High dimensional indexing for protein structure matching using bowties

生物化学复习资料Amino acid（氨基酸）: All proteins are made up from the same set of 20 standard amino acids. A typical amino acid has a primary amino group, a carboxyl group, a hydrogen atom and a side-chain(R group) attached to a central α-group atom(Cα). Proline（脯氨酸）is the exception to the rule in that it has a secondary amino group.Primary structure（一级结构）: The linear（线状的）sequence of amino acids joined together by peptide bonds is termed（被称为）the primary structure of the protein. The position of covalent disulfide bonds between cysteine（半胱氨酸）residues is also included in the primary structure.Secondary structure（二级结构）: Secondary structure is a protein refers to the regular folding of regions of the polypeptide chain. The two most common types of secondary structure are the αhelix and βpleated sheet（β折叠）.Tertiary structure（三级结构）: Tertiary structure in a protein refers to the three-dimensional（三维的）arrangement of all the amino acids in the polypeptide chain. This biologically active, native conformation（构造；形态）is maintained by multiple（多重的；多样的）noncovalent （非共价的）bonds.Quaternary structure（四级结构）: if a protein is made up of more than one polypeptide chain it is said to have quaternary structure. This refers to the spatial（空间的）arrangement of the polypeptide subunits（亚基；亚单位）and the nature of the interactions between them.Protein stability（蛋白质稳定性）: In addition to the peptide bonds between individual amino acid residues, the three-dimensional structure of a protein is maintained by a combination of noncovalent interactions(electrostatic forces（静电力）, van derWaals forces（范德华力）,hydrogen bonds（氢键）, hydrophobic forces（疏水作用力）) and covalent interactions(disulfide bonds（二硫键）).The Bohr effect(波尔效应): H+, CO2and 2,3-bisphosphoglycerate（2,3-二磷酸甘油酸）are allosteric effectors（变构效应剂）, promoting the release of O2 from hemoglobin（血红蛋白）. H+ and CO2 bind to different parts of the polypeptide chains, while 2,3-bisphosphoglycerate binds in the central cavity（凹穴）between the four subunits.Dialysis（透析）: Proteins can be separated from small molecules by dialysis through asemi-permeable membrane（半透膜）which has pores that allow small molecules to pass through but not proteins.Function and diversity of collagen（胶原蛋白）: Collagen is the name given to a family of structurally related proteins that form strong insoluble（不可溶的）fibers. Collagens consist of three polypeptide chains, the identity and distribution of which vary between collagen types.Gel filtration chromatography(凝胶过滤层析)Ion exchange chromatography(离子交换层析)Affinity chromatography(亲和层析)Enzymes as catalysts（作为催化剂的酶）: Enzymes are catalysts（催化剂）that change the rate of a reaction without being changed themselves. Enzymes are highly specific and their activity can be regulated. Virtually all enzymes are proteins, although some catalytically active RNAs have been identified.Active site（活性位点）: The active site is the region of the enzyme that binds the substrate, to form an enzyme-substrate complex, and transforms it into product. The active site is a three-dimensional entity（实体）, often a cleft（裂口）of crevice（缺口）on the surface of the protein, in which the substrate is bound by multiple weak interactions. Two models have been proposed to explain how an enzyme binds its substrate: the lock-and-key model and the induced-fit model（诱导契合模型）.Enzyme classification（酶的分类）: Enzymes are classified into six major groups on the basis of the type of reaction that they catalyze. Each enzyme has a unique four-digit classification number.Enzyme assays（酶的测定）: An enzyme assay measures the conversion of substrate to product, under conditions of cofactors, pH and temperature at which the enzyme is optimally active（最佳活性的）.Coenzymes（辅酶）and prosthetic group（辅基）: Some enzymes require the presence of cofactors, small nonprotein units, to function. Cofactors may be inorganic ions or complex organic molecules called coenzymes. A cofactor that is covalently attached to the enzyme is called a prosthetic group. A holoenzyme is the catalytically active form of the enzyme with its cofactor, whereas an apoenzyme（脱辅基酶蛋白）is the protein part on its own.Isoenzymes（同工酶）: Isoenzymes are different forms of an enzyme which catalyze the same reaction, but which exhibit different physical of kinetic（动力学的）properties（性质）.Activation energy and transition state: For a biochemical reaction to proceed, the energy barrierneeded to transform the substrate molecules into the transition state has to be overcome. The transition state has the highest free energy in the reaction pathway. The different in free energy between the substrate and the transition state is termed the Gibbs free energy of activation. An enzyme stabilizes the transition state and lowers Gibbs free energy（吉布斯自由能）, thus increasing the rate at which the reaction occurs.Michaelis-Menten equation（米氏方程）: v＝Vmax[S]/ (Km+[S])It describe these observations and predicts a hyperbolic curve（双曲线）of V0 against [S]. The Michaelis constant, Km, is equal to the sum of the rates of breakdown of the enzyme-substrate complex over its rate of formation, and is a measure of the affinity（亲和性）of an enzyme for its substrate.Enzyme inhibition（酶的抑制）: The catalytic rate of an enzyme can be lowered by inhibitor molecules. Many inhibitors exist, including normal body metabolites, foreign drugs and toxins. Enzyme inhibition can be of two main types: irreversible or reversible. Reversible inhibition can be subdivided into competitive and noncompetitive.Irreversible inhibition（不可逆抑制）: An irreversible inhibition binds tightly, often covalently, to amino acid residues at the active site of the enzyme, permanently inactivating the enzyme.Reversible competitive inhibition（可逆竞争抑制）: A competitive inhibitor competes with the substrate molecules for binding to the active site of the enzyme. At high substrate concentration, the effect of a competitive inhibitor can be overcome. the type of the inhibition can increase the Km ,but leave the Vmax unchanged.（Km变大，Vmax不变）Reversible noncompetitive inhibition（可逆非竞争性抑制）: A noncompetitive inhibitor binds at a site other than the active site of the enzyme and decreases its catalytic rate by causing a conformational change in the three-dimensional shape of the enzyme. The effect of a noncompetitive inhibitor cannot be overcome at high substrate concentrations. This type of the inhibition can decrease Vmax ,but leave the Km unchanged.（Km不变，Vmax变小）Reversible uncompetitive inhibition（可逆反竞争性抑制）: A uncompetitive inhibition binds at a site located in the substrate-enzyme complex. Thus, the type of the inhibition can decrease both the Vmax and the Km.（Km和Vmax都变小）Allosteric enzymes: A plot（绘制成的坐标图）of V0 against [S] for an allosteric enzyme gives a sigmoidal-shaped curve（S形的曲线）. Allosteric enzymes usually have more than one active site which cooperatively bind substrate molecules, such that the binding of substrate at one active site induces a conformational change in the enzyme which alters the affinity of the other active sites for substrate. Allosteric enzymes are often multisubunit proteins(多亚基蛋白质), with an active site on each subunit. Two models have been proposed to explain the allosteric behavior of enzyme, the concerted or the symmetry model（齐变模型）and the sequential model（序变模型）.Reversible covalent modification: The activity of many enzymes is altered by the reversiblemaking and breaking of a covalent bond between the enzyme and a small nonprotein group. The most common such modification is the addition and removal of a phosphate group（磷酸基）; phosphorylation（磷酸化）and dephosphorylation（去磷酸化）, respectively. Phosphorylation is catalyzed by protein kinases（蛋白激酶）, often using A TP as the phosphate donor, whereas dephosphorylation is catalyzed by protein phosphatases.（蛋白磷酸酯酶或蛋白磷酸化酶）Proteolytic activation（酶原激活）: Some enzymes are synthesized as larger inactive precursors （前体）called proenzymes or zymogens（酶原）. These are activated by the irreversible hydrolysis（水解）of one or more peptide bonds.DNA double helix（DNA双螺旋结构）: In a DNA double helix, the two strands（串；条）of DNA are wound round each other with the bases（碱基）on the inside and the sugar-phosphate backbones（磷酸糖主干）on the outside. The two DNA chains are held together by hydrogen bonds between pairs of bases; adenine（A）always pairs with thymine（T）and guanine（G）always pairs with cytosine（C）. In the double helix, the two strands are organized in an antiparallel arrangement（反平行的排布）(one is oriented in the 5’→3’ , the other is in the 3’→5’).Between the sugar-phosphate strand is the gap which could be divided into two types, that is, the wide named major groove（大沟）and the narrow named minor groove（小沟）.Glycogen（糖原）: glycogen is a branched-chained polysaccharide containing glucose residues linked by α-1-4 bonds（α-1,4糖苷键）with α1-6 branchpoints（α-1,6糖苷键分支点）. The branched nature of glycogen makes it more accessible to glycogen phosphorylase（糖原磷酸化酶）during degradation（降解）, since this enzyme degrades the molecule by sequential removal of glucose residues from the nonreducing ends.Starch（淀粉）: Starch is a mixture of unbranched amylase(glucose residues joined by α1-4 bonds) and branched amylopectin(glucose residues joined α1-4 but with some α1-6 branchpoints).Overview of pentose phosphate pathway（磷酸戊糖途径综述）: The two major products of the pathway are nicotinamide adenine dinucleotide and ribose 5-phosphate（核糖-5-磷酸酯）. Ribose 5-phosphate and its derivatives are components of important cellular molecules such as RNA, DNA, NAD+，flavine adenine dinucleotide(FAD), A TP and coenzyme A(CoA). NADPH is required for many biosynthetic pathways and particularly for synthesis of fatty acids and steroids （类固醇）.Four Biological roles of fatty acids（脂肪酸的四个生理功能）:1.They are components of membranes(sphingolipids and glycerophospholipids)2.Several proteins are covalently modified by fatty acids;3.They act as energy stores(triacylglycerols) and fuel molecules;4.Fatty acid derivatives serve as hormones and intracellular second messengers.Role of Citric acid cycle(柠檬酸循环，又名tricarboxylic acid cycle（三羧酸循环），简称TAC): The cycle oxidizes pyruvate(formed during the glycolytic breakdown of glucose) to CO2andH2O,with the concomitant production of energy. Acetyl CoA from fatty acid breakdown and amino acid degradation products are also oxidaized. In addition, the cycle has a role in producing precursors for biosynthetic pathways.Energy yield of Citric acid cycle（柠檬酸循环的能量产出）: For each turn of the cycle,10 ATP molecules are produced, one directly from the cycle and 9 from the re-oxidation of the three NADH and one FADH2 molecules produced by the cycle by oxidative phosphorylation（氧化磷酸化）.20种常见氨基酸的名称和结构式名称英文缩写结构式甘氨酸(α-氨基乙酸)Glycine Gly GCH2COONH3丙氨酸(α-氨基丙酸)Alanine Ala ACH COONH3CH3亮氨酸(γ-甲基-α-氨基戊酸)*Leucine Leu LCHCOONH3(CH3)2CHCH2异亮氨酸(β-甲基-α-氨基戊酸)* Isoleucine Ile ICHCOONH3CH3CH2CHCH3缬氨酸(β-甲基-α-氨基丁酸)*Valine Val VCHCOONH3(CH3)2CH脯氨酸(α-四氢吡咯甲酸)Proline Pro P COONH H苯丙氨酸(β-苯基-α-氨基丙酸)* Phenylalanine Phe FCHCOONH3CH2蛋(甲硫)氨酸(α-氨基-γ-甲硫基戊酸) * Methionine Met MCHCOONH3CH3SCH2CH2色氨酸[α-氨基-β-(3-吲哚基)丙酸]* Tryptophan Trp WNCH2CH COONH3H丝氨酸(α-氨基-β-羟基丙酸)Serine Ser SCHCOONH3HOCH2谷氨酰胺(α-氨基戊酰胺酸)Glutamine Gln Q CH2CH2CHCOONH3H2N CO苏氨酸(α-氨基-β-羟基丁酸)*Threonine Thr TCHCOONH3CH3CHOH半胱氨酸(α-氨基-β-巯基丙酸)Cysteine Cys CCHCOONH3HSCH2天冬酰胺(α-氨基丁酰胺酸)Asparagine Asn N CH2CHCOONH3H2N CO酪氨酸(α-氨基-β-对羟苯基丙酸) Tyrosine Tyr YCHCOONH3CH2HO天冬氨酸(α-氨基丁二酸) Aspartic acid Asp DNH3HOOCCH2CHCOO谷氨酸(α-氨基戊二酸) Glutamic acid Glu ECHCOONH3HOOCCH2CH2赖氨酸(α,ω-二氨基己酸)*Lysine Lys KCHCOONH2CH2CH2CH2CH2NH3精氨酸(α-氨基-δ-胍基戊酸)Arginine Arg RH2N C CHCOONH2NHCH2CH2CH2NH2组氨酸[α-氨基-β-(4-咪唑基)丙酸] Histidine His H NCH2CH COONH3HN* 为必需氨基酸中性脂肪族氨基酸：Gly (Gly也是二十种中唯一不含手性碳原子的氨基酸，不具有旋光性)Ala Val Lue Ile含羟基或者巯基：Ser Thr Cys Met酸性氨基酸及其酰胺：Asp Asn Glu Gln碱性氨基酸和杂环氨基酸：Lys Arg His Pro芳香族氨基酸：Phe Tyr Trp非极性R基氨基酸：Ala V al Leu Ile Pro Phe Trp Met带正电荷(碱性)R基氨基酸：Lys Arg His不带电荷的极性R基氨基酸：Gly Ser Thr Cys Tyr Asn Gln带负电（酸性）的R基氨基酸：Asp Glu在280nm附近有吸收峰的氨基酸是：Phe Tyr Trp ，其中Trp的吸收峰在280nm处最强等电点：在某一pH的溶液中，氨基酸解离成阳离子和阴离子的趋势及程度相等，所带净电荷为零，呈电中性，此时溶液的pH称为该氨基酸的等电点。

高内涵分析及Western blotting法在蛋白质核质分布研究中的应用及比较研究纪晓方;李丽英;常娜【摘要】目的使用不同方法对蛋白质的核质分布进行检测并比较差异.方法细胞经免疫荧光染色后使用高内涵分析(high content analysis,HCA)对蛋白质的细胞质或细胞核总荧光强度进行分析;分别提取胞质及胞核蛋白,使用Western blotting 法对核质组分中的蛋白质含量进行鉴定.结果两种方法均可检测到蛋白质的出核转运;经定量分析,HCA法检测到的HuR蛋白质/核比升高倍数小于Western blotting法.结论使用HCA法检测蛋白质核质定位具有准确、客观、成本低、快捷、多参数等特点,不失为Western blotting的有效补充及替代方法.【期刊名称】《首都医科大学学报》【年(卷),期】2016(037)005【总页数】5页(P616-620)【关键词】高内涵分析;Western blotting;蛋白质核质分布【作者】纪晓方;李丽英;常娜【作者单位】首都医科大学,北京100069;首都医科大学基础医学院细胞生物学系肝脏保护与再生调节北京市重点实验室,北京100069;首都医科大学基础医学院细胞生物学系肝脏保护与再生调节北京市重点实验室,北京100069【正文语种】中文【中图分类】Q26蛋白质作为一种生物大分子，是组成一切细胞、组织的基本有机物，同时也是生命活动的主要承担者和各种生物学功能的重要行使者。

在细胞中，蛋白质的核质定位是其发挥生物学功能的重要前提之一。

因此，如何分析鉴定蛋白质在细胞核及细胞质中的分布对于蛋白质功能的研究有着十分重要的意义。

高内涵分析(high content analysis, HCA)技术是一种基于成像的单细胞水平的多参数细胞分析方法。

随着技术的不断进步和发展，近年来，HCA在生命科学研究中开始得到了愈发广泛的应用[1-3]。

本实验以RNA结合蛋白HuR为研究对象，探索了使用HCA研究蛋白质核质分布的实验方法，并与Western blotting法进行比较。

高维蛋白质波谱癌症数据特征提取吴文峰;刘毅慧【期刊名称】《生物信息学》【年(卷),期】2015(000)002【摘要】The analysis of high⁃dimensional cancer protein mass spectrometry data is full of trouble from high⁃dimensional data.We propose method for selecting the feature of high⁃dimensional cancer protein mass spectrometry data based on the wavelet analysis and principal component analysis,and solving the faled problems when we reduce the dimensionality of high⁃dimensional cancer protein mass spectrometry data. After feature selection, we use the support Vector Machine ( SVM) for classification. We use wavelet decomposition on 8-7-02 data set at second level,use different waveletbasis(db1,db3,db4,db6,db8,db10,haar) and classify them with the SVM, then we get different recognition rates:98. 18%, 98. 35%, 98. 04%, 98. 36%, 97. 89%, 97. 96%, 98. 20%. Improve the classification accuracy and the efficiency of time simultaneously.%高维蛋白质波谱癌症数据分析，一直面临着高维数据的困扰。

2D Depiction of Nonbonding Interactions forProtein ComplexesPENG ZHOU,1FEIFEI TIAN,2ZHICAI SHANG11Institute of Molecular Design&Molecular Thermodynamics,Department of Chemistry,Zhejiang University,Hangzhou310027,China2College of Bioengineering,Chongqing University,Chongqing400044,ChinaReceived7May2008;Revised25June2008;Accepted22July2008DOI10.1002/jcc.21109Published online22October2008in Wiley InterScience().Abstract:A program called the2D-GraLab is described for automatically generating schematic representation of nonbonding interactions across the protein binding interfaces.The inputfile of this program takes the standard PDB format,and the outputs are two-dimensional PostScript diagrams giving intuitive and informative description of the protein–protein interactions and their energetics properties,including hydrogen bond,salt bridge,van der Waals interaction,hydrophobic contact,p–p stacking,disulfide bond,desolvation effect,and loss of conformational en-tropy.To ensure these interaction information are determined accurately and reliably,methods and standalone pro-grams employed in the2D-GraLab are all widely used in the chemistry and biology community.The generated dia-grams allow intuitive visualization of the interaction mode and binding specificity between two subunits in protein complexes,and by providing information on nonbonding energetics and geometric characteristics,the program offers the possibility of comparing different protein binding profiles in a detailed,objective,and quantitative manner.We expect that this2D molecular graphics tool could be useful for the experimentalists and theoreticians interested in protein structure and protein engineering.q2008Wiley Periodicals,Inc.J Comput Chem30:940–951,2009Key words:protein–protein interaction;nonbonding energetics;molecular graphics;PostScript;2D-GraLabIntroductionProtein–protein recognition and association play crucial roles in signal transduction and many other key biological processes. Although numerous studies have addressed protein–protein inter-actions(PPIs),the principles governing PPIs are not fully under-stood.1,2The ready availability of structural data for protein complexes,both from experimental determination,such as by X-ray crystallography,and by theoretical modeling,such as protein docking,has made it necessary tofind ways to easily interpret the results.For that,molecular graphics tools are usually employed to serve this purpose.3Although a large number of software packages are available for visualizing the three-dimen-sional(3D)structures(e.g.PyMOL,4GRASP,5VMD,6etc.)and interaction modes(e.g.MolSurfer,7ProSAT,8PIPSA,9etc.)of biomolecules,the options for producing the schematic two-dimensional(2D)representation of nonbonding interactions for PPIs are very scarce.Nevertheless,a few2D graphics programs were developed to depict protein-small ligand interactions(e.g., LIGPLOT,10PoseView,11MOE,12etc.).These tools,however, are incapable of handling the macromolecular complexes.Some other available tools presenting macromolecular interactions in 2D level mainly include DIMPLOT,10NUCPLOT,13and MON-STER,14etc.Amongst,only the DIMPLOT can be used for aesthetically visualizing the nonbinding interactions of PPIs. However,such a program merely provides a simple description of hydrogen bonds,hydrophobic interactions,and steric clashes across the binding interfaces.In this article,we describe a new molecular graphics tool, called the two-dimensional graphics lab for biosystem interac-tions(2D-GraLab),which adopts the page description language (PDL)to intuitively,exactly,and detailedly reproduce the non-bonding interactions and energetics properties of PPIs in Post-Script page.Here,the following three points are the emphasis of the2D-GraLab:(i)Reliability.To ensure the reliability,the pro-grams and methods employed in2D-GraLab are all widely used in chemistry and biology community;(ii)Comprehensiveness. 2D-GraLab is capable of handling almost all the nonbonding interactions(and even covalent interactions)across binding Additional Supporting Information may be found in the online version of this article.Correspondence to:Z.Shang;e-mail:shangzc@interface of protein complexes,such as hydrogen bond,salt bridge,van der Waals(vdW)interaction,hydrophobic contact, p–p stacking,disulfide bond,desolvation effect,and loss of con-formational entropy.The outputted diagrams are diversiform, including individual schematic diagram and summarized sche-matic diagram;(iii)Artistry.We elaborately scheme the layout, color match,and page style for different diagrams,with the goal of producing aesthetically pleasing2D images of PPIs.In addi-tion,2D-GraLab provides a graphical user interface(GUI), which allows users to interact with this program and displays the spatial structure and interfacial feature of protein complexes (see .Fig.S1).Identifying Protein Binding InterfacesAn essential step in understanding the molecular basis of PPIs is the accurate identification of interprotein contacts,and based upon that,subsequent works are performed for analysis and lay-out of nonbonding mon methods identifyingprotein–protein binding interfaces include a Voronoi polyhedra-based approach,changes in solvent accessible surface area(D SASA),and various radial cutoffs(e.g.,closest atom,C b,andcentroid,etc.).152D-GraLab allows for the identification of pro-tein–protein binding interfaces at residue and atom levels.Identifying Binding Interfaces at Residue LevelAll the identifying interface methods at residue level belong toradial cutoff approach.In the radial cutoff approach,referencepoint is defined in advance for each residue,and the residues areconsidered in contact if their reference points fell within thedefined cutoff ually,the C a,C b,or centroid are usedas reference point.16–18In2D-GraLab,cutoff distance is moreflexible:cutoff distance5r A1r B1d,where r A and r B are residue radii and d is set by users(as the default d54A˚,which was suggested by Cootes et al.19).Identifying Binding Interfaces at Atom LevelAt atom level,binding interfaces are identified using closestatom-based radial cutoff approach20and D SASA-basedapproach.21For the closest atom-based radial cutoff approach,ifthe distance between any two atoms of two residues from differ-ent chains is less than a cutoff value,the residues are consideredin contact;In the D SASA-based approach,the SASA is calcu-lated twice to identify residues involved in a binding interface,once for the monomers and once for the complex,if there is achange in the SASA(D SASA)of a residue when going from themonomers to the dimer form,then it is considered involved inthe binding interface.In2D-GraLab,three manners are provided for visualizing thebinding interfaces,including spatial structure exhibition,residuedistance plot,and residue-pair contact map(see .Figs.S2–S4).Analysis and2D Layout of NonbondingInteractionsThe inputfile of2D-GraLab is standard PDB format,and the outputs are two-dimensional PostScriptfile giving intuitive and informative representation of the PPIs and their strengths, including hydrogen bond,salt bridge,vdW interaction,desolva-tion effect,ion-pair,side-chain conformational entropy(SCE), etc.The outputs are in two forms as individual schematic dia-gram and summarized schematic diagram.The individual sche-matic diagram is a detailed depiction of each nonbonding profile,whereas the summarized schematic diagram covers all nonbonding interactions and disulfide bonds across the binding interface.To produce the aesthetically high quality layouts,which pos-sess reliable and accurate parameters,several widely used pro-grams listed in Table1are employed in2D-GraLab to perform the core calculations and analysis of different nonbonding inter-actions.2D-GraLab carries out prechecking procedure for pro-tein structures and warns the structural errors,but not providing revision and refinement functions.Therefore,prior to2D-GraLab analysis,protein structures are strongly suggested to be prepro-cessed by programs such as PROCHECK(structure valida-tion),27Scwrl3(side-chain repair),28and X-PLOR(structure refinement).29Individual Schematic DiagramHydrogen BondThe program we use for analyzing hydrogen bonds across bind-ing interfaces is HBplus,23which calculates all possible posi-tions for hydrogen atoms attached to donor atoms which satisfy specified geometrical criteria with acceptor atoms in the vicinity. In2D-GraLab,users can freely select desired hydrogen bonds involving N,O,and/or S atoms.Besides,the water-mediated hydrogen bond is also given consideration.Bond strength of conventional hydrogen bonds(except those of water-mediated Table1.Standalone Programs Employed in2D-GraLab.Program FunctionReduce v3.0322Adding hydrogen atoms for proteinsHBplus v3.1523Identifying hydrogen bonds and calculatingtheir geometric parametersProbe v2.1224Identifying steric contacts and clashes at atomlevelMSMS v2.6125Calculating SASA values of protein atoms andresiduesDelphi v4.026Calculating Coulombic energy and reactionfield energy,determining electrostatic energyof ion-pairsDIMPLOT v4.110Providing application programming interface,users can directly set and executeDIMPLOT in the2D-GraLab GUI9412D Depiction of Nonbonding Interactions for Protein ComplexesFigure1.(a)Schematic representation of a conventional hydrogen bond and a water-mediated hydro-gen bond across the binding interface of IGFBP/IGF complex(PDB entry:2dsr).This diagram was produced using2D-Gralab.The conventional hydrogen bond is formed between the atom N(at the backbone of residue Leu69in chain B)and the atom OE1(at the side-chain of residue Glu3in chain I);The water-mediated hydrogen bond is formed between the atom ND1(at the side-chain of residue His5in chain B)and the atom O(at the backbone of residue Asp20in chain I),and because hydrogen positions of water are almost never known in the PDBfile,the water molecule,when serving as hydrogen bond donor,is not yet determined for its H...A length and D—H...A angle,denoted as mark ‘‘????.’’In this diagram,chains,residues,and atoms are labeled according to the PDB format.(b)Spa-tial conformation of the conventional hydrogen bond.(c)Spatial conformation of the water-mediated hydrogen bond.hydrogen bonds)is calculated using Lennard-Jones 8-6potential with angle weighting.30D U HB¼E m 3d m 8À4d m6"#cos 4h ðh >90 Þ(1)where d is the separation between the heavy acceptor atom andthe donor hydrogen atom in angstroms;E m ,the optimum hydro-gen-bond energy for the particular hydrogen-bonding atoms con-sidered;d m ,the optimum hydrogen-bond length for the particu-lar hydrogen-bonding atoms considered.E m and d m vary accord-ing to the chemical type of the hydrogen-bonding atoms.The hydrogen bond potential is set to zero when angle h 908.31Hydrogen bond parameters are taken from CHARMM force field (for N and O atoms)and Autodock (for S atom).32,33Figure 1a is the schematic representation of a conventional hydrogen bond and a water-mediated hydrogen bond across the binding interface of insulin-like growth factor-binding protein (IGFBP)/insulin-like growth factor (IGF)complex.In this dia-gram,abundant information about the hydrogen bond geometry and energetics properties is presented in a readily acceptant manner.Figures 1b and 1c are spatial conformations of the cor-responding conventional hydrogen bond and water-mediated hydrogen bond.Van der Waals InteractionThe small-probe approach developed in Richardson’s laboratory enables us to detect the all atom contact profile in protein pack-ing.2D-GraLab uses program Probe 24to realize this method to identity steric contacts and clashes on the binding interfaces.Word et al.pointed out that explicit hydrogen atoms can effec-tively improve Probe’s performance.24However,considering calculations with explicit hydrogen atoms are time-consuming,and implicit hydrogen mode is also possibly used in some cases;therefore,in 2D-GraLab,both explicit and implicit hydrogen modes are provided for users.In addition,2D-GraLab uses the Reduce 22to add hydrogen atoms for proteins,and this programis also developed in Richardson’s laboratory and can be wellcompatible with Probe.According to previous definition,vdW interaction between two adjacent atoms is classified into wide contact,close contact,small overlap,and bad overlap.24Typically,vdW potential function has two terms,a repulsive term and an attractive term.In 2D-GraLab,vdW interaction is expressed as Lennard-Jones 12-6potential.34D U SI ¼E m d m d 12À2d md6"#(2)where E m is the Lennard-Jones well depth;d m is the distance at the Lennard-Jones minimum,and d is the distance between two atoms.The Lennard-Jones parameters between pairs of different atom types are obtained from the Lorentz–Berthelodt combina-tion rules.35Atomic Lennard-Jones parameters are taken from Probe and AMBER force field.24,36Figure 2a was produced using 2D-GraLab and gives a sche-matic representation of steric contacts and clashes (overlaps)between the heavy chain residue Tyr131and two light chain res-idues Ser121and Gln124of cross-reaction complex FAB (the antibody fragment of hen egg lysozyme).By this diagram,we can obtain the detail about the local vdW interactions around the residue Tyr131.In contrast,such information is inaccessible in the 3D structural figure (Fig.2b).Desolvation EffectIn 2D-GraLab,program MSMS 25is used to calculate the SASA values of interfacial residues at atom level,and four atomic radii sets are provided for calculating the SASA,including Bondi64,Chothia75,Li98,and CHARMM83.32,37–39Bondi64is based on contact distances in crystals of small molecules;Chothia75is based on contact distances in crystals of amino acids;Li98is derived from 1169high-resolution protein crystal structures;CHARMM83is the atomic radii set of CHARMM force field.Desolvation free energy of interfacial residues is calculated using empirical additive model proposed by Eisenberg andFigure 2.(a)Schematic representation of steric contacts and overlaps between the residue Tyr131in heavy chain (chain H)and the surrounding residues Ser121and Gln124in light chain (chain L)of cross-reaction complex FAB (PDB entry:1fbi).This diagram was produced using 2D-Gralab in explicit hydrogen mode.In this diagram,interface is denoted by the broken line;Wide contact,close contact,small overlap,and bad overlap are marked by blue circle,green triangle,yellow square,and pink rhombus,respectively;Moreover,vdW potential of each atom-pair is given in the histogram,with the value measured by energy scale,and the red and blue indicate favorable (D U \0)and unfav-orable (D U [0)contributions to the binding,respectively;Interaction potential 20.324kcal/mol in the center circle denotes the total vdW contribution by residue Tyr131;Chains,residues,and heavy atoms are labeled according to the PDB format,and hydrogen atoms are labeled in Reduce format.(b)Spatial conformation of chain H residue Tyr131and its local environment.Green or yellow stands forgood contacts (green for close contact and yellow for slight overlaps \0.2A˚),blue for wide contacts [0.25A˚,hot pink spikes for bad overlaps !0.4A ˚.It is revealed that Tyr131is in an intensive clash with chain L Gln124,while in slight contact with chain L Ser121,which is well consistent with the 2D schematic diagram.9432D Depiction of Nonbonding Interactions for Protein Complexes944Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure2.(Legend on page943.)Maclachlam,40and the conformation of interfacial residues is assumed to be invariant during the binding process.D G dslv¼Xic i D A i(3)where the sum is over all the atoms;c i and D A i are the atomic solvation parameter(ASP)and the changes in solvent accessible surface area(D SASA)of atom i,respectively.Juffer et al.41 found that although desolvation free energies calculated from different ASP sets are linear correlation to each other,the abso-lute values are greatly different.In view of that,2D-GraLab pro-vides four ASP sets published in different periods:Eisenberg86, Kim90,Schiffer93,and Zhou02.40,42–44As shown in Figure3,the D SASA and desolvation free energy of interfacial residues in chain A of HLA-A*0201pro-tein complex during the binding process are reproduced in a rotiform diagram form using2D-GraLab.In this diagram,the desolvation free energy contributed by chain A is28.056kcal/ mol,and moreover,the D SASA value of each interfacial residue is also presented clearly.Ion-PairThere are six types of residue-pairs in the ion-pairs:Lys-Asp, Lys-Glu,Arg-Asp,Arg-Glu,His-Asp,and ually,ion-pairs include three kinds:salt bridge,NÀÀO bridge,and longer-range ion-pair,and found that most of the salt bridges are stabi-lizing toward proteins;the majority of NÀÀO bridges are stabi-lizing;the majority of the longer-range ion-pairs are destabiliz-ing toward the proteins.45The salt bridge can be further distin-guished as hydrogen-bonded salt bridge(HB-salt bridge)and nonhydrogen-bonded salt bridge(NHB-salt bridge or salt bridge).46In2D-GraLab,the longer-range ion-pair is neglected, and for short-range ion-pair,four kinds are defined:HB-salt bridge,NHB-salt bridge or salt bridge,hydrogen-bonded NÀÀO bridge(HB-NÀÀO bridge),and nonhydrogen-bonded N-O bridge (NHB-NÀÀO bridge or NÀÀO bridge).Although both the N-terminal and C-terminal residues of a given protein are also charged,the large degree offlexibility usually experienced by the ends of a chain and the poor structural resolution resulting from it.47Therefore,we preclude these terminal residues in the 2D-GraLab.A modified Hendsch–Tidor’s method is used for calculating association energy of ion-pairs across binding interfaces.48D G assoc¼D G dslvþD G brd(4)where D G dslv represents the sum of the unfavorable desolvation penalties incurred by the individual ion-pairing residues due to the change in their environment from a high dielectric solvent (water)in the unassociated state;D G brd represents the favorable bridge energy due to the electrostatic interaction of the side-chain charged groups.We usedfinite difference solutions to the linearized Poisson–Boltzmann equations in Delphi26to calculate the D G dslv and D G brd.Centroid of the ion-pair system is used as grid center,with temperature of298.15K(in this way,1kT50.593kcal/mol),and the Debye-Huckel boundary conditions are applied.49Considering atomic parameter sets have a great influ-ence on the continuum electrostatic calculations of ion-pair asso-ciation energy,502D-GraLab provides three classical atomic parameter sets for users,including PARSE,AMBER,and CHARMM.51–53Figure4is the schematic representation of four ion-pairs formed across the binding interface of penicillin acylase enzyme complex.This diagram clearly illustrates the information about the geometries and energetics properties of ion-pairs,such as bond length,centroid distance,association energy,and angle. The ion-pair angle is defined as the angle between two unit vec-tors,and each unit vector joins a C a atom and a side-chain charged group centroid in an ion-pairing residue.54In this dia-gram,the four ion-pairs,two HB-salt bridges,and two HB-NÀÀO bridges formed across the binding interface are given out. Association energies of the HB-salt bridges are both\21.5 kcal/mol,whereas that of the HB-NÀÀO bridges are all[20.5 kcal/mol.Therefore,it is believed that HB-salt bridge is more stable than HB-NÀÀO bridge,which is well consistent with the conclusion of Kumar and Nussinov.45,46Side-Chain Conformational EntropyIn general,SCE can be divided into the vibrational and the con-formational.55Comparison of several sets of results using differ-ent techniques shows that during protein folding process,the mean conformational free energy change(T D S)is1kcal/mol per side-chain or0.5kcal/mol per bond.Changes in vibrational entropy appear to be negligible compared with the entropy change resulted from the loss of accessible rotamers.56SCE(S) can be calculated quite simply using Boltzmann’s formulation.57S¼ÀRXip i ln p i(5)where R is the universal gas constant;The sum is taken over all conformational states of the system and p i is the probability of being in state i.Typical methods used for SCE calculations, include self-consistent meanfield theory,58molecular dynam-ics,59Monte Carlo simulation,60etc.,that are all time-consum-ing,thus not suitable for2D-GraLab.For that,the case is sim-plified,when we calculate the SCE of an interfacial residue,its local surrounding isfixed(adopting crystal conformation).In this way,SCE of each interfacial residue is calculated in turn.For the20coded amino acids,Gly,Ala,Pro,and Cys in disulfide bonds are excluded.57For other cases,each residue’s side-chain conformation is modeled as a rotamer withfinite number of discrete states.61The penultimate rotamer library used was developed by Lovell et al.,62as recommended by Dun-brack for the study of SCE.63For an interfacial residue,the potential E i of each rotamer i is calculated in both binding state and unbinding state,and subsequently,rotamer’s probability dis-tribution(p)of this residue is resulted by Boltzmann’s distribu-tion law,then the SCE in different states are solved out using eq.(5).The situation of rotamer i is defined as serious clash or nonclash:serious clash is the clash score of rotamer i more than a given threshold value,and then E i511;whereas for the9452D Depiction of Nonbonding Interactions for Protein Complexes946Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure3.Schematic representation of desolvation effect for interfacial residues in chain A of HLA-A*0201complex(PDB entry:1duz).This diagram was produced using2D-GraLab.In this diagram,the pie chart is equally divided,with each section indicates an interfacial residue in chain A;In a sec-tor,red1blue is the SASA of corresponding residue in unbinding state,the blue is in binding state,and the red is thus of D SASA;The green polygonal line is made by linking desolvation free energy ofeach interfacial residue,and at the purple circle,desolvation free energy is0(D U50),beyond thiscircle indicates unfavorable contributions to binding(D U[0),otherwise is favorable(D U\0);Inthe periphery,residue symbols are colored in red,blue,and black in terms of favorable,unfavorable,and neutral contributions to the binding,respectively;The SASA and desolvation free energy for eachinterfacial residue can be measured qualitatively by the horizontally black and green scales.[Colorfigure can be viewed in the online issue,which is available at .]Figure4.Four ion-pairs formed across the binding interface of penicillin acylase enzyme complex (PDB entry:1gkf).In thisfigure,left is2D schematic diagram produced using2D-GraLab,and posi-tively and negatively charged residues are colored in blue and red,respectively;Bridge-bonds formed between the charged atoms of ion-pairs are colored in green,blue,and yellow dashed lines for the hydrogen-bonded bridge,nonhydrogen-bonded bridge,and long-range interactions,respectively;The three parameters in bracket are ion-pair type,angle,and association energy.The right in thisfigure is the spatial conformations of corresponding ion-pairs.[Colorfigure can be viewed in the online issue, which is available at .]Figure5.(a)Loss of side-chain conformational entropy of chain B interfacial residues in HIV-1 reverse transcriptase complex(PDB entry:1rt1).This diagram was produced using2D-GraLab.In this diagram,the pie chart is equally divided,with each section indicates an interfacial residue in chain B; In a sector,side-chain conformational entropies in unbinding and binding state are colored in yellow and blue,respectively;The green polygonal line is made by linking conformational free energy of each interfacial residue;The conformational entropy and conformational free energy for each interfa-cial residue can be measured qualitatively by the horizontally black and green scales,respectively;In the periphery,residue symbols are colored in yellow,blue,and black in terms of favorable,unfavora-ble,and neutral contributions to binding,respectively.(b)The rotamers of chain B interfacial residues Lys20,Lys22,Tyr56,Asn136,Ile393,and Trp401in HIV-1reverse transcriptase complex.These rotamers were generated using2D-GraLab.[Colorfigure can be viewed in the online issue,which is available at .]9472D Depiction of Nonbonding Interactions for Protein Complexes948Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure5.(Legend on page947.)Figure6.The summarized schematic diagram of nonbonding interactions and disulfide bond across the interface of AIV hemagglutinin H5complex(PDB entry:1jsm).Length of chain A and chain B are321and160,represented as two bold horizontal lines.Interface parts in the bold lines are colored in orange,and residue-pairs in interactions are linearly linked;Conventional hydrogen bond,water-mediated hydrogen bond,ionpair,hydrophobic force,steric clash,p–p stacking,and disulfide bond are colored in aqua,bottle green,red,blue,purple,yellow,and brown,respectively;In the‘‘dumbbell shape’’symbols,residue-pair types and distances are also presented.[Colorfigure can be viewed in the online issue,which is available at .]9492D Depiction of Nonbonding Interactions for Protein Complexescase of nonclash,four potential functions are used in2D-Gra-Lab:(i)E i5E0,a constant61;(ii)statistical potential,the poten-tial energy E i of rotamer i is calculated from database-derived probability61;(iii)coarse-grained model,E i of rotamer i is esti-mated by atomic contact energies(ACE)64;and(iv)Lennard-Jones potential.58Loss of binding entropy of chain B interfacial residues in HIV-1reverse transcriptase complex is schematically repre-sented in Figure5a.Similar to desolvation effect diagram,loss of binding entropy is also presented in a rotiform diagram form. This diagram reveals that during the process of forming HIV-1 reverse transcriptase complex,the total loss of conformational free energy of chain B is9.14kcal/mol,indicating a strongly unfavorable contribution to binding(D G[0),and the average loss of conformational free energy for each residue is about0.3 kcal/mol,much less than those in protein folding(about1kcal/ mol56).Figure5b shows the rotamers of six interfacial residues in chain B.Summarized Schematic DiagramFigure6illustrates nonbonding interactions and disulfide bond formed across the binding interface of avian influenza virus (AIV)hemagglutinin H5.This protein is a dimer linked by a disulfide bond.In this diagram,conventional hydrogen bond, water-mediated hydrogen bond,ion-pair,hydrophobic force, steric clash,p–p stacking,and disulfide bond are represented in different colors.Hydrogen bonds,colored in aqua,are calculated by program HBplus.23Data in this diagram are the separation between the acceptor atom and the heavy donor atom.Water-mediated hydrogen bonds are colored in bottle green, also calculated by HBplus.23Ion-pairs,colored in red,include salt bridge and NÀÀO bridge,determined by the Kumar’s rule.45,46Data in this dia-gram are centroid distance of ion-pair.Hydrophobic forces are colored in blue.According to the D SASA rule,if the two apolar and/or aromatic interfacial resi-dues(Leu,Ala,Val,Ile,Met,Cys,Pro,Tyr,Phe,and Trp)are within the distance d\r A1r B12.8(r A and r B are side-chain radii,2.8is the diameter of water molecule),they are considered in hydrophobic contact.Data in this diagram are centroid–cent-roid separation between the two residues.Steric clashes are colored in purple.Here,only bad overlaps calculated by Probe24are presented.In2D-GraLab,explicit and implicit hydrogen modes are provided,hydrogen atoms in explicit hydrogern mode are added using Reduce.22Data in this diagram are the centroid–centroid separation when the two atoms are badly overlapped.p–p stacking are colored in yellow.Presently,studies on pro-tein stacking interactions are in lack.In2D-GraLab,p–p stack-ing is identified using the McGaughey’s rule,65i.e.,if the cent-roid–centroid separation between two aromatic rings is within 7.5A˚,they are regarded as p–p stacking(aromatic residues are Phe,Tyr,Trp,and His).This rule has been successfully adopted to study the p–p stacking across protein interfaces by Cho et al.66Besides,2D-GraLab also sets the constraints of stacking angle(dihedral angel between the planes of two aromatic rings).Data in this diagram are centroid–centroid separations between two aromatic rings in stacking state.Disulfide bonds are colored in brown,taken from the PDB records.Data in this diagram are the separations of two sulfide atoms.ConclusionsMost,if not all,biological processes are regulated through asso-ciation and dissociation of protein molecules and essentially controlled by nonbonding energetics.67Graphically-intuitive vis-ualization of these nonbonding interactions is an important approach for understanding the mechanism of a complex formed between two proteins.Although a large number of software packages are available for visualizing the3D structures,the options for producing schematic2D summaries of nonbonding interactions for a protein complex are comparatively few.In practice,the2D and3D visualization methods are complemen-tary.In this article,we have described a new2D molecular graphics tool for analyzing and visualizing PPIs from spatial structures,and the intended goal is to schematically present the nonbonding interactions stabilizing the macromolecular complex in a graphically-intuitive manner.We anticipate that renewed in-terest in automated generation of2D diagrams will significantly reduce the burden of protein structure analysis and make insights into the mechanism of PPIs.2D-GraLab is written in C11and OpenGL,and the output-ted2D schematic diagrams of nonbinding interactions are described in PostScript.Presently,2D-GraLab v1.0is available to academic users free of charge by contacting us. 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