Understanding the relationship between the primary structure of proteins and their amyloidogenic propensity: clues from inclusion body formation

Amyloid formation is dependent to a considerable extent on the amino acid sequence of the protein. The present study delineates certain sequence-dependent features that are correlated with amyloidogenic propensity. The analyses indicate that amyloid formation is favored by lower thermostability and increased half-life of the protein. There seems to be a certain degree of bias in the composition of order-promoting amino acids in the case of amyloidogenic proteins. Based on these parameters, a prediction function for the amyloidogenic propensity of proteins has been created. The prediction function has been found to rationalize the reported effect of certain mutations on amyloid formation. It seems that a higher sheet propensity of residues that constitute the first seven residues of a helical structure in a protein might increase the propensity for a helix to sheet transition in that region under denaturing conditions.     

Amide inequivalence in the fibrillar assembly of islet amyloid polypeptide

Amyloid fibers are aggregated, yet highly ordered, beta-sheet-rich assemblies of misfolded proteins. Order is established in such systems following profiles indicative of nucleation-dependent assembly. Nucleation dependence suggests that specific interactions, such as long-range contacts and/or strand registration, are critical to establishing initial fiber structure. Here, we show that amino acids at selected positions participate in key interactions that modulate the pathway of amyloid fiber formation by the hormone, islet amyloid polypeptide (IAPP). Specifically, we investigated the role of amide side-chain interactions in the process of IAPP assembly. We mutated five of the asparagine side chains in IAPP and assessed their effects on the kinetics of assembly. We find that the asparagine amide side chains strongly dictate the ability of IAPP to form fibers. In particular, the elimination of two specific asparagines results in near and total loss of amyloid, respectively. Interestingly, the two asparagines are located in a recently identified domain with alpha-helical bias. These sensitivities are unusual for IAPP, as IAPP is generally tolerant to mutation. Here, we demonstrate this mutational tolerance by assessing 10 alterations at five distinct sites. In all cases, the constructs form fibers on timescales perturbed by less than a factor of two compared with wild-type protein. These findings indicate the presence of key specific interactions that are the determinants of IAPP amyloid formation.     

Molecular modeling of the amyloid-{beta}-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer'sbrain is the amyloid-ß-peptide (Aß), a proteolyticproduct of a membrane precursor protein. Previous structuralstudies have found different conformations for the Aßpeptide depending on the solvent and pH used. In general, theyhave suggested an -helix conformation at the N-terminal domainand a ß-sheet conformation for the C-terminal domain.The structure of the complete Aß peptide (residues 1–40)solved by NMR has revealed that only helical structure is presentin Aß. However, this result cannot explain the large ß-sheetAß aggregates known to form amyloid under physiologicalconditions. Therefore, we investigated the structure of Aßby molecular modeling based on extensive homology using theSmith and Waterman algorithm implemented in the MPsrch program(Blitz server). The results showed a mean value of 23% identitywith selected sequences. Since these values do not allow a clearhomology to be established with a reference structure in orderto perform molecular modeling studies, we searched for detailedhomology. A 28% identity with an /ß segment of a triosephosphateisomerase (TIM) from Culex tarralis with an unsolved three-dimensionalstructure was obtained. Then, multiple sequence alignment wasperformed considering Aß, TIM from C.tarralis and anotherfive TIM sequences with known three-dimensional structures.We found a TIM segment with secondary structure elements inagreement with previous experimental data for Aß. Moreover,when a synthetic peptide from this TIM segment was studied invitro, it was able to aggregate and to form amyloid fibrils,as established by Congo red binding and electron microscopy.The Aß model obtained was optimized by molecular dynamicsconsidering ionizable side chains in order to simulate Aßin a neutral pH environment. We report here the structural implicationsof this study.     

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrP^Sc is the only identifiablecomponent of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakobdisease in humans are transmissible, degenerative neurologicaldiseases caused by prions. Standard predictive strategies havebeen used to analyze the secondary structure of the prion proteinin conjunction with Fourier analysis of the primary sequencehydrophobicities to detect potential amphipathic regions. Severalhydrophobic segments, a proline- and glycine-rich repeat regionand putative glycosylation sites are incorporated into a modelfor the integral membrane topology of PrP. The complete aminoacid sequences of the hamster, human and mouse prion proteinsare compared and the effects of residue substitutions upon thepredicted conformation of the polypeptide chain are discussed.While PrP has a unique primary structure, its predicted secondarystructure shares some interesting features with the serum amyloidA proteins. These proteins undergo a post-translational modificationto yield amyloid A, molecules that share with PrP the abilityto polymerize into birefringent filaments. Our analyses mayexplain some experimental observations on PrP, and suggest furtherstudies on the properties of the scrapie and cellular PrP isoforms.     

Local polarity analysis: a sensitive method that discriminates between native proteins and incorrectly folded models

The evaluation of calculated protein structures is an importantstep in the protein design cycle. Known criteria for this assessmentof proteins are the polar and apolar, accessible and buriedsurface area, electrostatic interactions and other interactionsbetween the protein atoms (e.g. HO, S-S),atomic packing, analysisof amino acid environment and surface charge distribution. Weshow that a powerful test of accuracy of protein structure canbe derived by analysing the water contact of atoms and additionallytaking into account their polarity. On the basis of estimatedreference values of the polar fraction of typical globular proteinswith known structure (mean, SD and distribution), the evaluationof misfolded structures can be improved significantly. The referencevalues are derived by moving windows of different length (3–99amino acid residues) over the amino acid sequence. Model proteins,which are included in the Brookhaven protein structure databank,deliberately misfolded proteins, hypothetical proteins and predictedprotein structures are diagnosed as at least partially incorrectlyfolded. The local fault, mostly observed, is that polar groupsare buried too frequently in the interior of the protein. Thedatabase-derived quantities are useful in screening the designedproteins prior to experimentation and may also be useful inthe assessment of errors in the experimentally determined proteinstructures.     

Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction

De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction.     

Protein modelling using a chimera reference protein derived from exons

Bovine pancreatic /S-trypsin (PDB ID-code: 1TPO) which is registeredin the Brookhaven Protein Data Bank (PDB) consists of four exons.The results of homology searches for each exon in the PDB showedthat homologous proteins were tonin (PDB ID-code: 1TON), ratmast cell protease (PDB ID-code: 3RP2_A), kaffikrein A (PDBID-code: 2PKA_B) and kallikrein A (2PKA_B) respectively. Thus,for the three-dimensional structure prediction of 1TPO, a chimeraprotein was constructed from the three proteins mentioned aboveand the 3-D structure prediction was performed using this chimerareference protein. The modelled structure of 1TPO was energeticallyoptimized by molecular mechanics and molecular dynamics simulationand was compared with its X-ray crystal structure registeredin the PDB. The root mean square deviations (r.m.s.d.) of mainchain atoms and the neighbouring active site (5 sphere fromHis57, AsplO2 and Serl95) between the modelled structure andthe X-ray structure were 1.66 and 0.94 respectively. Porcinepancreatic elastase (PDB ID-code: 3EST) which is registeredin the PDB was used as the reference protein and the modelledstructure from 3EST was also compared with the X-ray data. Ther.m.s.d. of main chain atoms and that of the active site were2.14 and 1.18 respectively. These results dearly support thepropriety of this method using the chimera reference protein.     

The RPSP: Web server for prediction of signal peptides

The RPSP is a fast web service for detection of signal peptides in proteins. The method uses neural networks trained on known signal peptides from the Swiss-Prot protein database. The web server works either on prokaryotic and eukaryotic proteins or without specifying an organism type. The accuracy of the web server is similar to other available computational prediction web services, yet because of its speed and portability the method can be easily applied to whole proteomes. The RPSP web server is available at http://rpsp.bioinfo.pl.     

A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm

We present a novel method that predicts transmembrane domainsin proteins using solely information contained in the sequenceitself. The PRED-TMR algorithm described, refines a standardhydrophobicity analysis with a detection of potential termini(`edges', starts and ends) of transmembrane regions. This allowsone both to discard highly hydrophobic regions not delimitedby clear start and end configurations and to confirm putativetransmembrane segments not distinguishable by their hydrophobiccomposition. The accuracy obtained on a test set of 101 non-homologoustransmembrane proteins with reliable topologies compares wellwith that of other popular existing methods. Only a slight decreasein prediction accuracy was observed when the algorithm was appliedto all transmembrane proteins of the SwissProt database (release35). A WWW server running the PRED-TMR algorithm is availableat http://o2.db.uoa.gr/PRED-TMR/     

Non-polar nuclei in fungal microbial RNases

An application of a previously proposed method for the analysisof the non-polar structure of proteins is presented. A detailedanalysis of the composition and properties of non-polar nucleiand microclusters of fungal microbial ribonucleases has beenperformed on the basis of the 3-D structures of RNase T1 andrelated proteins. Three hydrophobic nuclei were found in thesestructures. It has been shown that all residues in non-polarnuclei have high homology ({small tilde}89%). Residues in thenuclei are practically fully buried in the interior of a molecule.Detailed analysis of non-polar nuclei properties shows thatthese nuclei determine the hydrophobic core of a protein andthe location and role of each residue in the non-polar interiorof proteins. In addition it was found that there are variableresidues not only on the surface of a protein but on the surfaceof the nuclei inside the protein and between the nuclei andthat there is a consistent region in all proteins, the hydrophobic-nuclei. An evaluation of the stability of non-polar nuclei,the conservation of their compositions and their positions inthe protein globule, allows one to assume that these three nucleiplay an important functional role in the stability and foldingof molecules of RNases and possibly can be considered as independentstructural elements of 3-D structures of these proteins.     

Detection of non-topological motifs in protein structures

We present an efficient technique for the comparison of proteinstructures. The algorithm uses a vector representation of thesecondary structure elements and searches for spatial configurationsof secondary structure elements in proteins. In such recurringprotein folds, the order of the secondary structure elementsin the protein chains is disregarded. The method is based onthe geometric hashing paradigm and implements approaches originatingin computer vision. It represents and matches the secondarystructure element vectors in a 3-D translation and rotationinvariant manner. The matching of a pair of proteins takes onaverage under 3 s on a Silicon Graphics Indigo2 workstation,allowing extensive all-against-all comparisons of the data setof non-redundant protein structures. Here we have carried outsuch a comparison for a data set of over 500 protein molecules.The detection of recurring topological and non-topological,secondary structure element order-independent protein foldsmay provide further insight into evolution. Moreover, as theserecurring folding units are likely to be conformationalHy favourable,the availability of a data set of such topological motifs canserve as a rich input for threading routines. Below, we describethis rapid technique and the results it has obtained. Whilesome of the obtained matches conserve the order of the secondarystructure elements, others are entirely order independent. Asan example, we focus on the results obtained for Che Y, a signaltransduction protein, and on the profilin-ß-actincomplex. The Che Y molecule is composed of a five-stranded,parallel ß-sheet flanked by five helices. Here weshow its similarity with the Escherichia coli elongation factor,with L-arabinose binding protein, with haloalkane dehalogenaseand with adenylate kinase. The profilin–ß-actincontains an antiparallel ß-pleated sheet with -helicaltermini. Its similarities to lipase, fructose disphosphataseand ß-lactamase are displayed.     

Molecular dynamics studies on the thermostability of family 11 xylanases

Twelve members of the family 11 xylanases, including both mesophilic and thermophilic proteins, were studied using molecular dynamics (MD). Simulations of xylanases were carried out in an explicit water environment at four different temperatures, 300, 400, 500 and 600 K. A difference in thermotolerance between mesophilic and thermophilic xylanases became clear: thermophilic xylanases endured heat in higher simulation temperatures better than mesophilic ones. The unfolding pathways seemed to be similar for all simulations regardless of the protein. The unfolding initiates at the N-terminal region or alternatively from the alpha-helix region and proceeds to the 'finger region'. Unfolding of these regions led to denaturated structures within the 4.5 ns simulation at 600 K. The results are in agreement with experimental mutant studies. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. The MD analysis suggests regions in the protein structure which are more unstable and thus potential targets for mutation experiments to improve thermostability.     

A novel search method for protein sequence-structure relations using property profiles

In protein engineering and design it is very important thatresidues can be inspected in their specific environment. A standardrelational database system cannot serve this purpose adequatelybecause it cannot handle relations between individual residues.With SCAN3D we introduce a new database system for integratedsequence and structure analysis of proteins. It uses the relationalparadigm wherever possible. Its main power, however, stems fromthe ability to retrieve stretches of consecutive residues withcertain properties by comparing a property profile with allstretches of residues in the database, exploiting the orderedcharacter of proteins. In doing so, it bypasses the large numberof join operations that would be required by relational databasesystems. An additional advantage of using property profile matchingis that searches can be carried out allowing a pre-set numberof mismatches. Also, as the database is read-only, SCAN3D doesnot need interactive data update mechanisms. Queries typicalof a molecular engineering environment are demonstrated withspecific examples: analysis of peptides that induce local structure,analysis of site-dependent rotamers and residue-residue contactanalysis     

An object-oriented database for protein structure analysis

An object-oriented database system has been developed whichis being used to store protein structure data. The databasecan be queried using the logic programming language Prolog orthe query language Daplex. Queries retrieve information by navigatingthrough a network of objects which represent the primary, secondaryand tertiary structures of proteins. Routines written in bothProlog and Daplex can integrate complex calculations with theretrieval of data from the database, and can also be storedin the database for sharing among users. Thus object-orienteddatabases are better suited to prototyping applications andanswering complex queries about protein structure than relationaldatabases. This system has been used to find loops of varyinglength and anchor positions when modelling homologous proteinstructures.     

Secondary structure prediction for modelling by homology

An improved method of secondary structure prediction has beendeveloped to aid the modelling of proteins by homology. Selecteddata from four published algorithms are scaled and combinedas a weighted mean to produce consensus algorithms. Each consensusalgorithm is used to predict the secondary structure of a proteinhomologous to the target protein and of known structure. Bycomparison of the predictions to the known structure, accuracyvalues are calculated and a consensus algorithm chosen as theoptimum combination of the composite data for prediction ofthe homologous protein. This customized algorithm is then usedto predict the secondary structure of the unknown protein. Inthis manner the secondary structure prediction is initiallytuned to the required protein family before prediction of thetarget protein. The method improves statistical secondary structureprediction and can be incorporated into more comprehensive systemssuch as those involving consensus prediction from multiple sequencealignments. Thirty one proteins from five families were usedto compare the new method to that of Garnier, Osguthorpe andRobson (GOR) and sequence alignment. The improvement over GORis naturally dependent on the similarity of the homologous protein,varying from a mean of 3% to 7% with increasing alignment significancescore.     

Families of proteins with similar total structure identified using statistical geometry

Two quantities, herein defined as the displacement and the uniqueness,describe quantitatively the total structural difference betweentwo proteins. All possible pairs of protein chains in the Brookhavendatabase are characterized in terms of these quantities. Pairsof proteins with small values of both displacement and uniqueness,in secondary and super-secondary configuration spaces, havesimilar total structure. Proteins related in this fashion aregrouped into 158 families of similar total structure. The radialdisplacement function is herein defined to characterize therelative displacement of a residue from the center of the massof its protein. In addition, the residue backbone structurefunction is also defined to characterize the local configurationof the protein main chain in the vicinity of the residue. Thevalues of polynomial convolutions of these two functions arecharacteristic of a particular tertiary structure type. Thesepolynomial convolutions, together with other structural parameters,are used to verify the structural similarity of proteins belongingto the families indicated above. Variations in these polynomialconvolutions illustrate the amount and sequence location ofstructural deviations between proteins of the same family.     

Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through Hydrophobic Cluster Analysis and molecular modelling

A 21 kDa protein purified from bovine brain cytosol was previouslydescribed as a hydrophobic ngand binding protein; however, itsaccurate biological function remained still uncertain. In orderto get further information about its potential biological role,an extended prediction of its secondary and three dimensionalstructures was undertaken. We describe here a process whichpermitted us to discover a structural homology between the 21kDa protein and the N-domain of yeast phosphoglycerate kinase(PGK). This process is based on comparing the 21 kDa proteinwith all the proteins presenting a slight homology, by usingthe Hydrophobic Cluster Analysis (HCA) method. According tothe observed similarity between the N-domaln of yeast PGK andthe 21 kDa protein, we built a model which was shown to possessa potential binding site for nucleotides. Moreover, the modelobtained presents three-dimensional (3D) structure similaritywith adenylate kinase. These results suggest two main hypotheses:(0 the 21 kDa protein may belong to the kinase family; (ii)the binding of a nucleotide could imply a modification of the3D structure of the 21 kDa protein that can promote the transferof hydrophobic ligands to the plasma membrane. Meanwhile, verificationof these hypotheses has been in part performed experimentally:the 21 kDa protein binds MgATP as well as, to a lesser extent,phosphoglycerate.     

Analysis of protein conformational characteristics related to thermostability

The thermal stability of proteins was studied, 195 single aminoacid residue replacements reported elsewhere being analysedfor several protein conformational characteristics: type ofresidue replacement; conservative versus nonconservative substitution;replacement being in a homologous stretch of amino acid residues;change in hydrogen bond, van der Waals and secondary structurepropensities; solvent-accessible versus inaccessible replacement;type of secondary structure involved in the substitution; thephysico-chemical characteristics to which the thermostabilityenhancement can be attributed; and the relationship of the replacementsite to the folding intermediates of the protein, when known.From the above analyses, some general rules arise which suggestwhere amino acid substitutions can be made to enhance proteinthermostability: substitutions are conservative according tothe Dayhoff matrix; mainly occur on conserved stretches of residues;preferentially occur on solvent-accessible residues; maintainor enhance the secondary structure propensity upon substitution;contribute to neutralize the dipole moment of the caps of helicesand strands; and tend to increase the number of potential hydrogenbonding or van der Waals contacts or improve hydrophobic packing.