Protein Engineering Design and Selection - recent issues

The V119I polymorphism in protein L-isoaspartate O-methyltransferase alters the substrate-binding interface

Rutherford, K., Daggett, V. — Fri, 13 Nov 2009 10:03:13 PST

Protein L-isoaspartate O-methyltransferase (PIMT) repairs isoaspartate residues in damaged proteins, and it contains a Val–Ile polymorphismin in 5, ~13 Å from its active site. Val119 has lower activity and thermal stability but increased affinity for endogenous substrates. Studies suggest that heterozygosity for Val/Ile favors efficient isoaspartate repair. We have performed multiple molecular dynamics simulations of 119I and 119V PIMT. Both V119 and I119 interact with the same residues throughout all of the simulations. However, the larger Ile altered the orientations of 5 and β5, both of which have co-substrate binding residues on their distal ends. I119 increases the flexibility of several residues, loosening up the S-adenosylmethionine (SAM)-binding site. These subtle changes are propagated towards the isoaspartate-docking site via residues common to both active sites. The increased mobility in 119I PIMT reorients 3, resulting in a salt-bridge network at the substrate-binding interface that disrupts several key side-chain interactions in the isoaspartate site. In contrast, 119V PIMT remains quite rigid with little change to the co-substrate binding site, which could hinder SAM's binding and release, accounting for the decreased activity. These results shed light on the molecular basis behind the decreased activity and increased specificity for endogenous substrates of 119V PIMT relative to the 119I variant. 119I PIMT catalyzes the methylation reaction but may have difficulties recognizing and orienting specific substrates due to its distorted substrate-binding site. Heterozygosity for both the Ile and Val alleles may provide the best of both worlds, allowing the fast and specific methylation of damaged proteins.

Engineering and characterization of a baculovirus-expressed mouse/human chimeric antibody against transferrin receptor

Fri, 13 Nov 2009 10:03:13 PST

Transferrin receptor (TfR) has been explored as a target for antibody-based therapy of cancer. In the previous study, we reported a murine anti-TfR monoclonal antibody (mAb) 7579 had good anti-tumor activities in vitro. In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effector mechanism in vivo, we herein developed its chimera in the baculovirus/insect cell expression system based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The chimeric light and heavy chains, containing human IgG1 constant regions, were correctly processed and assembled in insect cells, and then secreted into the mediums as heterodimeric H₂L₂ immunoglobulins. Furthermore, analyses of antigen-binding assay and competitive binding assay indicated that the chimeric antibody possessed specificity and affinity similar to that of its parental murine antibody. Results of the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assay verified that the chimeric antibody could efficiently mediate ADCC and CDC against TfR-overexpressing tumor cells. These results suggested that this baculovirus-expressed chimeric anti-TfR IgG1 might have the potential to be used for cancer immunotherapy. Meanwhile, the MAGIC strategy, facilitating the rapid generation of chimeric mAbs, could be one of the efficient strategies for antibody engineering.

Engineering of a novel hybrid enzyme: an anti-inflammatory drug target with triple catalytic activities directly converting arachidonic acid into the inflammatory prostaglandin E2

Ruan, K.-H., Cervantes, V., So, S.-P. — Fri, 13 Nov 2009 10:03:13 PST

Cyclooxygenase isoform-2 (COX-2) and microsomal prostaglandin E₂ synthase-1 (mPGES-1) are inducible enzymes that become up-regulated in inflammation and some cancers. It has been demonstrated that their coupling reaction of converting arachidonic acid (AA) into prostaglandin (PG) E₂ (PGE₂) is responsible for inflammation and cancers. Understanding their coupling reactions at the molecular and cellular levels is a key step toward uncovering the pathological processes in inflammation. In this paper, we describe a structure-based enzyme engineering which produced a novel hybrid enzyme that mimics the coupling reactions of the inducible COX-2 and mPGES-1 in the native ER membrane. Based on the hypothesized membrane topologies and structures, the C-terminus of COX-2 was linked to the N-terminus of mPGES-1 through a transmembrane linker to form a hybrid enzyme, COX-2-10aa-mPGES-1. The engineered hybrid enzyme expressed in HEK293 cells exhibited strong triple-catalytic functions in the continuous conversion of AA into PGG₂ (catalytic-step 1), PGH₂ (catalytic-step 2) and PGE₂ (catalytic-step 3), a pro-inflammatory mediator. In addition, the hybrid enzyme was also able to directly convert dihomo-gamma-linolenic acid (DGLA) into PGG₁, PGH₁ and then PGE₁ (an anti-inflammatory mediator). The hybrid enzyme retained similar K_d and V_max values to that of the parent enzymes, suggesting that the configuration between COX-2 and mPGES-1 (through the transmembrane domain) could mimic the native conformation and membrane topologies of COX-2 and mPGES-1 in the cells. The results indicated that the quick coupling reaction between the native COX-2 and mPGES-1 (in converting AA into PGE₂) occurred in a way so that both enzymes are localized near each other in a face-to-face orientation, where the COX-2 C-terminus faces the mPGES-1 N-terminus in the ER membrane. The COX-2-10aa-mPGES-1 hybrid enzyme engineering may be a novel approach in creating inflammation cell and animal models, which are particularly valuable targets for the next generation of NSAID screening.

Shear flow promotes amyloid-{beta} fibrilization

Dunstan, D. E., Hamilton-Brown, P., Asimakis, P., Ducker, W., Bertolini, J. — Fri, 13 Nov 2009 10:03:13 PST

The rate of formation of amyloid fibrils in an aqueous solution of amyloid-β (Aβ) is greatly increased when the solution is sheared. When Aβ solution is stirred with a magnetic stirrer bar at 37°C, a rapid increase in thioflavin T fluorescence is observed. Atomic Force Microscopy (AFM) images show the formation of aggregates, the growth of fibrils and the intertwining of the fibrils with time. Circular dichroism (CD) spectroscopy of samples taken after stirring shows a transition from random coil to -helix to β-sheet secondary structure over 20 h at 37°C. The fluorescence, AFM and CD measurements are all consistent with the formation of amyloid fibrils. Quiescent, non-stirred solutions incubated at 37°C showed no evidence of amyloid formation over a period of 3 days. Couette flow was found to accelerate the formation of amyloid fibrils demonstrating that the primary effect of stirring is not mixing but shearing. Only very small shear forces are applied to individual molecules in our experiments. Simple calculation suggests that the force is too small to support a hypothesis that shearing promotes partial unfolding of the protein as is observed.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis

Fri, 13 Nov 2009 10:03:13 PST

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca²⁺-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

Novel regulation of HIV-1 replication and pathogenicity: Rev inhibition of integration

Fri, 13 Nov 2009 10:03:13 PST

Following fusion of the human immunodeficiency virus type-1 (HIV-1) with host cells' membrane and reverse transcription of the viral RNA, the resulted cDNA is integrated into the host genome by the viral integrase enzyme (IN). Quantitative estimations have revealed that only 1–2 copies are integrated per infected cell, although many copies of the viral RNA are reverse-transcribed. The molecular mechanism that restricts the integration degree has not, so far, been elucidated. Following integration, expressed partially spliced and unspliced transcripts are exported from the nuclei by the viral Rev protein. Here, we show that in virally infected cells, the Rev interacts with the IN forming a Rev–IN complex and consequently limits the number of integration events. Disruption of the Rev–IN complex by selected IN-derived peptides or infection by a Rev-deficient virus stimulate integration resulting in large numbers of integration event/cell. Conversely, infection of Rev-expression cells blocks integration and inhibits virus production. Increased integration appears to correlate with increased cell death of infected cultures. Our results thus demonstrate a new regulatory function of Rev and probably establish a link between Rev restriction of HIV-1 integration and protection of HIV-1-infected cells from premature cell death.

Expression and purification of active recombinant equine lysozyme in Escherichia coli

Mon, 19 Oct 2009 07:37:29 PDT

Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and -lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni²⁺ affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.

Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis

Riahi-Madvar, A., Hosseinkhani, S. — Mon, 19 Oct 2009 07:37:29 PDT

Firefly luciferase (EC.1.13.12.7) from Photinus pyralis is a single polypeptide chain (62 kDa), responsible for emission of yellow-green (557 nm) light, known to be most efficient bioluminescence system that make it an excellent tool for reporter in nano-system biology. However, it is very sensitive to proteolytic degradation, which reduces its intracellular half-life, leads to loss in sensitivity and precision in analytic applications. In order to generate more stable luciferases against protease digestion, we substituted two tryptic sites: R²¹³, R³³⁷ and also next residue to it (Q³³⁸) with another amino acids. Overall, all mutations brought about structural changes that indicated more compact structure upon mutation, which revealed by enhancement of tryptophan fluorescence, decreases flexibility and less surface hydrophobic pockets. In general, structural changes associated with a clear improvement in thermostability and resistance against trypsin hydrolysis. In particular, R337Q mutant shows higher light stability in mammalian cell culture, which makes it as a suitable reporter for imaging.

Modeling the functional consequences of single residue replacements in bacteriophage f1 gene V protein

Masso, M., Mathe, E., Parvez, N., Hijazi, K., Vaisman, I. I. — Mon, 19 Oct 2009 07:37:29 PDT

A computational mutagenesis methodology utilizing a four-body, knowledge-based, statistical contact potential is applied toward globally quantifying relative environmental perturbations (residual scores) in bacteriophage f1 gene V protein (GVP) due to single amino acid substitutions. We show that residual scores correlate well with experimentally measured relative changes in protein function upon mutation. Residual scores also distinguish between GVP amino acid positions grouped according to protein structural or functional roles or based on similarities in physicochemical characteristics. For each mutant, the in silico mutagenesis additionally yields local measures of environmental change (EC scores) occurring at every residue position (residual profile) relative to the native protein. Implementation of the random forest (RF) algorithm, utilizing experimental GVP mutants whose feature vector components include EC scores at the mutated position and at six structurally nearest neighbors, correctly classifies mutants based on function with up to 77% cross-validation accuracy while achieving 0.82 area under the receiver operating characteristic curve. A control experiment highlights the effectiveness of mutant feature vector signals, and a variety of learning curves are generated to analyze the impact of GVP mutant data set size on performance measures. An optimally trained RF model is subsequently used for inferring function for all the remaining unexplored GVP mutants.

Towards evolving a better repressor

Daber, R., Lewis, M. — Mon, 19 Oct 2009 07:37:29 PDT

Transcriptional regulation is an essential component of all metabolic pathways. At the most basic level, a protein binds to a particular DNA sequence (operator) on the genome and either positively or negatively alters the level of transcription. Together, the protein and its operator form an epigenetic switch that regulates gene expression. In an effort to produce a ‘better’ switch, we have discovered novel facets of the lac operon that are responsible for optimal functionality. We have uncovered a relationship between operator binding affinity and inducibility and demonstrated that the operator DNA is not a passive component of a genetic switch; it is responsible for establishing binding affinity, specificity as well as translational efficiency. In addition, an operator's directionality can indirectly affect gene expression. Unraveling the basic properties of this classical epigenetic switch demonstrates that multiple factors must be optimized in designing a better switch.

Accelerating phage-display library selection by reversible and site-specific biotinylation

Koide, A., Wojcik, J., Gilbreth, R. N., Reichel, A., Piehler, J., Koide, S. — Mon, 19 Oct 2009 07:37:29 PDT

Immobilization of a target molecule to a solid support is an indispensable step in phage display library sorting. Here we describe an immobilization method that addresses shortcomings of existing strategies. Our method is based on the use of a polyhistidine-tagged (His-tagged) target molecule and ^BTtris-NTA, a high-affinity capture reagent for His-tags that also contains a biotin moiety. ^BTtris-NTA provides a stable and reversible linkage between a His-tag and a streptavidin-coated solid support. Because His-tags are the de facto standard for recombinant protein purification, this method dramatically simplifies target preparation for phage display library sorting. Here, we demonstrate the utility of this method by selecting high-affinity binding proteins based on the fibronectin type III (FN3) scaffold to two His-tagged protein targets, yeast small ubiquitin-like modifier and maltose-binding protein. Notably, a significant number of FN3 clones binding either targets selected using the new immobilization method exhibited only very weak binding when the same target was immobilized by coating on a polystyrene surface. This suggests that the His-tag-mediated immobilization exposes epitopes that are masked by commonly used passive adsorption methods. Together, these results establish a method with the potential to streamline and enhance many binding-protein engineering experiments.

An in vitro selection strategy for conferring protease resistance to ligand binding peptides

Mon, 19 Oct 2009 07:37:29 PDT

One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.

GPCR-GIA: a web-server for identifying G-protein coupled receptors and their families with grey incidence analysis

Lin, W.-Z., Xiao, X., Chou, K.-C. — Mon, 19 Oct 2009 07:37:29 PDT

G-protein-coupled receptors (GPCRs) play fundamental roles in regulating various physiological processes as well as the activity of virtually all cells. Different GPCR families are responsible for different functions. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to develop an automated method to address the two problems: given the sequence of a query protein, can we identify whether it is a GPCR? If it is, what family class does it belong to? Here, a two-layer ensemble classifier called GPCR-GIA was proposed by introducing a novel scale called ‘grey incident degree’. The overall success rate by GPCR-GIA in identifying GPCR and non-GPCR was about 95%, and that in identifying the GPCRs among their nine family classes was about 80%. These rates were obtained by the jackknife cross-validation tests on the stringent benchmark data sets where none of the proteins has ≥50% pairwise sequence identity to any other in a same class. Moreover, a user-friendly web-server was established at http://218.65.61.89:8080/bioinfo/GPCR-GIA. For user's convenience, a step-by-step guide on how to use the GPCR-GIA web server is provided. Generally speaking, one can get the desired two-level results in around 10 s for a query protein sequence of 300–400 amino acids; the longer the sequence is, the more time that is needed.

Prediction of palmitoylation sites using the composition of k-spaced amino acid pairs

Wang, X.-B., Wu, L.-Y., Wang, Y.-C., Deng, N.-Y. — Mon, 19 Oct 2009 07:37:29 PDT

Palmitoylation is an important hydrophobic protein modification activity that participates many cellular processes, including signaling, neuronal transmission, membrane trafficking and so on. So it is an important problem to identify palmitoylated proteins and the corresponding sites. Comparing with the expensive and time-consuming biochemical experiments, the computational methods have attracted much attention due to their good performances in predicting palmitoylation sites. In this paper, we develop a novel automated computational method to perform this work. For a sequence segment in a given protein, the encoding scheme based on the composition of k-spaced amino acid pairs (CKSAAP) is introduced, and then the support vector machine is used as the predictor. The proposed prediction model CKSAAP-Palm outperforms the existing method CSS-Palm2.0 on both cross-validation experiments and some independent testing data sets. These results imply that our CKSAAP-Palm is able to predict more potential palmitoylation sites and increases research productivity in palmitoylation sites discovery. The corresponding software can be freely downloaded from http://www.aporc.org/doc/wiki/CKSAAP-Palm.

Computational design-based molecular engineering of the glycosyl hydrolase family 11 B. subtilis XynA endoxylanase improves its acid stability

Belien, T., Joye, I. J., Delcour, J. A., Courtin, C. M. — Fri, 11 Sep 2009 09:10:38 PDT

Rational protein engineering was applied to improve the limited stability of the glycosyl hydrolase family 11 (GH11) endo-β-1,4-xylanase from Bacillus subtilis under acidic conditions. Since the pH dependence of protein stability is governed by the ionisation states of the side chains of its titrable amino acid residues, we explored the strategy of changing pH-stability profiles by altering pK_a values of key residues through in silico designed mutations. To this end, computational predictions and molecular modelling were carried out using the recently developed pKD software package. Four endoxylanase variants, in which the pK_a values of either Asp4 and Asp11 or His149 were targeted to shift downwards through incorporation of three to five point mutations, were generated and recombinantly expressed in the cytoplasm of Escherichia coli. All four mutants showed considerably increased functional stability at acid pH levels. They retained ~30–70% and ~75–95% of their activity after incubation at pH 3 and 4, respectively, in comparison with only ~23% and ~57%, respectively, for the wild-type enzyme under the experimental conditions. No acidophilic adaptation of the catalytic activity had occurred. In addition, their functional stability and catalytic activity profiles under different temperature and ionic strength conditions were significantly altered. These findings contribute to general understanding of the molecular mechanisms governing the pH-dependent stability of GH11 proteins, and hence they can be applied to enhance the stability and effectiveness of many GH11 endoxylanases used in industry today.

Evaluation of folding co-operativity of a chimeric protein based on the molecular recognition between polyproline ligands and SH3 domains

Candel, A. M., Cobos, E. S., Conejero-Lara, F., Martinez, J. C. — Fri, 11 Sep 2009 09:10:38 PDT

In previous work, we designed a chimeric protein, named SPCp41, to evaluate the thermodynamics of the interaction between SH3 domains and proline-rich ligands by combining thermal unfolding measurements and mutagenesis. Here, we have investigated the energetic integrity of the chain extension corresponding to the ligand sequence into the native structure, since the opposite will produce changes in the folding mechanism of the SH3 domain that may give rise to undesirable contributions to the thermodynamic parameters. We have analysed the folding–unfolding kinetics under standard conditions (50 mM phosphate pH 7). Kinetic evolutions are well described by a bi-exponential where, on top of the main kinetic phase, a low-populated slower phase appears as a consequence of cis–trans isomerisation of Pro39, as demonstrated by the influence of prolyl isomerases and by mutational analysis. There is also a burst phase possibly due to a productive formation of some helical ensembles. The main evolution, accounting for the true folding kinetics of SPCp41, can be considered as a two-state process, where the folding transition state produces essentially the same picture shown by the circular permutant S19-P20s (the ‘nucleus’ of the design) and the ligand will dock at the latter stages of the two-state process. Thus, all conclusions argue in favour of the effectiveness of SPCp41 to study energetic, dynamic and structural aspects of SH3–ligand interactions.

Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins

Whitehead, T. A., Bergeron, L. M., Clark, D. S. — Fri, 11 Sep 2009 09:10:38 PDT

Recombinant proteins often suffer from poor expression because of proteolysis. Existing genetic engineering or fermentation strategies work for only a subset of cases where higher recombinant protein expression is needed. In this paper, we describe the use of circular permutation, wherein the original termini of a protein are concatenated and new termini are generated elsewhere with the sequence, as a general protein engineering strategy to produce full-length, active recombinant protein. We show that a circularly permuted variant of the thermosome (Group II chaperonin) from Methanocaldococcus jannaschii exhibited reduced proteolysis and increased expression in three different strains of Escherichia coli. Circular permutation of a different protein, TEM-1 β-lactamase, by a similar method increased the expression lifetime of the protein in the periplasm of E. coli. Both circularly permuted proteins maintained activity near their wild-type counterparts and design criteria for selecting the sites for circular permutation are discussed. It is expected that this method will find broad utility for enhanced expression of recombinant proteins when proteolysis is a factor.

Enzyme stabilization by domain insertion into a thermophilic protein

Kim, C.-S., Pierre, B., Ostermeier, M., Looger, L. L., Kim, J. R. — Fri, 11 Sep 2009 09:10:38 PDT

Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzyme's desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 37°C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 37°C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.

Tubulin isotype specificity and identification of the epitope for antibody Tub 2.1

Yang, H., Cabral, F., Bhattacharya, R. — Fri, 11 Sep 2009 09:10:38 PDT

Tub 2.1, a monoclonal antibody commonly used to measure cellular tubulin content, is widely believed to recognize all β-tubulin isotypes. Though it has been used for more than two decades, the epitope for this antibody is not well established. We report for the first time that contrary to common belief, this antibody does not react with all isotypes of β-tubulin. Of the seven vertebrate β-tubulins, the more divergent class V and VI isotypes are not recognized by this antibody. Among the isotypes that do react, binding is similar for β2, β3, β4a and β4b but lower for β1, the most abundant isotype. Expression of chimeric tubulins verified that the epitope for Tub 2.1 is near the C-terminal end of β-tubulin. Site-directed mutagenesis of this region in nonreactive β5 to match the sequence of β4b resulted in strong reaction to Tub 2.1 and narrowed the epitope to amino acids 431–436.

Mutations in fd phage major coat protein modulate affinity of the displayed peptide

Fri, 11 Sep 2009 09:10:38 PDT

Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying β-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12–19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around –1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to -helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with β-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed.

Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase

Fri, 11 Sep 2009 09:10:38 PDT

Predicting mutations that enhance protein–protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, we redesigned fasciculin's sequence to enhance its interactions with Torpedo californica acetylcholinesterase. Mutations were predicted in 5 out of 13 interfacial residues on fasciculin, preserving most of the polar inter-molecular contacts seen in the wild-type toxin/enzyme complex. To experimentally characterize fasciculin mutants, we developed an efficient strategy to over-express the toxin in Escherichia coli, followed by refolding to the native conformation. Despite our predictions, a designed quintuple fasciculin mutant displayed reduced affinity for the enzyme. However, removal of a single mutation in the designed sequence produced a quadruple mutant with improved affinity. Moreover, one designed mutation produced 7-fold enhancement in affinity for acetylcholinesterase. This led us to reassess our criteria for enhancing affinity of the toxin for the enzyme. We observed that the change in the predicted inter-molecular energy, rather than in the total energy, correlates well with the change in the experimental free energy of binding, and hence may serve as a criterion for enhancement of affinity in protein–protein complexes.