A novel fingerprint for the characterization of protein folds

A novel fingerprint, defined without the use of distances, isintroduced to characterize protein folds. It is of the formof binary matrices whose elements are defined by angles betweenthe C=O direction, the backbone axis and the line connectingthe -carbons of the various residues. It is shown that matchesin the fingerprint matrices correspond to low r.m.s.d. Received November 15, 2002; revised July 25, 2003; accepted August 31, 2003.     

An empirical approach for structure-based prediction of carbohydrate-binding sites on proteins

A computer program system was developed to predict carbohydrate-bindingsites on three-dimensional (3D) protein structures. The programssearch for binding sites by referring to the empirical rulesderived from the known 3D structures of carbohydrate–proteincomplexes. A total of 80 non-redundant carbohydrate–proteincomplex structures were selected from the Protein Data Bankfor the empirical rule construction. The performance of theprediction system was tested on 50 known complex structuresto determine whether the system could detect the known bindingsites. The known monosaccharide-binding sites were detectedamong the best three predictions in 59% of the cases, whichcovered 69% of the polysaccharide-binding sites in the targetproteins, when the performance was evaluated by the overlapbetween residue patches of predicted and known binding sites. Received April 24, 2003; revised June 2, 2003; accepted June 10, 2003.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

Reduction of protein sequence complexity by residue grouping

It is well known that there are some similarities among variousnaturally occurring amino acids. Thus, the complexity in proteinsystems could be reduced by sorting these amino acids with similaritiesinto groups and then protein sequences can be simplified byreduced alphabets. This paper discusses how to group similaramino acids and whether there is a minimal amino acid alphabetby which proteins can be folded. Various reduced alphabets areobtained by reserving the maximal information for the simplifiedprotein sequence compared with the parent sequence using globalsequence alignment. With these reduced alphabets and simplifiedsimilarity matrices, we achieve recognition of the protein foldbased on the similarity score of the sequence alignment. Thecoverage in dataset SCOP40 for various levels of reduction onthe amino acid types is obtained, which is the number of homologouspairs detected by program BLAST to the number marked by SCOP40.For the reduced alphabets containing 10 types of amino acids,the ability to detect distantly related folds remains almostat the same level as that by the alphabet of 20 types of aminoacids, which implies that 10 types of amino acids may be thedegree of freedom for characterizing the complexity in proteins. Received November 20, 2002; revised March 10, 2003; accepted April 4, 2003.     

Prediction of protein secondary structure with a reliability score estimated by local sequence clustering

Most algorithms for protein secondary structure prediction arebased on machine learning techniques, e.g. neural networks.Good architectures and learning methods have improved the performancecontinuously. The introduction of profile methods, e.g. PSI-BLAST,has been a major breakthrough in increasing the prediction accuracyto close to 80%. In this paper, a brute-force algorithm is proposedand the reliability of each prediction is estimated by a z-scorebased on local sequence clustering. This algorithm is intendedto perform well for those secondary structures in a proteinwhose formation is mainly dominated by the neighboring sequencesand short-range interactions. A reliability z-score has beendefined to estimate the goodness of a putative cluster foundfor a query sequence in a database. The database for predictionwas constructed by experimentally determined, non-redundantprotein structures with <25% sequence homology, a list maintainedby PDBSELECT. Our test results have shown that this new algorithm,belonging to what is known as nearest neighbor methods, performedvery well within the expectation of previous methods and thatthe reliability z-score as defined was correlated with the reliabilityof prediction. This led to the possibility of making very accuratepredictions for a few selected residues in a protein with anaccuracy measure of Q₃ > 80%. The further development ofthis algorithm, and a nucleation mechanism for protein foldingare suggested. Received March 27, 2003; revised June 30, 2003; accepted August 22, 2003.     

SMoS: a database of structural motifs of protein superfamilies

The Structural Motifs of Superfamilies (SMoS) database providesinformation about the structural motifs of aligned protein domainsuperfamilies. Such motifs among structurally aligned multiplemembers of protein superfamilies are recognized by the conservationof amino acid preference and solvent inaccessibility and areexamined for the conservation of other features like secondarystructural content, hydrogen bonding, non-polar interactionand residue packing. These motifs, along with their sequenceand spatial orientation, represent the conserved core structureof each superfamily and also provide the minimal requirementof sequence and structural information to retain each superfamilyfold. Received April 25, 2003; revised September 9, 2003; accepted September 24, 2003.     

Have we seen all structures corresponding to short protein fragments in the Protein Data Bank? An update

Assembling short fragments from known structures has been awidely used approach to construct novel protein structures.To what extent there exist structurally similar fragments inthe database of known structures for short fragments of a novelprotein is a question that is fundamental to this approach.This work addresses that question for seven-, nine- and 15-residuefragments. For each fragment size, two databases, a query databaseand a template database of fragments from high-quality proteinstructures in SCOP20 and SCOP90, respectively, were constructed.For each fragment in the query database, the template databasewas scanned to find the lowest r.m.s.d. fragment among non-homologousstructures. For seven-residue fragments, there is a 99% probabilitythat there exists such a fragment within 0.7 Å r.m.s.d.for each loop fragment. For nine-residue fragments there isa 96% probability of a fragment within 1 Å r.m.s.d., whilefor 15-residue fragments there is a 91% probability of a fragmentwithin 2 Å r.m.s.d.. These results, which update previousstudies, show that there exists sufficient coverage to modeleven a novel fold using fragments from the Protein Data Bank,as the current database of known structures has increased enormouslyin the last few years. We have also explored the use of a gridsearch method for loop homology modeling and make some observationsabout the use of a grid search compared with a database searchfor the loop modeling problem. Received October 23, 2002; revised March 3, 2003; accepted March 30, 2003.     

A grafting approach to obtain site-specific metal-binding properties of EF-hand proteins

The EF-hand calcium-binding loop III from calmodulin was insertedwith glycine linkers into the scaffold protein CD2.D1 at threelocations to study site-specific calcium binding propertiesof EF-hand motifs. After insertion, the host protein retainsits native structure and forms a 1:1 metal–protein complexfor calcium and its analog, lanthanum. Tyrosine-sensitized Tb³⁺energy transfer exhibits metal binding and La³⁺ and Ca²⁺ competefor the metal binding site. The grafted EF-loop III in differentenvironments has similar La³⁺ binding affinities, suggestingthat it is largely solvated and functions independently fromthe host protein. Received May 25, 2002; revised January 23, 2003; accepted April 25, 2003.     

A long insertion reverts the functional effect of a substitution in acetylcholinesterase

Proteins are thought to undertake single substitutions, deletionsand insertions to explore the fitness landscape. Nevertheless,the ways in which these different kind of mutations act togetherto alter a protein phenotype remain poorly described. We introducedincrementally the single substitution W290A and a 26 amino acidlong insertion at the 297 location in the Nippostrongylus brasiliensisacetylcholinesterase B sequence and analysed in vitro the inducedchanges in the hydrolysis rate of three hemi-substrates: pirimicarb,paraoxon methyl and omethoate. The substitution decreased thehydrolysis rate of the three hemi-substrates. The insertiondid not influence this kinetic alteration induced by the substitutionfor the former hemi-substrate, but reverted it for the two others.These results show that two different kinds of mutations caninteract together to influence the direction of a protein’sadaptative walk on the fitness landscape. Received January 28, 2003; revised April 25, 2003; accepted June 6, 2003.     

Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases

Expression of recombinant proteins as translational fusionsis commonly employed to enhance stability, increase solubilityand facilitate purification of the desired protein. In general,such fusion proteins must be cleaved to release the mature proteinin its native form. The usefulness of the procedure dependson the efficiency and precision of cleavage and its cost perunit activity. We report here the development of a general procedurefor precise and highly efficient cleavage of recombinant fusionproteins using the protease chymosin. DNA encoding a modifiedpro-peptide from bovine chymosin was fused upstream of hirudin,carp growth hormone, thioredoxin and cystatin coding sequencesand expressed in a bacterial Escherichia coli host. Each ofthe resulting fusion proteins was efficiently cleaved at thejunction between the pro-peptide and the desired protein bythe addition of chymosin, as determined by activity, N-terminalsequencing and mass spectrometry of the recovered protein. Thesystem was tested further by cleavage of two fusion proteins,cystatin and thioredoxin, sequestered on oilbody particles obtainedfrom transgenic Arabidopsis seeds. Even when the fusion proteinwas sequestered and immobilized on oilbodies, precise and efficientcleavage was obtained. The precision, efficiency and low costof this procedure suggest that it could be used in larger scalemanufacturing of recombinant proteins which benefit from expressionas fusions in their host organism. Received June 5, 2003; revised August 1, 2003; accepted August 20, 2003.     

Structural diversity in the small heat shock protein superfamily: control of aggregation by the N-terminal region

The small heat shock protein superfamily, extending over allkingdoms, is characterized by a common core domain with variableN- and C-terminal extensions. The relatively hydrophobic N-terminusplays a critical role in promoting and controlling high-orderaggregation, accounting for the high degree of structural variabilitywithin the superfamily. The effects of N-terminal volume onaggregation were studied using chimeric and truncated proteins.Proteins lacking the N-terminal region did not aggregate abovethe tetramers, whereas larger N-termini resulted in large aggregates,consistent with the N-termini packing inside the aggregates.Variation in an extended internal loop differentiates typicalprokaryotic and plant superfamily members from their animalcounterparts; this implies different geometry in the dimericbuilding block of high-order aggregates. Received March 15, 2003; revised May 20, 2003; accepted September 4, 2003.     

循环流化床中颗粒旋转特性

利用由高速数字摄影设备及大功率激光构成的测试系统在一截面为200 mm×200 mm、高为4 m的冷态循环流化床实验台上进行了床内颗粒旋转特性的实验研究.对在距布风板3.54 m高度的稀相区的1/4截面内13个测试点拍摄获得的图像序列利用Matlab、PhotoShop和ACDSee软件进行分析处理,采用人工直接判别获得颗粒转速,用双帧频验证法进行颗粒转速校验.结果表明：循环流化床气固两相流中固相颗粒普遍存在旋转现象,截面边壁区内的颗粒平均转速高于中心区域;粒径小或径向速度大的颗粒,其平均转速较大,反之亦然;不规则颗粒的平均转速明显高于球[JP2]形颗粒;当空截面气体速度V_g=5 m&#8226;s^-1,固体质量循环流率G_s=1.5 kg&#8226;m^-2&#8226;s^-1,玻璃珠颗粒平均粒径d_p=0.5 mm时,颗粒转速最高可达2000 r&#8226;s^-1,平均转速300 r&#8226;s^-1.     

Immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragments

Three foldases—the apical domain of GroEL (mini-chaperone)and two oxidoreductases (DsbA and DsbC) from Escherichia coli—werestudied in refolding a protein with immunoglobulin fold (immunoglobulin-foldedprotein) that had been produced as inclusion bodies in E.coli.The foldases promoted the refolding of single-chain antibodyfragments from denaturant-solubilized and reduced inclusionbodies in vitro, and also effectively functioned as alternativesfor labilizing agent and oxidizing reagent in the stepwise dialysissystem. Immobilization of the oxidoreductases enhanced refoldingand recovery of functional single-chain antibody in the dialysissystem, suggesting that immobilized oxidoreductases can be usedas an effective additive for refolding immunoglobulin-foldedproteins in vitro. Received April 7, 2003; revised June 5, 2003; accepted June 6, 2003.     

Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potentialin biotechnology and medicine. We prepared several histidineanalogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine.These histidine analogues were assayed for translational activityin histidine-auxotrophic Escherichia coli strain UTH780. Weobserved that several histidine analogues, including our novelhistidine analogue, were efficiently incorporated into the proteinin vivo; however, other analogues were rejected. These resultssuggest that the hydrogen atom at a specific position seriouslyaffects incorporation. Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003.     

PROSPECT II: protein structure prediction program for genome-scale applications

A new method for fold recognition is developed and added tothe general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/).The new method (PROSPECT II) has four key features. (i) We havedeveloped an efficient way to utilize the evolutionary informationfor evaluating the threading potentials including singletonand pairwise energies. (ii) We have developed a two-stage threadingstrategy: (a) threading using dynamic programming without consideringthe pairwise energy and (b) fold recognition considering allthe energy terms, including the pairwise energy calculated fromthe dynamic programming threading alignments. (iii) We havedeveloped a combined z-score scheme for fold recognition, whichtakes into consideration the z-scores of each energy term. (iv)Based on the z-scores, we have developed a confidence index,which measures the reliability of a prediction and a possiblestructure–function relationship based on a statisticalanalysis of a large data set consisting of threadings of 600query proteins against the entire FSSP templates. Tests on severalbenchmark sets indicate that the evolutionary information andother new features of PROSPECT II greatly improve the alignmentaccuracy. We also demonstrate that the performance of PROSPECTII on fold recognition is significantly better than any othermethod available at all levels of similarity. Improvement inthe sensitivity of the fold recognition, especially at the superfamilyand fold levels, makes PROSPECT II a reliable and fully automatedprotein structure and function prediction program for genome-scaleapplications. Received March 20, 2003; revised June 28, 2003; accepted July 8, 2003.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspectsof protein design and engineering. Previously, we designed aseries of consensus ankyrin repeat proteins containing one,two, three and four identical repeats (1ANK, 2ANK, 3ANK and4ANK). These proteins, particularly 4ANK, are intended for useas a universal scaffold on which specific binding sites canbe constructed. Despite being well folded and extremely stable,4ANK is soluble only under acidic conditions. Designing interactionswith naturally occurring proteins requires the designed proteinto be soluble at physiological pH. Substitution of six leucineswith arginine on exposed hydrophobic patches on the surfaceof 4ANK resulted in increased solubility over a large pH range.Study of the pH dependence of stability demonstrated that 4ANKis one of the most stable ankyrin repeat proteins known. Inaddition, analogous leucine to arginine substitutions on thesurface of 2ANK allowed the partially folded protein to assumea fully folded conformation. Our studies indicate that replacementof surface-exposed hydrophobic residues with positively chargedresidues can significantly improve protein solubility at physiologicalpH. Received June 23, 2003; revised August 22, 2003; accepted August 28, 2003.     

The role of loop 7 in mediating calcineurin regulation

Calcineurin (CN) is a heterodimer protein consisting of a 61kDa catalytic subunit A and a 19 kDa regulatory subunit B. Itplays a critical role in T-cell activation and is involved inmany cellular processes. Regulation of CN is rather complex,including a number of factors such as divalent metal ions (primarilyCa²⁺ and Mn²⁺), calmodulin (CaM) and autoinhibition (AI) segment.Previously, we reported that a loop 7 deletion mutant (V314)in subunit A exhibited high phosphatase activity, although themechanism for the surprising activity enhancement and whetherthe activity change applies to other loop 7 residues were notknown. In order to probe the role of loop 7, we have carriedout extensive mutagenesis experiments, followed by systematicactivity assays under a number of regulatory conditions. Allmutants, including single deletion mutants Y315, N316 and doubledeletion mutant V314Y315, showed increased phosphatase activity.Significantly, activities of the mutants containing the V314deletion, namely V314 and V314Y315, were no longer regulatedby regulatory subunit B. These results, along with the structureanalysis, suggest that loop 7 as a whole plays an importantrole in mediating CN’s regulation through bridging theregulatory subunit and catalytic core and interaction with theAI segment of CN. Received April 30, 2003; revised September 9, 2003; accepted September 12, 2003.     

Using multiple sequence correlation analysis to characterize functionally important protein regions

Protein co-evolution under structural and functional constraintsnecessitates the preservation of important interactions. Identifyingfunctionally important regions poses many obstacles in proteinengineering efforts. In this paper, we present a bioinformatics-inspiredapproach (residue correlation analysis, RCA) for predictingfunctionally important domains from protein family sequencedata. RCA is comprised of two major steps: (i) identifying pairsof residue positions that mutate in a coordinated manner, and(ii) using these results to identify protein regions that interactwith an uncommonly high number of other residues. We hypothesizethat strongly correlated pairs result not only from contactingpairs, but also from residues that participate in conformationalchanges involved during catalysis or important interactionsnecessary for retaining functionality. The results show thathighly mobile loops that assist in ligand association/dissociationtend to exhibit high correlation. RCA results exhibit good agreementwith the findings of experimental and molecular dynamics studiesfor the three protein families that are analyzed: (i) DHFR (dihydrofolatereductase), (ii) cyclophilin, and (iii) formyl-transferase.Specifically, the specificity (percentage of correct predictions)in all three cases is substantially higher than those obtainedby entropic measures or contacting residue pairs. In addition,we use our approach in a predictive fashion to identify importantregions of a transmembrane amino acid transporter protein forwhich there is limited structural and functional informationavailable. Received December 1, 2002; revised February 7, 2003; accepted April 15, 2003.     

Simple consensus procedures are effective and sufficient in secondary structure prediction

We have analyzed the performance of majority voting on minimalcombination sets of three state-of-the-art secondary structureprediction methods in order to obtain a consensus prediction.Using three large benchmark sets from the EVA server, our resultsshow a significant improvement in the average Q₃ predictionaccuracy of up to 1.5 percentage points by consensus formation.The application of an additional trivial filtering procedurefor predicted secondary structure elements that are too short,does not significantly affect the prediction accuracy. Our analysisalso provides valuable insight into the similarity of the resultsof the prediction methods that we combine as well as the higherconfidence in consistently predicted secondary structure. Received March 7, 2003; revised May 24, 2003; accepted June 6, 2003.