (41)7.7.1 Backing Material Fixture Preparation (41)7.7.2 Mounting Armor for Ballistic Testing (41)7.8 Perforation and Backface Signature Test (P-BFS) (43)7.8.1 Shot Location Marking (43)7.8.2 Armor Submersion (45)7.8.3 Test Threats for P-BFS Tests (45)7.8.4 Test Duration (45)7.8.5 Requirements for Number of Shots and Number of Armor Samples (45)7.8.6 P-BFS Test for Special Type Armor (49)7.8.7 P-BFS Test for Accessory Ballistic Panels (49)7.8.8 P-BFS Performance Requirements (49)7.9 Ballistic Limit (BL) Determination Test (50)7.9.1 Requirements for Number of BL Tests and Test Samples (51)7.9.2 Test Procedure Requirements (51)7.9.3 Backing Material Conditioning (52)7.9.4 Data Set Tabulation (52)7.9.5 Ballistic Limit Performance Requirements (52)8. References (53)APPENDIX A –Acceptable Bullets for Handloading (55)APPENDIX B –Common Special Type Threats (57)APPENDIX C –Armor Sizing Templates (59)APPENDIX D –Analysis of Backface Signature Data (65)APPENDIX E –Analysis of Ballistic Limit Data (69)APPENDIX F –Explanatory Materials (71)T ABLESTable 1. Armor samples required for each test threat (flexible vests and jackets) (11)Table 2. Uniform thermal exposure conditions (33)Table 3. Thermal cycle testing conditions (34)Table 4. P-BFS performance test summary (46)Table 5. Number of shots and fair hits on each size of new condition armor samples (47)Table 6. Number of shots and fair hits on each size of conditioned samples (47)Table 7. Angle of incidence locations (48)Table 8. Baseline ballistic limit determination test summary (50)Table 9. Test parameters and requirements for ballistic limit test (51)Table 10. Acceptable bullets (55)Table 11. Special type threats of particular concern to law enforcement (58)Table 12. Surface areas of armor sizing templates (59)Table 13. Minimum allowable surface areas for production armor (59)Table 14. Maximum allowable surface areas for production armor (59)Table 15. k Factors for 80 % Probability with 95 % Confidence (67)F IGURESFigure 1. Angle of incidence (5)Figure 2. Examples of BFS measurements (6)Figure 3. Sample quantity and utilization for armor Types IIA, II, and IIIA (14)Figure 4. Sample quantity and utilization for flexible armor of Type III, Type IV, and Type Special (15)Figure 5. Sample quantity and utilization for hard armors and plate inserts of Type III (16)Figure 6. Sample quantity and utilization for hard armors and plate inserts of Type IV (16)Figure 7. Example label for test samples (19)Figure 8. Test range configuration (21)Figure 9. General pretest drop locations (25)Figure 10. Flexible armor conditioning test interruption flow chart (31)Figure 11. Test interruption flowchart for hard armor (37)Figure 12. Proposed mechanical durability test apparatus (38)Figure 13. Acceptable strapping methods (42)Figure 14. General armor panel impact locations (front and back) (44)S TANDARD S PECIFIC A BBREVIATIONSACP =Automatic Colt Pistol LR = Long Rifle ANSI =American National Standards Institute LRN = Lead Round Nose AP =Armor Piercing NLECTC = BFS =Backface Signature National Law Enforcement and Corrections Technology Center BL =Ballistic Limit P-BFS = Perforation and Backface Signature CPO =Compliance Program Office RN = Round Nose CTP =Compliance Testing Program S&W = Smith & Wesson CTR =Compliance Test Report SAAMI = FMJ =Full Metal Jacket Sporting Arms and Ammunition Manufacturers’ Institute ISO =International Standards Organization JHP =Jacketed Hollow Point SJHP = Semi Jacketed Hollow Point JSP =Jacketed Soft Point SJSP = Semi Jacketed Soft PointC OMMONLY U SED S YMBOLS A ND A BBREVIATIONSA ampereH henry nm nanometer ac alternating currenth hour No. number AM amplitude modulationhf high frequency o.d. outside diameter cd candelaHz hertz Ω ohm cm centimeteri.d. inside diameter p. page CP chemically purein inch Pa pascal c/s cycle per secondIR infrared pe probable error d dayJ joule pp. pages dB decibelL lambert ppm parts per million dc direct currentL liter qt quart °C degree CelsiusLb pound rad radian °F degree Fahrenheitlbf pound force rf radio frequency diam diameterlbf ⋅in pound force inch rh relative humidity emf electromotive forcelm lumen s second eq equationln logarithm (base e) SD standard deviation F faradlog logarithm (base 10) sec. section fc footcandleM molar SWR standing wave ratio fig. figurem meter uhf ultrahigh frequency FM frequency modulationmin. minute UV ultraviolet ft footmm millimeter V volt ft/s foot per secondmph miles per hour vhf very high frequency g accelerationm/s meter per second W watt g gramN newton λ wavelength gr grainN ⋅m newton meter wt weightarea = unit 2 (e.g., ft 2, in 2, etc.); volume = unit 3 (e.g., ft 3, m 3, etc.)P REFIXES d deci (10-1) da deka (10) c centi (10-2) h hecto (102) m milli (10-3) k kilo (103) µ micro (10-6) M mega (106) n nano (10-9) G giga (109)p pico (10-12) T tera (1012) C OMMON C ONVERSIONS(See ASTM E380)0.30480 m = 1 ft 4.448222 N = 1 lbf 2.54 cm = 1 in 1.355818 J = 1 ft ⋅lbf 0.4535924 kg = 1 lb 0.1129848 N.m = 1 lbf ⋅in 0.06479891 g = 1 gr 14.59390 N/m = 1 lbf/ft 0.9463529 L = 1 qt 6894.757 Pa = 1 lbf/in 2 3600000 J = 1 kW ⋅h1.609344 km/h = 1 mphTemperature: T °C = (T °F - 32) × 5/9Temperature: T °F = (T °C × 9/5) + 32This page intentionally left blank.NIJ STANDARD–0101.06FORBALLISTIC RESISTANCE OF BODY ARMOR1.P URPOSE A ND S COPEThe purpose of this standard is to establish minimum performance requirements and test methods for the ballistic resistance of personal body armor intended to protect against gunfire.This standard is a revision of NIJ Standard–0101.04, dated September 2000. It supersedes the NIJ 2005 Interim Requirements, dated September 2005, NIJ Standard–0101.04, and all other revisions and addenda to NIJ Standard–0101.04.The scope of the standard is limited to ballistic resistance only; this standard does not address threats from knives and sharply pointed instruments, which are different types of threats and are addressed in the current version of NIJ Standard–0115 Stab Resistance of Personal Body Armor.Body armor manufacturers and purchasers may use this standard to help to determine whether specific armor models meet the minimum performance standards and test methods identified in this document. However, NIJ strongly encourages body armor manufacturers to participate in the NIJ Voluntary Compliance Testing Program (CTP) and encourages purchasers to insist that the armor model(s) they purchase be tested by the NIJ CTP and be listed on the NIJ Compliant Products List. This will help to assure that the armor models will meet the minimum performance standards for use by the criminal justice community.The ballistic tests described in this standard have inherent hazards. Adequate safeguards for personnel and property must be employed when conducting these tests.This page intentionally left blank.2.NIJ B ODY A RMOR C LASSIFICATIONPersonal body armor covered by this standard is classified into five types (IIA, II, IIIA, III, IV) by level of ballistic performance. In addition, a special test class is defined to allow armor to be validated against threats that may not be covered by the five standard classes.The classification of an armor panel that provides two or more levels of NIJ ballistic protection at different locations on the ballistic panel shall be that of the minimum ballistic protection provided at any location on the panel.2.1Type IIA (9 mm; .40 S&W)Type IIA armor that is new and unworn shall be tested with 9 mm Full Metal Jacketed Round Nose (FMJ RN) bullets with a specified mass of 8.0 g (124 gr) and a velocity of 373 m/s ± 9.1 m/s (1225 ft/s ± 30 ft/s) and with .40 S&W Full Metal Jacketed (FMJ) bullets with a specified mass of 11.7 g (180 gr) and a velocity of 352 m/s ± 9.1 m/s (1155 ft/s ± 30 ft/s).Type IIA armor that has been conditioned shall be tested with 9 mm FMJ RN bullets with a specified mass of 8.0 g (124 gr) and a velocity of 355 m/s ± 9.1 m/s (1165 ft/s ± 30 ft/s) and with .40 S&W FMJ bullets with a specified mass of 11.7 g (180 gr) and a velocity of 325 m/s ± 9.1 m/s (1065 ft/s ± 30 ft/s).2.2Type II (9 mm; .357 Magnum)Type II armor that is new and unworn shall be tested with 9 mm FMJ RN bullets with a specified mass of 8.0 g (124 gr) and a velocity of 398 m/s ± 9.1 m/s (1305 ft/s ± 30 ft/s) and with .357 Magnum Jacketed Soft Point (JSP) bullets with a specified mass of 10.2 g (158 gr) and a velocity of 436 m/s ± 9.1 m/s (1430 ft/s ± 30 ft/s).Type II armor that has been conditioned shall be tested with 9 mm FMJ RN bullets with a specified mass of 8.0 g (124 gr) and a velocity of 379 m/s ±9.1 m/s (1245 ft/s ± 30 ft/s) and with .357 Magnum JSP bullets with a specified mass of 10.2 g (158 gr) and a velocity of 408 m/s ±9.1 m/s (1340 ft/s ± 30 ft/s).2.3Type IIIA (.357 SIG; .44 Magnum)Type IIIA armor that is new and unworn shall be tested with .357 SIG FMJ Flat Nose (FN) bullets with a specified mass of 8.1 g (125 gr) and a velocity of 448 m/s ± 9.1 m/s (1470ft/s ± 30 ft/s) and with .44 Magnum Semi Jacketed Hollow Point (SJHP) bullets with a specified mass of 15.6 g (240 gr) and a velocity of 436 m/s ± 9.1 m/s (1430 ft/s ± 30 ft/s).Type IIIA armor that has been conditioned shall be tested with .357 SIG FMJ FN bullets with a specified mass of 8.1 g (125 gr) and a velocity of 430 m/s ± 9.1 m/s (1410 ft/s ± 30 ft/s) and with .44 Magnum SJHP bullets with a specified mass of 15.6 g (240 gr) and a velocity of 408 m/s ± 9.1 m/s (1340 ft/s ± 30 ft/s).2.4Type III (Rifles)Type III hard armor or plate inserts shall be tested in a conditioned state with 7.62 mm FMJ, steel jacketed bullets (U.S. Military designation M80) with a specified mass of 9.6 g (147 gr) and a velocity of 847 m/s ± 9.1 m/s (2780 ft/s ± 30 ft/s).Type III flexible armor shall be tested in both the “as new” state and the conditioned state with 7.62 mm FMJ, steel jacketed bullets (U.S. Military designation M80) with a specified mass of 9.6 g (147 gr) and a velocity of 847 m/s ± 9.1 m/s (2780 ft/s ± 30 ft/s).For a Type III hard armor or plate insert that will be tested as an in conjunction design, the flexible armor shall be tested in accordance with this standard and found compliant as a stand-alone armor at its specified threat level. The combination of the flexible armor and hard armor/plate shall then be tested as a system and found to provide protection at the system’s specified threat level. NIJ-approved hard armors and plate inserts must be clearly labeled as providing ballistic protection only when worn in conjunction with the NIJ-approved flexible armor system with which they were tested.2.5Type IV (Armor Piercing Rifle)Type IV hard armor or plate inserts shall be tested in a conditioned state with .30 caliber armor piercing (AP) bullets (U.S. Military designation M2 AP) with a specified mass of 10.8 g (166 gr) and a velocity of 878 m/s ± 9.1 m/s (2880 ft/s ± 30 ft/s).Type IV flexible armor shall be tested in both the “as new” state and the conditioned state with .30 caliber AP bullets (U.S. Military designation M2 AP) with a specified mass of 10.8 g (166 gr) and a velocity of 878 m/s ± 9.1 m/s (2880 ft/s ± 30 ft/s).For a Type IV hard armor or plate insert that will be tested as an in conjunction design, the flexible armor shall be tested in accordance with this standard and found compliant as a stand-alone armor at its specified threat level. The combination of the flexible armor and hard armor/plate shall then be tested as a system and found to provide protection at the system’s specified threat level. NIJ-approved hard armors and plate inserts must be clearly labeled as providing ballistic protection only when worn in conjunction with the NIJ-approved flexible armor system with which they were tested.2.6Special TypeA purchaser having a special requirement for a level of protection other than one of the above standard types and threat levels should specify the exact test round(s) and reference measurement velocities to be used and indicate that this standard shall govern all other aspects. Guidance on common special type threats and the appropriate threat velocities is provided in appendix B, along with a methodology for determining the correct reference velocity for other threats.3.D EFINITIONS3.1Absolute Humidity: The quantity of water in a given volume of air, often reported ingrams per cubic meter.3.2Accessory Ballistic Panels: Accessory panels are armor components that are detachable orremovable from the primary body armor sample and are intended to provide comparable ballistic protection. Examples of accessory panels include groin, coccyx, and sideprotection panels, which are attached to or inserted into the external armor carrier but are not integral to the armor panels or armor sample.3.3Angle of Incidence: The angle between the bullet’s line of flight and the perpendicular tothe front surface of the backing material fixture as shown in figure 1.Figure 1. Angle of incidence3.4Armor Carrier: A component of the armor sample or armor panel whose primary purposeis to retain the ballistic panel and provide a means of supporting and securing the armor garment to the user. Generally, the carrier is not ballistic resistant.3.5Armor Conditioning: Environmental and mechanical conditioning of armor prior toballistic testing, which consists of exposure to specified conditions of temperature,humidity, and mechanical damage.3.6Armor Panel or Panel: The portion of an armor sample that consists of an externalballistic cover and its internal ballistic panel. The word “panel,” if not preceded by theword “ballistic,” refers to an armor panel in this standard.3.7Armor Sample:One complete armor garment. Typically, a front armor panel, a backarmor panel, and the armor carrier comprise a body armor sample. The armor sample may be a single wraparound style or consist of multiple parts that are worn around the body. 3.8Backface Signature (BFS): The greatest extent of indentation in the backing materialcaused by a nonperforating impact on the armor. The BFS is the perpendicular distance between two planes, both of which are parallel to the front surface of the backing materialfixture. One plane contains the reference point on the original (pretest) backing material surface that is co-linear with the bullet line of flight. (If armor were not present, the bulletFigure 2. Examples of BFS measurementswould strike this point.) The other plane contains the point that represents the deepestindentation in the backing material. Depending on bullet–armor–backing materialinteractions, the two points that define the locations of the measurement planes may not be co-linear with the bullet line of flight. Examples of how BFS is measured are shown infigure 2.3.9Backing Material: A homogenous block of nonhardening, oil-based modeling clay placedin contact with the back of the armor panel during ballistic testing.3.10Backing Material Fixture: A box-like rigid frame, with a removable back, which containsthe backing material. The removable back is used for perforation-backface signature testing but is not used for ballistic limit testing.3.11Ballistic Limit: For a given bullet type, the velocity at which the bullet is expected toperforate the armor 50 % of the time. The ballistic limit is typically denoted as the V50 or V50 value.3.12Ballistic Panel: The protective component of an armor sample or armor panel, consistingprimarily of ballistic resistant materials. The ballistic panel is usually enclosed in a non-removable panel cover, which is considered part of the ballistic panel. The ballistic panel is normally enclosed within an armor carrier.3.13Baseline Ballistic Limit: The experimentally derived ballistic limit of an armor modelwhen new.3.14Body Armor: An item of personal protective equipment that provides protection againstspecific ballistic threats within its coverage area. In this standard, the term body armorrefers to that which provides coverage primarily for the torso.3.15Complete Penetration: This nomenclature is no longer used in this standard. See“perforation.” Perforation replaces complete penetration. Although the terms partialpenetration and complete penetration are no longer used in this standard, they may still be used by test laboratories for compatibility with military standards.3.16Compliance Test Group: A group of armor samples submitted for testing according to thisstandard.3.17Condensation: Precipitation of water vapor on a surface whose temperature is lower thanthe dew point of the ambient air. The dew point depends on the quantity of water vapor in the air. The dew point, the absolute humidity, and the vapor pressure are directlyinterdependent. Condensation occurs on a test item when the temperature at the surface of the item placed in the test chamber is lower than the dew point of the air in the chamber. Asa result, the item may need to be preheated to prevent condensation.3.18Dewpoint (or dew point): The temperature to which a parcel of air must be cooled (atconstant barometric pressure) for water vapor present in the air to condense into water(dew).3.19Fair Hit: The impact of a bullet on an armor panel that meets the shot spacing and velocityrequirements of section 7.6.3.20Flexible Body Armor: Body armor constructed of pliable, textile-based materials such thatthe complete system is capable of being flexed. Such systems are typically in the form of vests or jackets that provide greater coverage area than rigid plate armor. Generally, these armors provide protection against handgun threats.3.21Full Metal Jacketed Bullet (FMJ): A bullet consisting of a lead core completely covered,except for the base, with copper alloy (approximately 90 % copper and 10 % zinc). “Total Metal Jacket (TMJ),” “Totally Enclosed Metal Case (TEMC),” and other commercialterminology for bullets with electro-deposited copper and copper alloy coatings have been tested and are considered comparable to FMJ bullets for this standard.3.22Hard Armor or Rigid Armor: Rigid armor systems, plates, inserts, accessories, or semi-rigid armor systems constructed with rigid plates that are typically designed to provideprotection against rifle threats.。