Showing papers in "Protein Engineering in 2002"

An analysis of protein domain linkers: their classification and role in protein folding

Abstract: Recent advances in protein engineering have come from creating multi-functional chimeric proteins containing modules from various proteins. These modules are typically joined via an oligopeptide linker, the correct design of which is crucial for the desired function of the chimeric protein. Here we analyse the properties of naturally occurring inter-domain linkers with the aim to design linkers for domain fusion. Two main types of linker were identified; helical and non-helical. Helical linkers are thought to act as rigid spacers separating two domains. Non-helical linkers are rich in prolines, which also leads to structural rigidity and isolation of the linker from the attached domains. This means that both linker types are likely to act as a scaffold to prevent unfavourable interactions between folding domains. Based on these results we have constructed a linker database intended for the rational design of linkers for domain fusion, which can be accessed via the Internet at http://mathbio.nimr.mrc.ac.uk.

Prediction of proteasome cleavage motifs by neural networks.

Abstract: We present a predictive method that can simulate an essential step in the antigen presentation in higher vertebrates, namely the step involving the proteasomal degradation of polypeptides into fragments which have the potential to bind to MHC Class I molecules. Proteasomal cleavage prediction algorithms published so far were trained on data from in vitro digestion experiments with constitutive proteasomes. As a result, they did not take into account the characteristics of the structurally modified proteasomes--often called immunoproteasomes--found in cells stimulated by gamma-interferon under physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC Class I ligands, i.e. the specificity of the immunoproteasome is different than the constitutive proteasome. The tools developed in this study in combination with a predictor of MHC and TAP binding capacity should give a more complete prediction of the generation and presentation of peptides on MHC Class I molecules. Here we demonstrate that such an approach produces an accurate prediction of the CTL the epitopes in HIV Nef. The method is available at www.cbs.dtu.dk/services/NetChop/.

Protein Design is NP-hard

Abstract: Biologists working in the area of computational protein design have never doubted the seriousness of the algorithmic challenges that face them in attempting in silico sequence selection. It turns out that in the language of the computer science community, this discrete optimization problem is NP-hard. The purpose of this paper is to explain the context of this observation, to provide a simple illustrative proof and to discuss the implications for future progress on algorithms for computational protein design.

The consensus concept for thermostability engineering of proteins: further proof of concept

Abstract: Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.

Occurrence, conformational features and amino acid propensities for the pi-helix.

Abstract: The most abundant helix type in proteins is the alpha-helix, accounting for about 31% of amino acid secondary structure states, while the 3(10)-helix accounts for about 4%. The pi-helix appears to be extremely rare and is considered to be unstable. Existing secondary structure definition methods find very few within the Protein Data Bank. Using an improved pi-helix definition algorithm to search a non-redundant subset of high-resolution and well-refined protein structures, we found that almost every tenth protein contained a pi-helix. This enabled us to show for the first time that the pi-helix has structural parameters that are different from the hypothesized model values. It also has distinctive amino acid preferences and it is conserved within functionally related proteins. Features that may contribute to the stability of the pi-helical structure have also been identified. In addition to hydrogen bonds, several other factors contribute to the stability of pi-helices. The pi-helix may have some functional advantages over other helical structures. Thus, we describe cases where the side chains of functionally important residues at every fourth position within a pi-helix could be aligned and brought close together in a way that would not be allowed by any other helix type.

Molecular dynamics simulations as a tool for improving protein stability

Abstract: Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.

On filtering false positive transmembrane protein predictions.

Abstract: While helical transmembrane (TM) region prediction tools achieve high (>90%) success rates for real integral membrane proteins, they produce a considerable number of false positive hits in sequences of known nontransmembrane queries. We propose a modification of the dense alignment surface (DAS) method that achieves a substantial decrease in the false positive error rate. Essentially, a sequence that includes possible transmembrane regions is compared in a second step with TM segments in a sequence library of documented transmembrane proteins. If the performance of the query sequence against the library of documented TM segment-containing sequences in this test is lower than an empirical threshold, it is classified as a non-transmembrane protein. The probability of false positive prediction for trusted TM region hits is expressed in terms of E-values. The modified DAS method, the DAS-TMfilter algorithm, has an unchanged high sensitivity for TM segments ( approximately 95% detected in a learning set of 128 documented transmembrane proteins). At the same time, the selectivity measured over a non-redundant set of 526 soluble proteins with known 3D structure is approximately 99%, mainly because a large number of falsely predicted single membrane-pass proteins are eliminated by the DAS-TMfilter algorithm.

Geometric criteria of hydrogen bonds in proteins and identification of "bifurcated" hydrogen bonds.

Abstract: Empirical criteria for identification of hydrogen bonds were analyzed to produce a set of geometrically consistent criteria. For a data set of 30 structures, application of a set of purely geometrical criteria, along with exclusion of abnormal backbone conformations, also excluded a common interaction of Ser/Thr side chains with Asp/Glu side chains ([ST]/[DE] pairs). These interactions were termed ‘bifurcated hydrogen bonds’, which implies delocalization of a positively charged hydrogen of hydroxyl between the two acceptor atoms of the carboxylic group. These ‘bifurcated’ interactions are among the most common packing patterns for [ST]/[DE] pairs of side chains. Therefore, the identification of hydrogen bonds cannot be based on geometrical criteria only and requires introduction of some physico-chemical criteria.

Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-β-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH

Abstract: We studied the effects of increase in the number of surface arginines on the enzyme activity and stability of Trichoderma reesei endo-1,4-beta-xylanase II. The number of arginines was increased in two mutant series. The first set contained six arginines on different sides of the protein surface. These arginines had no significant effect on the thermostability. However, the optimal pH region became narrower. Another series of five arginines was engineered into the 'Ser/Thr surface', formed of part of the double-layered beta-sheet located on one side of the 'right-hand-like' xylanase. These mutations shifted the activity profile to the alkaline region by approximately 0.5-1.0 pH units. In addition, the arginines on the Ser/Thr surface increased the enzyme activity at high temperature, although the enzyme stability in the absence of substrate decreased significantly at 50-55 degrees C. In the presence of the substrate, the thermostability increased 4-5-fold at 60-65 degrees C. Thus, the substrate neutralized the destabilizing effect of Ser/Thr surface arginines and revealed a stabilizing effect of the same mutations. The stabilizing effect of arginines at high temperatures was seen clearly only when five arginines were introduced into the Ser/Thr surface.

Aromatic-aromatic interactions in and around α-helices

Abstract: To understand the role of aromatic-aromatic interactions in imparting specificity to the folding process, the geometries of four aromatic residues with different sequence spacing, located in alpha-helices or five residues from helical ends, interacting with each other have been elucidated. The geometry is found to depend on the sequence difference. Specific interactions (C-H...pi and N-H...pi) which result from this geometry may cause a given pair of residues (such as Phe-His) with a particular sequence difference to occur more than expected. The most conspicuous residue in an aromatic pair in the context of helix stability is His, which is found at the last (C1) position or the two positions (Ncap and Ccap) immediately flanking the helix. An alpha-helix and a contiguous 3(10)-helix or two helices separated by a non-helical residue can have interacting aromatic pairs, the geometry of interaction and the relative orientation between the helices being rather fixed. Short helices can also have interacting residues from either side.

Gene expression response to misfolded protein as a screen for soluble recombinant protein.

Abstract: Proper protein folding is key to producing recombinant proteins for structure determination. We have examined the effect of misfolded recombinant protein on gene expression in Escherichia coli. Comparison of expression patterns indicates a unique set of genes responding to translational misfolding. The response is in part analogous to heat shock and suggests a translational component to the regulation. We have further utilized the expression information to generate reporters responsive to protein misfolding. These reporters were used to identify properly folded recombinant proteins and to create soluble domains of insoluble proteins for structural studies.

Quantifying the accessible surface area of protein residues in their local environment

Abstract: The quantification of the packing of residues in proteins and docking of ligands to macromolecules is important in understanding protein stability and drug design The number of atoms in contact (within a distance of 45 A) can be used to describe the local environment of a residue As this number increases, the accessible surface area (ASA) of the residue decreases exponentially and the variation can be described in terms of an exponential equation of the form y = a 1 exp(-x/a 2 ), each residue having its own set of parameters a 1 and a 2 , which also depend on whether the whole residue or just the side chain is considered Hydrophobic and hydrophilic residues can be distinguished on the basis of both the average number of surrounding atoms and the variation of ASA For a given number of partner atoms, a comparison of the observed ASA with the expected value obtained from the equation provides a method of assessing the goodness of packing of the residue in a protein structure or its importance in the binding of a ligand The equation provides a method to estimate the ASA of a protein molecule and the average relative accessibilities of different residues, the latter being inversely correlated with hydrophobicity values

Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity.

Abstract: The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.

Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain.

Abstract: The tenth fibronectin type III domain of human fibronectin (FNfn10) is a small, monomeric β-sandwich protein, similar to the immunoglobulins. We have developed small antibody mimics, ‘monobodies’, using FNfn10 as a scaffold. We initially altered two loops of FNfn10 that are structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. In order to assess the possibility of utilizing other loops in FNfn10 for target binding, we determined the effects of the elongation of each loop on the conformational stability of FNfn10. We found that all six loops of FNfn10 allowed the introduction of four glycine residues while retaining the global fold. Insertions in the AB and FG loops exhibited very small degrees of destabilization, comparable to or less than predicted entropic penalties due to the elongation, suggesting the absence of stabilizing interactions in these loops in wildtype FNfn10. Insertions in the BC, CD and DE loops, respectively, resulted in modest destabilization. In contrast, the EF loop elongation was highly destabilizing, consistent with previous studies showing the presence of stabilizing interactions in this loop. These results suggest that all loops, except for the EF loop, can be used for engineering a binding site, thus demonstrating excellent properties of the monobody scaffold.

Impact of single-residue mutations on the structure and function of ovispirin/novispirin antimicrobial peptides.

Abstract: We studied three model antibacterial peptides that resembled the N-terminal 18 amino acids of SMAP-29, an alpha-helical, antimicrobial peptide of sheep. Although the parent compound, ovispirin-1 (KNLRR IIRKI IHIIK KYG), was potently antimicrobial, it was also highly cytotoxic to human epithelial cells and hemolytic for human erythrocytes. Single residue substitutions to ovispirin-1 yielded two substantially less cytotoxic peptides (novispirins), with intact antimicrobial properties. One of these, novispirin G-10, differed from ovispirin-1 only by containing glycine at position 10, instead of isoleucine. The other, novispirin T-7, contained threonine instead of isoleucine at position 7. We determined the three-dimensional solution structures of all three peptides by circular dichroism spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy. Although all retained an amphipathic helical structure in 2,2,2-trifluoroethanol, they manifested subtle fine-structural changes that evidently impacted their activities greatly. These findings show that simple structural modifications can 'fine-tune' an antimicrobial peptide to minimize unwanted cytotoxicity while retaining its desired activity.

A conformational analysis of Walker motif A (GXXXXGKT (S)) in nucleotide-binding and other proteins

Abstract: The sequence GXXXXGKT/S, popularly known as Walker motif A, is widely believed to be the site for binding nucleotides in many proteins. Examination of the crystal structures in the Protein Data Bank showed that about half of the examples having these sequences do not bind or use nucleotides. Data analyses showed 92 different Walker sequences of the variable quartet (XXXX). Ramachandran angles in this segment revealed conformational similarity in the group of 45 proteins, known to bind or utilize nucleotides. The conformations of this segment in other proteins differ widely and it is not known whether they play any role in their functions. A flip of a peptide unit at different locations, with little change in the backbone conformation was noted in nine pairs of these proteins having same Walker sequence. An examination of the immediate neighborhood of the Walker sequence indicates that this region is preceded by a $\beta$-strand and followed by an $\alpha$-helix, resulting in the motif $\beta–W–\alpha$, an invariant feature amongst nucleotide-binding proteins.

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis.

Abstract: Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.

Acetylcholinesterase engineering for detection of insecticide residues

Abstract: To detect traces of insecticides in the environment using biosensors, we engineered Drosophila acetylcholinesterase (AChE) to increase its sensitivity and its rate of phosphorylation or carbamoylation by organophosphates or carbamates. The mutants made by site-directed mutagenesis were expressed in baculovirus. Different strategies were used to obtain these mutants: (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site, (iii) Ala-scan analysis of amino acids lining the active site gorge, (iv) mutagenesis at positions detected as important for sensitivity in the Ala-scan analysis and (v) combination of mutations which independently enhance sensitivity. The results highlighted the difficulty of predicting the effect of mutations; this may be due to the structure of the site, a deep gorge with the active serine at the bottom and to allosteric effects between the top and the bottom of the gorge. Nevertheless, the use of these different strategies allowed us to obtain sensitive enzymes. The greatest improvement was for the sensitivity to dichlorvos for which a mutant was 300-fold more sensitive than the Drosophila wild-type enzyme and 288 000-fold more sensitive than the electric eel enzyme, the enzyme commonly used to detect organophosphate and carbamate.

Prediction of the structure of human Janus kinase 2 (JAK2) comprising JAK homology domains 1 through 7

Abstract: A theoretical model of human Janus kinase 2 (JAK2) comprising all seven Janus homology domains is presented. The model was generated by application of homology modelling approaches. The three-dimensional structure contains, starting from the N-terminus, FERM (4.1, ezrin, radixin, moesin), SH2 (Src homology region 2), tyrosine kinase-like, and tyrosine kinase domains. The predicted inter-domain orientation in JAK2 is discussed and the currently existing mutational data for Janus kinases are evaluated. Structural details of the SH2 and the FERM domains are presented. The predictions indicate that the SH2 domain is not fully functional. A number of hydrophobic amino acids of the FERM domain that are predicted to be involved in the constitutive association with the cytokine receptors are highlighted. The model gives new insights into the structure-function relationship of this important protein, and areas that could be investigated by mutation studies are highlighted.

Protein sequence comparison based on the wavelet transform approach

Abstract: A protein's chemical properties, the chain conformation, the function of the protein and its species specificity are determined by the information contained in the amino acid sequence. Proteins of similar functions have at some level sequential identical amino acid sequences. The closer the phylogenetic relationship, the more similar are the sequences. To find the similarities between two or more protein sequences is of great importance for protein sequence analysis. The differences in the amino acid sequences permit the construction of a family tree of evolution. In this work, a comparison method was devised that is capable of analysing a protein sequence 'hierarchically', i.e. it can examine a protein sequence at different spatial resolutions. Based on a wavelet decomposition of protein sequences and a cross-correlation study, a sequence-scale similarity concept is proposed for generating a similarity vector, which renders the comparison of two sequences feasible at different spatial resolutions (scales). This new similarity concept is an expansion of the conventional sequence similarity, which only takes into account the local pairwise amino acid match and ignores the information contained in coarser spatial resolutions.

Prediction of heterodimerization interfaces of G-protein coupled receptors with a new subtractive correlated mutation method

Abstract: Recent studies employing differential epitope tagging, selective immunoprecipitation of receptor complexes and fluorescence or bioluminescence resonance energy transfer techniques provide direct evidence for heterodimerization between both closely and distantly related members of the G-protein coupled receptor (GPCR) family. Since heterodimerization appears to play a role in modulating agonist affinity, efficacy and/or trafficking properties, the molecular models of GPCRs required to understand receptor function must consider these oligomerization hypotheses. To advance knowledge in this field, we present here a computational approach based on correlated mutation analysis and the structural information contained in three-dimensional molecular models of the transmembrane regions of GPCRs built using the rhodopsin crystal structure as a template. The new subtractive correlated mutation method reveals likely heterodimerization interfaces amongst the different alternatives for the positioning of two tightly packed bundles of seven transmembrane domains next to each other in contact heterodimers of GPCRs. Predictions are applied to GPCRs in the class of opioid receptors. However, in the absence of a known structure of any GPCR dimer, the features of the method and predictions are also illustrated and analyzed for a dimeric complex of known structure.

An engineered IN-1 Fab fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity

Abstract: The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/κ antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure‐function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 F ab fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 Fab fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 µM. The affinity of the unmutated IN-1 Fab fragment was 8-fold lower. The engineered Fab fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered Fab fragment as a reagent for promoting axonal regeneration in vivo.

Modifying the chain-length selectivity of the lipase from Burkholderia cepacia KWI-56 through in vitro combinatorial mutagenesis in the substrate-binding site.

Abstract: The mature lipase of Burkholderia cepacia KWI-56 was synthesized in an enzymatically active form using an in vitro Escherichia coli S30 coupled transcription/translation system by expressing the mature lipase gene (rlip) in the presence of its specific activator. To investigate the substrate specificity of the lipase comprehensively, a large number of mutant lipases were constructed and analyzed in a high throughput manner by combining overlapping PCR and in vitro protein synthesis. In this paper, Phe119 and Leu167, which are located in the acyl portion of the substratebinding pocket of the lipase of B.cepacia KWI-56, were substituted with six hydrophobic amino acid residues by the in vitro combinatorial mutagenesis. The wild-type and 35 mutant genes amplified by PCR were directly used as templates for the in vitro transcription/translation. The acyl chain-length selectivity of the in vitro expressed lipases against p-nitrophenyl butyrate, p-nitrophenyl caprylate and p-nitrophenyl palmitate, was compared by their relative hydrolysis rates. Two mutant lipases, L167V and F119A/ L167M, which showed a significant shift in substrate selectivity were further expressed in vivo and refolded in vitro. It was found that L167V raised its preference for the short-chain ester, whereas F119A/L167M improved its selectivity for the long-chain ester.

Sequence clustering strategies improve remote homology recognitions while reducing search times

Abstract: Sequence databases are rapidly growing, thereby increasing the coverage of protein sequence space, but this coverage is uneven because most sequencing efforts have concentrated on a small number of organisms. The resulting granularity of sequence space creates many problems for profile-based sequence comparison programs. In this paper, we suggest several strategies that address these problems, and at the same time speed up the searches for homologous proteins and improve the ability of profile methods to recognize distant homologies. One of our strategies combines database clustering, which removes highly redundant sequence, and a two-step PSI-BLAST (PDB-BLAST), which separates sequence spaces of profile composition and space of homology searching. The combination of these strategies improves distant homology recognitions by more than 100%, while using only 10% of the CPU time of the standard PSI-BLAST search. Another method, intermediate profile searches, allows for the exploration of additional search directions that are normally dominated by large protein sub-families within very diverse families. All methods are evaluated with a large fold-recognition benchmark.

Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling

Abstract: Evolutionary protein engineering is now proceeding to a new stage in which novel technologies, besides the conventional point mutations, to generate a library of proteins, are required. In this context, a novel method forshuffling and rearranging DNA blocks (leading to protein libraries) is reported. A cycle of processes for producing combinatorial diversity was devised and designated Y-ligation-based block shuffling (YLBS). Methodological refinement was made by applying it to the shuffling of module-sized and amino acid-sized blocks. Running three cycles of YLBS with module-sized GFP blocks resulted in a high diversity of an eight-block shuffled library. Partial shuffling of the central four blocks of GFP was performed to obtain in-effect shuffled protein, resulting in an intact arrangement. Shuffling of amino acid monomer-sized blocks by YLBS was also performed and a diversity of more than 10 1 0 shuffled molecules was attained. The deletion problems encountered during these experiments were shown to he solved by additional measures which tame type IIS restriction enzymes. The frequency of appearance of each block was skewed but was within a permissible range. Therefore, YLBS is the first general method for generating a huge diversity of shuffled proteins, recombining domains, exons and modules with ease.

In silico identification, structure prediction and phylogenetic analysis of the 2′-O-ribose (cap 1) methyltransferase domain in the large structural protein of ssRNA negative-strand viruses

Abstract: The Escherichia coli RrmJ gene product has recently been shown to be the 23S rRNA:U2552 specific 2'-O-ribose methyltransferase (MTase) (RrmJ). Its structure has been solved and refined to 1.5 A resolution, demonstrating conservation of the three-dimensional fold and key catalytic side chains with the vaccinia virus VP39 protein, which functions as an mRNA 5'm(7)G-cap-N-specific 2'-O-ribose MTase. Using the amino acid sequence of RrmJ as an initial probe in an iterative search of sequence databases, we identified a homologous domain in the sequence of the L protein of non-segmented, negative-sense, single-stranded RNA viruses. The plausibility of the prediction was confirmed by homology modeling and checking whether important residues at substrate/ligand-binding sites were conserved. The predicted structural compatibility and the conservation of the active site between the novel putative MTase domain and genuine 2'-O-ribose MTases, together with the available results of biochemical studies, strongly suggest that this domain is a 5'm(7)G-cap-N-specific 2'-O-ribose MTase (i.e. the cap 1 MTase). Evolutionary relationships between these proteins are also discussed.

Proteomics analysis of carbon-starved Mycobacterium smegmatis: induction of Dps-like protein.

Abstract: Mycobacterium tuberculosis is a globally successful pathogen, infecting more than one third of total world's population. These bacteria have the remarkable ability to persist in the host for long periods of time unrecognized by the immune system and then to re-emerge later in life causing the disease. The physiology of such persistent or dormant bacilli is not very well characterized. Some evidence suggests that the dormant bacilli survive in a nutrient-deprived state that is similar to the stationary phase of the bacteria with respect to gene expression and physiology. Under this assumption we have studied the survival of Mycobacterium smegmatis in carbon starvation conditions as a model for mycobacterial persistence. M.smegmatis, being a fast-growing strain, serves as a good model to study starvation responses. Using the two-dimensional electrophoresis-based proteomics approach, we identified a protein which was found to be expressed preferentially under starvation conditions. This protein is homologous to a family of proteins called Dps (DNA binding Protein from Starved cells) that are known to protect DNA under various kinds of environmental stresses and its existence has, so far, not been reported in mycobacteria. Upon expression and purification of this protein, we observed that it has non-specific DNA-binding ability. Formation of a cage-like dodecamer structure is a characteristic feature of Dps. Using comparative modelling we were able to show that Dps from M.smegmatis could form a dodecamer structure similar to the crystal structure of Dps from Escherichia coli.

Engineering streptococcal protein G for increased alkaline stability

Abstract: Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.

Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase.

Abstract: The Bacillus subtilis strain ATCC 21332 produces the lipoheptapeptide surfactin, a highly potent biosurfactant synthesized by a large multimodular peptide synthetase. We report the genetic engineering of the surfactin biosynthesis resulting in the production of a novel lipohexapeptide with altered antimicrobial activities. A combination of in vitro and in vivo recombination approaches was used to construct a modified peptide synthetase by eliminating a large internal region of the enzyme containing a complete amino acid incorporating module. The remaining modules adjacent to the deletion were recombined at different highly conserved sequence motifs characteristic of amino acid incorporating modules of peptide synthetases. The primary goal of this work was to identify permissive fusion sites suitable for the engineering of peptide synthetase genes by genetic recombination. Analysis of the rearranged enzymes after purification from B.subtilis and from the heterologous host Escherichia coli revealed that the selection of the recombination site is of crucial importance for a successful engineering. Only the recombination at a specific HHIIDGVS sequence motif resulted in an active peptide synthetase. The expected lipohexapeptide was produced in vivo and first evidence of a reduced toxicity against erythrocytes and an enhanced lysis of Bacillus licheniformis cells was shown.

A divide and conquer approach to fast loop modeling

Abstract: We describe a fast ab initio method for modeling local segments in protein structures. The algorithm is based on a divide and conquer approach and uses a database of precalculated look-up tables, which represent a large set of possible conformations for loop segments of variable length. The target loop is recursively decomposed until the resulting conformations are small enough to be compiled analytically. The algorithm, which is not restricted to any specific loop length, generates a ranked set of loop conformations in 20-180 s on a desktop PC. The prediction quality is evaluated in terms of global RMSD. Depending on loop length the top prediction varies between 1.06 A RMSD for three-residue loops and 3.72 A RMSD for eight-residue loops. Due to its speed the method may also be useful to generate alternative starting conformations for complex simulations.