A new application of the yeast two-hybrid system in protein engineering

Cytochromes P450 are involved in the biosynthesis of steroid hormones in mitochondria of the adrenal gland. The electrons required for these reactions are provided via a redox chain consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). A prerequisite for a fast and efficient electron transfer as well as high catalytic activity is the formation of functional complexes between the different redox partners. To improve the protein-protein interactions by directed evolution, we developed a new in vivo selection system. This high-throughput screening method is based on the yeast two-hybrid system. It enables a background-free screening for increased protein-protein interactions between stable and functional species including cofactor-containing proteins (FAD, [2Fe-2S], heme). The method was successfully applied for the directed evolution of Adx and selected variants were analyzed biochemically and biophysically. All analyzed proteins exhibit typical characteristics of [2Fe-2S]-cluster-type ferredoxins. Adx-dependent substrate conversion assays with different cytochromes demonstrated that the improved ability of the mutants to form complexes results in an enhanced catalytic efficiency of the cytochrome P450 system.     

Sequence divergence analysis for the prediction of seven-helix membrane protein structures: II. A 3-D model of human rhodopsin

A three-dimensional (3-D) model of the transmembrane domainof human rhodopsin was predicted from the sequence divergenceanalysis of 42 sequences of rhodopsins and visual pigments withouta template. The prediction steps include multiple sequence alignment,calculation of a variability profile of the aligned sequences,use of the variability profile to identify the boundaries oftransmembrane regions, their secondary structure and packingshape in a helix bundle, prediction of side-chain conformationsand structure refinement. The identification of the retinalbinding site was assisted by its known covalent linkage withK296. The structural features of the predicted 3-D model arein good agreement with a low resolution electron density mapof bovine rhodopsin and with residues in contact with retinalas determined experimentally.     

Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.     

The elusive role of the N-terminal extension of {beta}A3- and {beta}Al-crystallin

ß-Crystallins are structural lens proteins with aconserved two-domain structure and variable N- and C-terminalextensions. These extensions are assumed to be involved in quaternaryinteractions within the ß-crystallin oligomers orwith other lens proteins. Therefore, the production of ßA3-and ßAl-crystallin from the single ßA3/A1mRNA by dual translation initiation is of interest. These crystallinsare identical, except that ßAl has a much shorterN-terminal extension than ßA3. This rare mechanismhas been conserved for over 250 million years during the evolutionof the ßA3/A1 gene, suggesting that the generationof different N-terminal extensions confers a selective advantage.We therefore compared the stability and association behaviourof recombinant ßA3- and ßAl-crystallin.Both proteins are equally stable in urea- and pH-induced denaturationexperiments. Gel filtration and analytical ultracentrifugationestablished that ßA3 and ßA1 both form homodimers.In the water-soluble proteins of bovine lens, ßA3and ßA1 are present in the same molecular weight fractions,indicating that they oligomerize equally with other ß-crystallins.¹H-NMR spectroscopy showed that residues Met1 to Asn22 of theN-terminal extension of ßA3 have great flexibilityand are solvent exposed, excluding them from protein interactionsin the homodimer. These results indicate that the differentN-terminal extensions of ßA3 and ßA1 donot affect their homo- or heteromeric interactions.     

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance.     

Incorporation of crystallographic temperature factors in the statistical analysis of protein tertiary structures

A method to identify statistically significant differences betweenequivalent atoms in two closely related protein X-ray crystallographicstructures is described. This method uses the linear relationshipfound between the logarithm of the distance between equivalentatoms and their mean temperature factor to determine, by linearregression, the expected difference and variance.     

FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolylisomerase that binds the macrolides FK506 and rapamycin. Wehave examined the role of the binding pocket residues of FKBP12in protein–ligand interactions by making conservativesubstitutions of 12 of these residues by site-directed mutagenesis.For each mutant FKBP12, we measured the affinity for FK506 andrapamycin and the catalytic efficiency in the cis–transpeptidyl-prolyl isomerase reaction. The mutation of Trp59 orPhe99 generates an FKBP12 with a significantly lower affinityfor FK506 than wild-type protein. Tyr26 and Tyr82 mutants areenzymatically active, demonstrating that hydrogen bonding bythese residues is not required for catalysis of the cis–transpeptidyl-prolyl isomerase reaction, although these mutationsalter the substrate specificity of the enzyme. We conclude thathydrophobic interactions in the active site dominate in thestabilization of FKBP12 binding to macrolide ligands and tothe twisted-amide peptidyl-prolyl substrate intermediate.     

An approach to protein homology modelling based on an ensemble of NMR structures: application to the Sox-5 HMG-box protein

A new approach has been developed to reduce multiple proteinstructures obtained from NMR structure analysis to a smallernumber of representative structures which still reflect thestructural diversity of the data sets. The method, based onthe clustering of similar structures, has been tested in thehomology model building of the structure of Sox-5, a sequence-specificDNA-binding protein belonging to the high mobility group (HMG)nuclear proteins family. Sox (SRY box) genes are the autosomalgenes related to the sex-determining SRY, Y chromosomal gene.The Sox-5 protein, encoded by one of the SRY-related genes,displays a 29% sequence identity with the HMG1 B-box domainwhose structure, determined previously by NMR, has been usedin our study to predict the structure of Sox-5. Two independentensembles of HMG1 structures, each represented by closely relatedcoordinate sets, were used. Nine representative structures forHMG1 were subsequently selected as starting points for the modellingof Sox-5. The model of the protein shows close similarity tothe HMG1 fold, with differences at the secondary structure levellocated mainly in a-helices 1 and 3. A left-handed, three residueper turn polyproline II helix, forming a conserved polyprolineII/-helix supersecondary motif, was identified in the N-terminalregion of Sox-5 and other HMG boxes.     

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

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

Sequence divergence analysis for the prediction of seven-helix membrane protein structures: I. Comparison with bacteriorhodopsin

A method using protein sequence divergence to predict the three-dimensionalstructure of the transmembrane domain of seven-helix membraneproteins is described. The key component in the multistep procedureis the calculation of a hydrophilic and lipophilic variabilityindex for each amino acid in an alignment of a family of homologousproteins. The variability profile, a plot of the calculatedvariability index versus alignment position, can be used topredict a tertiary model of the backbone conformation of thetransmembrane domain. This method was applied to bacteriorhodopsin(BR) and the model obtained was compared with the known structureof this protein. Using an alignment of the amino acid sequencesof BR and closely related (20% identity) proteins, the boundariesof the transmembrane regions, their secondary structures andorientations inside the membrane bilayer were predicted basedon the variability profile. Additional information about theshape of the helix bundle was also obtained from the averagevariability of each transmembrane helix with the assumptionthat the helices are packed sequentially and form a closed helixbundle. Correct features of the known structure of BR were foundin the model structure, suggesting that a similar strategy canbe used to predict transmembrane helices and the packing shapeof other membrane proteins with seven transmembrane helices,such as the opsins and other G-protein coupled receptors.     

A new approach to artificial and modified proteins: theory-based design, synthesis in a cell-free system and fast testing of structural properties by radiolabels

A novel approach to the creation of artificial and modifiedproteins has been elaborated. The approach includes a sequencedesign based on the molecular theory of protein secondary structureand folding patterns, gene expression in a cell-free systemand testing of structural properties of the synthesized polypeptidesat a nanogram level using radiolabelled chains. The approachhas been applied to a new synthetic protein albebetin whichhas been designed to form a 3-D fold which does not contradictany structural rule but has been never observed up to now innatural proteins. Using size-exclusion chromatography, urea-gradientelectrophoresis and limited proteolysis of a radiolabelled chain,it has been shown that the artificial protein is nearly as compactas natural proteins, cooperatively unfolds at high urea concentrationsand has some structural features of a definite structure consistentwith the designed one. As albebetin has been designed as consistingof two structural repeats, a ‘halfalbebetin’ (oneof these repeats) has also been synthesized and studied. Itwas shown that ‘half-albebetin’ is also compact     

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy

The stability and unfolding of an immunoglobulin (Ig) G bindingprotein based upon the B domain of protein A (SpAB) from Staphylococcusaureus were studied by substituting tryptophan residues at strategiclocations within each of the three a-helical regions (al-a3)of the domain. The role of the C-terminal helix, a3, was investigatedby generating two protein constructs, one corresponding to thecomplete SpAB, the other lacking a part of ct3; the Trp substitutionswere made in both one-and two-domain versions of each of theseconstructs. The fluorescence properties of each of the single-tryptophanmutants were studied in the native state and as a function ofguanidine-HCl-mediated unfolding, and their IgG binding activitieswere determined by a competitive enzyme-linked immunosorbentassay. The free energies of folding and of binding to IgG foreach mutant were compared with those for the native domains.The effect of each substitution upon the overall structure andupon the IgG binding interface was modelled by molecular graphicsand energy minimization. These studies indicate that (i) 3 contributesto the overall stability of the domain and to the formationof the IgG binding site in l and 2, and (ii) al unfolds first,followed by 2 and 3 together.     

Relationship between thermal stability and 3-D structure in a homology model of 3-isopropylmalate dehydrogenase from Escherichia coli

To reveal the structural basis of the increased thermal stabilityof 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus,an extreme thermophile, the homology-based structural modelof one mesophilic (Escherichia coli) counterpart, was constructed.Both IPMDHs are homodimeric proteins. We built a model of onesubunit using the 3-D structures of the Th.thermophilus IPMDHand the homologous E.coli isocitrate dehydrogenase. Energy minimizationand molecular dynamics simulated annealing were performed onthe dimer, including a surrounding solvation shell. No seriouserrors were detected in the refined model using the 3-D profilemethod. The resulting structure was scrutinized and comparedwith the structure of the Th.thermophilus IPMDH. Significantdifferences were found in the non-specific interactions includingthe hydrophobic effect. The model predicts a higher number ofion pairs in the Th.thermophilus than hi the E.coli enzyme.An increase was observed in the stabilities of -helical regionshi the thermophilic protein. The preliminary X-ray coordinatesof the E.coli IPMDH were received after the completion of thiswork, allowing an assessment of the model in terms of the X-raystructure. The comparison proved that most of the structuralfeatures underlying the stability differences between the twoenzymes were predicted correctly.     

Calculations of antibody-antigen interactions: microscopic and semi-microscopic evaluation of the free energies of binding of phosphorylcholine analogs to McPC603

The study of antibody-antigen interactions should greatly benefitfrom the development of quantitative models for the evaluationof binding free energies in proteins. The present work addressesthis challenge by considering the test case of the binding freeenergies of phosphorylcholine analogs to the murine myelomaprotein McPC603. This includes the evaluation of the differentialbinding energy as well as the absolute binding energies andtheir corresponding electrostatic contributions. Four differentapproaches are examined: the Protein Dipoles Langevin Dipoles(PDLD) method, the semi-microscopic PDLD (PDLD/S) method, afree energy perturbation (FEP) method based on an adiabaticcharging procedure and a linear response approximation thataccelerates the FEP calculation. The PDLD electrostatic calculationsare augmented by estimates of the relevant hydrophobic and stericcontributions. The determination of the hydrophobic energy involvesan approach which considers the modification of the effectivesurface area of the solute by local field effects. The stericcontributions are analyzed in terms of the corresponding reorganizationenergies. This treatment, which considers the protein as a harmonicsystem, views the steric forces as the restoring forces forthe electrostatic interactions. The FEP method is found to giveunreliable results with regular cut-off radii and starts togive quantitative results only in very expensive treatment withvery large cut-off radii. The PDLD and PDLD/S methods are muchfaster than the FEP approach and give reasonable results forboth the relative and absolute binding energies. The speed andsimplicity of the PDLD/S method make it an effective strategyfor interactive docking studies and indeed such an option isincorporated in the program MOLARIS. A component analysis ofthe different energy contributions of the FEP treatment anda similar PDLD analysis indicate that electrostatic effectsprovide the largest contribution to the differential bindingenergy, while the hydrophobic and steric contributions are muchsmaller. This finding lends further support to the idea thatelectrostatic interactions play a major role in determiningthe antigen specificity of McPC603.     

A functional antibody mutant with an insertion in the framework region 3 loop of the VH domain: implications for antibody engineering

We have studied the effects of a four residue insertion intothe FR3 loop of the heavy chain variable region from the anti-NPantibody Bl-8. The insertion mutant is obtained as secretedantibody without major defects in biosynthesis, indicating thatantibody variable domains can accommodate length variation notonly in complementarity determining regions (CDRs), but alsoin framework region (FR) loops. The Bl-8 antigen binding siteis not affected by the change in a neighbouring loop. FR3 insertionsrepresent a new method of antibody engineering with a potentialto obtain strong antigen binding by designing additional antigencontacting residues.     

The role of the flap residue, threonine 77, in the activation and catalytic activity of pepsin A

Flexible loops, often referred to as flaps, have been shownto play a role in catalytic mechanisms of different enzymes.Flaps at the active site regions have been observed in the crystalstructures of aspartic proteinases and their residues implicatedin the catalytic processes. This research investigated the roleof the flap residue, threonine 77, in the activation of pepsinogenand the catalytic mechanism of pepsin. Three mutants, T77S,T77V and T77G, were constructed. Differences in amino acid polarityand hydrogen bonding potential were shown to have an influenceon the activation and catalytic processes. T77S activated atthe same rate and had similar catalytic parameters as the wild-typepepsin. The activation rates of T77V and T77G were slower andtheir catalytic efficiencies lower than the wild-type. The resultsdemonstrated that the threonine 77 polar side chain played arole in a proteolysis. The contribution of the side chain tozymogen activation was associated with the proteolytic cleavageof the prosegment. It was postulated that the hydroxyl groupat position 77 provided an essential hydrogen bond that contributedto proper substrate alignment and, indirectly, to a catalyticallyfavorable geometry of the transition state.     

A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants

A novel gene fusion system to express and purify small recombinantproteins in Escherichia coli has been constructed. The conceptallows for affinity purification of soluble gene products bysequential albumin- and Zn²+-affinity chromatography. The dual-affinitysystem is well suited for expression of unstable proteins asonly full-length protein is obtained after purification andproteins gain proteolytic stability in the fusion protein. Herewe show that the dual-affinity approach can be used for theexpression of various unstable derivatives of a single IgG-bindingdomain based on staphylococcal protein A. Analysis of the proteolyticstabilities and the IgG-binding properties of the differentmutant proteins suggest that the model for the structure ofan IgG-binding domain must be re-evaluated.     

Engineered disulfide bonds in recombinant human interferon-{gamma}: the impact of the N-terminal helix A and the AB-loop on protein stability

Insertion sites for cysteines with optimal stereochemistry forthe formation of unstrained disulfide bridges were identifiedin recombinant human interferon- (rhu-IFN-) by computer modelling.We have engineered two different disulfide cross-linked mutants,containing a pair of symmetry-related disulfide bonds, whichstabilize the N-termini of both monomers of the homodimenc protein.Mutations E7C and S69C allow the formation of an intramonomerdisuffide bond between helices A and D. In contrast, the A17Cand H111C mutations lead to a covalent cross-link between bothmonomers. The AB-loop is linked to helix F. The fluorescenceproperties of native and disulfide cross-linked proteins werestudied as a function of guanidine hydrochloride concentration.Melting temperatures (T_m) were calculated from the decreasein CD ellipticity at 220 nm. The induction of the antiviraleffect was measured using A549 fibroblast cells infected withencephalomyocarditis virus. The ability to induce the expressionof the HLA-DR antigen in Colo 205 cells was determined by fluorescence-activatedcell scanning analysis. The stability of both mutants was stronglyenhanced against temperature- and cosolvent-induced unfolding.The T_m of mutant IFN- E7C/S69C was 15°C. All measured biologicalactivities of this mutant were equal to wild type. In the caseof the other mutant IFN- A17C/H111C, the T_m value was 25°C.This mutation abolishes nearly the entire biological activity(<1%) with no detectable changes of secondary structure inthe CD spectrum. Our results illustrate the importance of theN-terminal helix A and the AB-loop for the unfolding pathwayand thermodynamic stability of rhu-IFN-.     

A mini-protein designed by removing a module from barnase: molecular modeling and NMR measurements of the conformation

A globular domain can be decomposed into compact modules consistingof contiguous 10–30 amino acid residues. The correlationbetween modules and exons observed in different proteins suggeststhat each module was encoded by an ancestral exon and that moduleswere combined into globular domains by exon fusion. Barnaseis a single domain RNase consisting of 110 amino acid residuesand was decomposed into six modules. We designed a mini-proteinby removing the second module, M2, from barnase in order togain an insight into the structural and functional roles ofthe module. In the molecular modeling of the mini-protein, weevaluated thermodynamic stability and aqueous solubility togetherwith mechanical stability of the model. We chemically synthesizeda mini-barnase with ¹⁵N-labeling at 10 residues, whose correspondingresidues in barnase are all found in the region around the hydrophobiccore. Circular dichroism and NMR measurements revealed thatmini-barnase takes a non-random specific conformation that hasa similar hydrophobic core structure to that of barnase. Thisresult, that a module could be deleted without altering thestructure of core region of barnase, supports the view thatmodules act as the building blocks of protein design.     

Mutational and structural analysis of the lectin activity in binding domain 2 of ricin B chain

The study of the lectin binding sites of ricin B chain and ofother homologous members of the small gene family that makeup ricin-like molecules has revealed a number of key contactresidues involved in sugar binding. In particular, on the basisof data generated by the X-ray crystallographic structure ofricin, comparisons of sequence homologies to other ricin-likemolecules and substrate binding studies with these molecules,it has been proposed that His248 of Ricinus communis agglutinin(RCA) B chain may interfere with galactose binding in the secondbinding domain of that lectin. To test that hypothesis, singlebinding domain 2 (SBD2) of ricin B chain was expressed as agene 3 fusion protein on the surface of fd phage to measuredirectly the effect of mutational changes on this binding site.Replacement of tyrosine with histidine at amino acid position248 of SBD2 of ricin B chain was shown to reduce lectin activity.The sequences of RCA and ricin B chains were aligned and comparedwith the tertiary structure of ricin B chain to select variousmutations that were introduced as controls in the study. Oneof these controls, Leu247 to Val247, displayed increased affinityfor galactosides. The role of sequence changes is discussedin relation to the structural and functional divergence in thesemolecules.