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.     

Sequence and structural analysis of two designed proteins with 88% identity adopting different folds

Protein folding is a natural phenomenon by which a sequence of amino acids folds into a unique functional three-dimensional structure. Although the sequence code that governs folding remains a mystery, one can identify key inter-residue contacts responsible for a given topology. In nature, there are many pairs of proteins of a given length that share little or no sequence identity. Similarly, there are many proteins that share a common topology but lack significant evidence of homology. In order to tackle this problem, protein engineering studies have been used to determine the minimal number of amino acid residues that codes for a particular fold. In recent years, the coupling of theoretical models and experiments in the study of protein folding has resulted in providing some fruitful clues. He et al. have designed two proteins with 88% sequence identity, which adopt different folds and functions. In this work, we have systematically analysed these two proteins by performing pentapeptide search, secondary structure predictions, variation in inter-residue interactions and residue-residue pair preferences, surrounding hydrophobicity computations, conformational switching and energy computations. We conclude that the local secondary structural preference of the two designed proteins at the Nand C-terminal ends to adopt either coil or strand conformation may be a crucial factor in adopting the different folds. Early on during the process of folding, both proteins may choose different energetically favourable pathways to attain the different folds.     

Systematic fold recognition analysis of the sequences encoded by the genome of Mycoplasma pneumoniae

A robust tool for fold recognition was applied to the systematic analysis of the sequences below 200 residues encoded by the genome of Mycoplasma pneumoniae. The goal was to determine the additional information gain achievable in genome analysis by fold recognition, beyond the intrinsic limits of homology studies. A list of 124 sequences encoding for soluble proteins or domains not homologous to each other, or to proteins with known three-dimensional structure, was analyzed, resulting in significant Z scores for the energy of the structural models in 12 of these cases. This result indicates that systematic application of fold recognition techniques to the analysis of structurally unassigned soluble proteins can lead to high-confidence structural predictions with an efficiency of about 10%, a relevant contribution besides the complementary approach of homology analysis. Four of the predictions presented include mapping of the putative active site of the target sequence and lead to the detection of probable catalytic and binding residues. The data are discussed with reference to the functional implications of the structural models and to the results reported for the homologous genome of Mycoplasma genitalium.      

Global folding of proteins using a limited number of distance constraints

A Monte Carlo method is presented which can obtain the correcttertiary fold of a protein given the secondary structure andas few as three interactions between each secondary structureunit. This method was used to fold hemerythrin, Qavodoxin, bovinepancreatic trypsin inhibitor and a variable light domain froman immunoglobulin using the known secondary structures of theseproteins. Each of the proteins was successfully folded to obtaina structure resembling the initial X-ray structure. Reasonablesuccess was also achieved when using a secondary structure predictionalgorithm to assign secondary structure. The r.m.s. deviationsbetween the folded proteins and the crystal structures are inthe order of 3–5 A for the backbone coordinates. Evaluationof the r.m.s. deviations between members of the globin familyindicates that two equivalent overall folds may have r.m.s.deviations of this or even larger magnitude. The limiting numberof constraints necesssary to achieve the correct fold is discussed.     

Protein fold recognition by threading: comparison of algorithms and analysis of results

Optimal sequence threading can be used to recognize membersof a library of protein folds which are closely related in 3-Dstructure to the native fold of an input test sequence, evenwhen the test sequence is not significantly homologous to thesequence of any member of the fold library. The methods providean alignment between the residues of the test sequence and theresidue positions in a template fold. This alignment optimizesa score function, and the predicted fold is the highest scoringmember of the library of folds. Most score functions containa pairwise interaction energy term. This, coupled with the needto introduce gaps into the alignment, means that the optimizationproblem is NP hard. We report a comparison between two heuristicoptimization algorithms used in the literature, double dynamicprogramming and an iterative algorithm based on the so-calledfrozen approximation. These are compared in terms of both theranking of likely folds and the quality of the alignment produced.     

Homology modelling and molecular dynamics studies of human placental tissue protein 13 (galectin-13)

The primary structure of the newly sequence analysed placentaltissue protein 13 (PP13) was highly homologous to several membersof the ß-galactoside-binding S-type lectin (galectin)family. By homology modelling, the three-dimensional structureof PP13 was built based on high-resolution crystal structuresof homologues and also their characteristic `jellyroll' foldwas found in the case of PP13. Our model has been depositedin the Brookhaven Protein Data Bank. By multiple sequence alignmentand structure-based secondary structure prediction, we underlinedthe structural similarity of PP13 with its homologues. The secondarystructure of PP13 was identical with `proto-type' galectinsconsisting of a five- and a six-stranded ß-sheet,joined by two     

Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus

The hyperthermophilic Archaeon Archaeoglobus fulgidus has agene (AF1763) which encodes a thermostable carboxylesterasebelonging to the hormone-sensitive lipase (HSL)-like group ofthe esterase/lipase family. Based on secondary structure predictionsand a secondary structure-driven multiple sequence alignmentwith remote homologous proteins of known three-dimensional structure,we previously hypothesized for this enzyme the     

5‐Hydroxymethyl‐2′‐Deoxyuridine Residues in the Thrombin Binding Aptamer: Investigating Anticoagulant Activity by Making a Tiny Chemical Modification

We report an investigation into analogues of the thrombin binding aptamer (TBA). Individual thymidines were replaced by the unusual residue 5‐hydroxymethyl‐2′‐deoxyuridine (hmU). This differs from the canonical thymidine by a hydroxyl group on the 5‐methyl group. NMR and CD data clearly indicate that all TBA derivatives retain the ability to fold into the “chair‐like” quadruplex structure. The presence of the hmU residue does not significantly affect the thermal stability of the modified aptamers compared to the parent, except for analogue H9 , which showed a marked increase in melting temperature. Although all TBA analogues showed decreased affinities to thrombin, H3 , H7 , and H9 proved to have improved anticoagulant activities. Our data open up the possibility to enhance TBA biological properties, simply by introducing small chemical modifications.     

PPII Helical Peptidomimetics Templated by Cation–π Interactions

Poly‐proline type II (PPII) helical PXXP motifs are the recognition elements for a variety of protein–protein interactions that are critical for cellular signaling. Despite development of protocols for locking peptides into α‐helical and β‐strand conformations, there remains a lack of analogous methods for generating mimics of PPII helical structures. We describe herein a strategy to enforce PPII helical secondary structure in the 19‐residue TrpPlexus miniature protein. Through sequence variation, we showed that a network of cation–π interactions could drive the formation of PPII helical conformations for both peptide and N‐substituted glycine peptoid residues. The achievement of chemically diverse PPII helical scaffolds provides a new route towards discovering peptidomimetic inhibitors of protein–protein interactions mediated by PXXP motifs.     

Filtering remote homologues using predicted structural information

Finding homologues for a given protein plays a major role in predicting the protein's structure and function. However, it is still difficult to find remote homologues with low sequence similarity, even with advanced sequence search methods. We propose a simple filtering method that uses predicted structural information, pertaining to secondary structures and solvent accessibilities. It filters the more promising homologues from the many candidate proteins obtained by PSI-BLAST with a less stringent threshold E-value. The final decision is made by a simple linear discrimination method, considering the E-value of PSI-BLAST and the statistical significance scores of structural matches. An in-house neural network program is used for the prediction of secondary structures and solvent accessibilities for both the query and library proteins. The performance of our filtering method was evaluated by the cross-validation method, using the SCOP superfamily relationship as the correct standard. Coverage-reliability plots show that our filtering method clearly improves the performance of PSI-BLAST. The secondary structure improves PSI-BLAST better than the solvent accessibilities, but the combination of these two features with PSI-BLAST leads to the best result. The advantage of our method is its easy implementation with fewer parameters to be tuned and faster computation. We also discuss its performance with predicted and observed secondary structures.     

Identifying sequence-structure pairs undetected by sequence alignments

We examine how effectively simple potential functions previouslydeveloped can identify compatibilities between sequences andstructures of proteins for database searches. The potentialfunction consists of pairwise contact energies, repulsive packingpotentials of residues for overly dense arrangement and short-rangepotentials for secondary structures, all of which were estimatedfrom statistical preferences observed in known protein structures.Each potential energy term was modified to represent compatibilitiesbetween sequences and structures for globular proteins. Pairwisecontact interactions in a sequence–structure alignmentare evaluated in a mean field approximation on the basis ofprobabilities of site pairs to be aligned. Gap penalties areassumed to be proportional to the number of contacts at eachresidue position, and as a result gaps will be more frequentlyplaced on protein surfaces than in cores. In addition to minimumenergy alignments, we use probability alignments made by successivelyaligning site pairs in order by pairwise alignment probabilities.The results show that the present energy function and alignmentmethod can detect well both folds compatible with a given sequenceand, inversely, sequences compatible with a given fold, andyield mostly similar alignments for these two types of sequenceand structure pairs. Probability alignments consisting of mostreliable site pairs only can yield extremely small root meansquare deviations, and including less reliable pairs increasesthe deviations. Also, it is observed that secondary structurepotentials are usefully complementary to yield improved alignmentswith this method. Remarkably, by this method some individualsequence–structure pairs are detected having only 5–20%sequence identity.     

Melectin: a novel antimicrobial peptide from the venom of the cleptoparasitic bee Melecta albifrons

A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-His-Met-Lys-NH(2) by Edman degradation and ESI-QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both gram-positive and -negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content alpha-helices, which indicates that melectin can adopt an amphipathic alpha-helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the alpha-helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N- and C-terminal-shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity.     

BioS2Net: Holistic Structural and Sequential Analysis of Biomolecules Using a Deep Neural Network

Background: For decades, the rate of solving new biomolecular structures has been exceeding that at which their manual classification and feature characterisation can be carried out efficiently. Therefore, a new comprehensive and holistic tool for their examination is needed. Methods: Here we propose the Biological Sequence and Structure Network (BioS2Net), which is a novel deep neural network architecture that extracts both sequential and structural information of biomolecules. Our architecture consists of four main parts: (i) a sequence convolutional extractor, (ii) a 3D structure extractor, (iii) a 3D structure-aware sequence temporal network, as well as (iv) a fusion and classification network. Results: We have evaluated our approach using two protein fold classification datasets. BioS2Net achieved a 95.4% mean class accuracy on the eDD dataset and a 76% mean class accuracy on the F184 dataset. The accuracy of BioS2Net obtained on the eDD dataset was comparable to results achieved by previously published methods, confirming that the algorithm described in this article is a top-class solution for protein fold recognition. Conclusions: BioS2Net is a novel tool for the holistic examination of biomolecules of known structure and sequence. It is a reliable tool for protein analysis and their unified representation as feature vectors.     

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.     

Simple intrasequence difference (SID) analysis: an original method to highlight and rank sub-structural interfaces in protein folds. Application to the folds of bovine pancreatic trypsin inhibitor, phospholipase A2, chymotrypsin and carboxypeptidase A

We present Simple Intrasequence Difference (SID) analysis, anovel bioinformatic technique designed to help comprehend theproperties of protein fold topologies. The analysis grades numericallyevery residue position in a given protein 3D structure accordingto the topological situation of the position in the folded chain.This results in an expression of the potential contributionof each residue position and its vicinity towards the integrityof the molecular conformation. Contiguous highly graded residuesdelineate the sub-structural interfaces that arise from thepresence within the molecular fold of discrete domains and sub-domains.This comprehensive rendering of the internal arrangement ofchain interfacing helps predict the potential for site-specificinductions (e.g. via mutations or ligand binding) of conformationalchange in the fold. Whereas SID analysis of single folds canconvey an idea of the basic potential for topological adjustmentin the protein family, comparative SID analysis of related foldsfocuses attention on those areas of the family fold where evolutionarychanges, activation events and ligand binding have had the mosttopological impact. For demonstration, SID analysis is appliedto the folds of pancreatic trypsin inhibitor (Kunitz), phospholipaseA₂, chymotrypsin and carboxypeptidase A. We find that many ofthe potentially vulnerable sub-structural interfaces tend tobe protected in the fold interior, in many cases stabilisedby disulfide bridges spanning the interface. However, the mostprominent interfaces tend to be externally accessible, withoutremedial stabilisation by disulfide bridges. These latter interfacesare associated so closely with the known functional sites thatalterations to the interfacial juxtapositions should influencerecognition and catalytic behaviour directly. This shows howside chain mutations, chemical modifications and binding eventsremote from the sites can nevertheless adjust, via interfacialrealignment, the conformations and emergent properties of thesites. The close association also provides clear opportunitiesfor interfacial rearrangements to follow intermolecular recognitionevents in the sites, facilitating translation of the bindinginto adjustment of the molecular conformation in areas distantfrom the sites. As a direct consequence of the topological arrangements,a large proportion of the molecular structure has the capacityto shape the character of the functional sites and, conversely,binding at these sites has the potential to radiate influenceto the rest of the molecule. For the enzymes considered, theevidence is consistent with the possibility that primary andsecondary binding by the substrate enhances catalytic efficiencyby imposing conformational change upon the catalytic centrevia adjustments to the fold. This influence may be expressedas favourable adjustment of the catalytic geometry, transitionstate ensemble, energy propagation pathway, or as a physicalstrain exerted on the substrate bond to be cleaved. The scaleof the adjustments, and their importance to the mechanisms,may have been seriously underestimated.     

Halogenation of drugs enhances membrane binding and permeation

Halogenation of drugs is commonly used to enhance membrane binding and permeation. We quantify the effect of replacing a hydrogen residue by a chlorine or a trifluoromethyl residue in position C-2 of promazine, perazine, and perphenazine analogues. Moreover, we investigate the influence of the position (C-6 and C-7) of residue CF(3) in benzopyranols. The twelve drugs are characterized by surface activity measurements, which yield the cross-sectional area, the air-water partition coefficient, and the critical micelle concentration. By using the first two parameters (A(D) and K(aw)) and the appropriate membrane packing density, the lipid-water partition coefficients, are calculated in excellent agreement with the lipid-water partition coefficients measured by means of isothermal titration calorimetry for small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Replacement of a hydrogen residue by a chlorine and a trifluoromethyl residue enhances the free energy of partitioning into the lipid membrane, on average by deltaG(lw) approximately -1.3 or -4.5 kJ mol(-1), respectively, and the permeability coefficient by a factor of approximately 2 or approximately 9, respectively. Despite exhibiting practically identical hydrophobicities, the two benzopyranol analogues differ in their permeability coefficients by almost an order of magnitude; this is due to their different cross-sectional areas at the air-water and lipid-water interfaces.     

Transmembrane helix prediction: a comparative evaluation and analysis

The prediction of transmembrane (TM) helices plays an important role in the study of membrane proteins, given the relatively small number (approximately 0.5% of the PDB) of high-resolution structures for such proteins. We used two datasets (one redundant and one non-redundant) of high-resolution structures of membrane proteins to evaluate and analyse TM helix prediction. The redundant (non-redundant) dataset contains structure of 434 (268) TM helices, from 112 (73) polypeptide chains. Of the 434 helices in the dataset, 20 may be classified as 'half-TM' as they are too short to span a lipid bilayer. We compared 13 TM helix prediction methods, evaluating each method using per segment, per residue and termini scores. Four methods consistently performed well: SPLIT4, TMHMM2, HMMTOP2 and TMAP. However, even the best methods were in error by, on average, about two turns of helix at the TM helix termini. The best and worst case predictions for individual proteins were analysed. In particular, the performance of the various methods and of a consensus prediction method, were compared for a number of proteins (e.g. SecY, ClC, KvAP) containing half-TM helices. The difficulties of predicting half-TM helices suggests that current prediction methods successfully embody the two-state model of membrane protein folding, but do not accommodate a third stage in which, e.g., short helices and re-entrant loops fold within a bundle of stable TM helices.     

Structures of the Apo and FAD‐Bound Forms of 2‐Hydroxybiphenyl 3‐monooxygenase (HbpA) Locate Activity Hotspots Identified by Using Directed Evolution

The FAD‐dependent monooxygenase HbpA from Pseudomonas azelaica HBP1 catalyses the hydroxylation of 2‐hydroxybiphenyl (2HBP) to 2,3‐dihydroxybiphenyl (23DHBP). HbpA has been used extensively as a model for studying flavoprotein hydroxylases under process conditions, and has also been subjected to directed‐evolution experiments that altered its catalytic properties. The structure of HbpA has been determined in its apo and FAD‐complex forms to resolutions of 2.76 and 2.03 Å, respectively. Comparisons of the HbpA structure with those of homologues, in conjunction with a model of the reaction product in the active site, reveal His48 as the most likely acid/base residue to be involved in the hydroxylation mechanism. Mutation of His48 to Ala resulted in an inactive enzyme. The structures of HbpA also provide evidence that mutants achieved by directed evolution that altered activity are comparatively remote from the substrate‐binding site.     

基于Modbus-GPRS的换热站无线远程监控系统设计及应用

由GPRS通信网络和PLC控制器组成的无线远程自动监控系统,解决了居民供、换热控制系统无人值守的问题。PLC现场控制站实时采集换热站温度、流量及压力等参数,按照预先设定的目标实现二次网供热和补水自动控制,并通过GPRS无线数据传输网络与调度中心进行通信,从而实现调度中心对热网各换热站进行远程自动化控制和管理。