Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

Greek key jellyroll protein motif design: expression and characterization of a first-generation molecule

A protein designed de novo to fold into the Greek key jellyrollstructural motif has been studied. Theoretical analyses haveindicated that the designed sequence should adopt the ß-strandarrangement of the Greek key jellyroll rather than any otherarrangement. A synthetic gene was constructed and the proteinexpressed in Escherichia coli. Circular dichroism spectroscopyis consistent with the protein folding into the designed conformationand also suggests the presence of tertiary structure. Fluorescencespectroscopy showed the single tryptophan to be partially buried,while denaturation studies showed changes in fluorescence toprecede alterations in secondary structure.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli

For the first time the pro-form of a recombinant cysteine proteinasehas been expressed at a high level in Escherichia coli. Thisinactive precursor can subsequently be processed to yield activeenzyme. Sufficient protein can be produced using this systemfor X-ray crystallographic structure studies of engineered proteinases.A cDNA clone encoding propapain, a precursor of the papaya proteinase,papain, was expressed in E.coli using a T7 polymerase expressionsystem. Insoluble recombinant protein was solubilized in 6 Mguanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6.A protein-glutathione mixed disulphide was formed by dilutioninto oxidized glutathione and 6 M GuHCl, also at pH 8.6. Finalrefolding and disulphide bond formation was induced by dilutioninto 3 mM cysteine at pH 8.6. Renatured propapain was processedto active papain at pH 4.0 in the presence of excess cysteine.Final processing could be inhibited by the specific cysteineproteinase inhibitors E64 and leupeptin, but not by pepstatin,PMSF or EDTA. This indicates that final processing was due toa cysteine proteinase and suggests that an autocatalytic eventis required for papain maturation.     

Protein modelling using a chimera reference protein derived from exons

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

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

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

Redesigning a sweet protein: increased stability and renaturability

Monellin is one of two natural proteins from African berrieswith potent sweet taste. Monellin is the smaller of the two,and consists of two peptides. The protein loses sweetness whenheated above 50°C under acidic pH. Based on the crystalstructure of monellin we have fused the two chains into a singlechain using several different linkers copied and ‘transplanted’from the same molecule. One of the newly designed proteins isas potently sweet as the natural one, is more stable upon temperatureor pH changes, and renatures easily even after heating to 100°Cat low pH.     

The prediction and characterization of metal binding sites in proteins

The rational engineering of novel functions into proteins canonly be attempted when the underlying structural scaffold onwhich the new function is displayed and the structure of thetarget protein are both well understood. To introduce functionsmediated by metals it is therefore necessary to identify theprincipal liganding residues for the chosen metal, the requiredarchitecture of the metal-ligand complex and sites within thetarget protein that could accommodate such sites. Here we presenta method that applies structural information from the proteindata bank to the ab initio design and characterization of novelmetal binding sites. The prediction method has been tested on28 metalloprotein structures from the Brookhaven Protein DataBank. It successfully identified >90% of the metal bindingsites. In addition, we have used the method to design and characterizezinc binding sites in two antibody structures. Metal bindingstudies on one of these putative metalloantibodies showed metalbinding, confirming the predictive power of the method.     

Protein structural domain identification

A simple method for the definition of protein structural domainsis described that requires only -carbon coordinate data. Thebasic method, which encodes no specific aspects of protein structure,captures the essence of most domains but does not give highenough priority to the integrity of ß-sheet structure.This aspect was encouraged both by a bias toward attaining intactß-sheets and also as an acceptance condition on the finalresult. The method has only one variable parameter, reflectingthe granularity level of the domains, and an attempt was madeto set this level automatically for each protein based on thebest agreement attained between the domains predicted on thenative structure and a set of smoothed coordinates. While notperfect, this feature allowed some tightly packed domains tobe separated that would have remained undivided had the bestfixed granularity level been used. The quality of the resultswas high and, when compared with a large collection of accepteddomain definitions, only a few could be said to be clearly incorrect.The simplicity of the method allowed its easy extension to thesimultaneous definition of domains across related structuresin a way that does not involve loss of detail through averagingthe structures. This was found to be a useful approach to reconcilingdifferences among structural family members. The method is fast,taking less than 1 s per 100 residues for medium-sized proteins.     

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     

Structure-guided SCHEMA recombination of distantly related beta-lactamases

We constructed a library of beta-lactamases by recombining three naturally occurring homologs (TEM-1, PSE-4, SED-1) that share 34-42% sequence identity. Most chimeras created by recombining such distantly related proteins are unfolded due to unfavorable side-chain interactions that destabilize the folded structure. To enhance the fraction of properly folded chimeras, we designed the library using SCHEMA, a structure-guided approach to choosing the least disruptive crossover locations. Recombination at seven selected crossover positions generated 6561 chimeric sequences that differ from their closest parent at an average of 66 positions. Of 553 unique characterized chimeras, 111 (20%) retained beta-lactamase activity; the library contains hundreds more novel beta-lactamases. The functional chimeras share as little as 70% sequence identity with any known sequence and are characterized by low SCHEMA disruption (E) compared to the average nonfunctional chimera. Furthermore, many nonfunctional chimeras with low E are readily rescued by low error-rate random mutagenesis or by the introduction of a known stabilizing mutation (TEM-1 M182T). These results show that structure-guided recombination effectively generates a family of diverse, folded proteins even when the parents exhibit only 34% sequence identity. Furthermore, the fraction of sequences that encode folded and functional proteins can be enhanced by utilizing previously stabilized parental sequences.     

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.     

Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins

We have designed and constructed a DNA sequence encoding humantissue plasminogen activator (tPA) with convenient restrictionsites that flank each of the domains of the heavy chain. Toaccomplish this, the first 1095 bases of the gene coding forthe mature protein were synthesized with unique restrictionsites engineered into the interdomainal regions. This syntheticconstruction was then ligated to a cDNA fragment of the tPAgene that encoded the active site, thus generating a full-lengthtPA gene. The gene products produced by Chinese hamster ovary(CHO) cells transfected with either the tPA cassette gene orthe tPA cDNA gene were then compared with the tPA produced byBowes melanoma cells to determine whether or not synthetic interdomainalamino acid changes had an effect on the biochemical characteristicsof the molecule. Specifically, molecular weight, specific activity,enhancement by fibrinogen fragments and kinetic constants wereanalysed. None of the properties examined were significantlydifferent from those of the native melanoma tPA. Therefore,the cassette gene described herein should provide considerableversatility and precision in the construction of tPA mutantsby facilitating the manipulation of the finger, growth factorand kringle domains, and likewise should be useful in assessingthe function of these domains within the tPA molecule. We presentthis cassette gene system as a model for the analysis of proteindomain function applicable to other multi-domain proteins.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcalprotein A, designated domain B, was chemically synthesized.This gene was tandemly repeated to give dimeric and tetramericdomain B genes by the use of two restriction enzymes which gaveblunt ends. The genes were highly expressed in Escherichia colito afford a large amount of dimeric and tetrameric domain Bproteins. The single domain B protein was efficiently producedas a fusion protein with a salmon growth hormone fragment. Thefusion protein was converted to monomeric domain B by cyanogenbromide cleavage. The CD spectra of the monomeric, dimeric andtetrameric domain B proteins were essentially the same as thatof native form protein A, showing that their secondary structureswere very similar. The dimeric and tetrameric domain B proteinsformed precipitates with IgG as protein A. This system permitsthe efficient production of mutated single and multiple IgG-bindingdomains which can be used to study structural changes and proteinA–immunoglobulin interactions.     

Improved genetic algorithm-based protein structure comparisons: pairwise and multiple superpositions

Three major improvements to a previously described method forautomatic protein structure comparison are described. First,a limit to translations for the rigid-body superposition isnow assigned according to the dimensions of the structures beingcompared. Second, examination of the effect of the gap penaltyon the derivation of a sequence alignment corresponding to agiven structure superposition has led to a method to evaluatealternative structure-based sequence alignments. Third, thepairwise procedure has been generalized to multiple structurealignment. This implementation of rigid-body superposition canrecognize well documented distant relationships which hithertohave required consideration of additional features and propertiesas well as those relationships between proteins of differentsizes. A much larger common scaffold or framework between sixglobins can be extracted than that obtained using a standardalgorithm for multiple structure superposition     

Polarity as a criterion in protein design

Hypothetical proteins can be tested computationally by determiningwhether or not the designed sequence-structure pair has thecharacteristics of a typical globular protein. We have developedsuch a test by deriving quantities with approximately constantvalue for all globular proteins, based on empirical analysisof the exposed and buried surfaces of 128 structurally knownproteins. The characteristic quantities that best appear tosegregate badly designed or deliberately misfolded proteinsfrom their properly folded natural relatives are the polar fractionof side chains on the protein surface and, independently, inthe protein interior. Three of the seven hypothetical structurestested here can be rejected as having too many polar side-chaingroups in the interior or too few on the protein surface. Inaddition, a recently designed nutritional protein is identifiedas being very much unlike globular proteins. These database-derivedcharacteristic quantities are useful in screening designed proteinsprior to experiment and may be useful in screening experimentallydetermined (X-ray, NMR) protein structures for possible errors.     

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.     

Stabilization of TRAIL, an all-beta-sheet multimeric protein, using computational redesign

Protein thermal stability is important for therapeutic proteins, both influencing the pharmacokinetic and pharmacodynamic properties and for stability during production and shelf-life of the final product. In this paper we show the redesign of a therapeutically interesting trimeric all-beta-sheet protein, the cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), yielding variants with improved thermal stability. A combination of tumor necrosis factor (TNF) ligand family alignment information and the computational design algorithm PERLA was used to propose several mutants with improved thermal stability. The design was focused on non-conserved residues only, thus reducing the use of computational resources. Several of the proposed mutants showed a significant increase in thermal stability as experimentally monitored by far-UV circular dichroism thermal denaturation. Stabilization of the biologically active trimer was achieved by monomer subunit or monomer-monomer interface modifications. A double mutant showed an increase in apparent T(m) of 8 degrees C in comparison with wild-type TRAIL and remained biologically active after incubation at 73 degrees C for 1 h. To our knowledge, this is the first study that improves the stability of a large multimeric beta-sheet protein structure by computational redesign. A similar approach can be used to alter the characteristics of other multimeric proteins, including other TNF ligand family members.     

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

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

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

Structural basis for the extreme thermostability of D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: analysis based on homology modelling

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a hyperthermophiliceubacterium, Thermotoga maritima, is remarkably heat stable(T_m = 109°C). In this work, we have applied homology modellingto predict the 3-D structure of Th.maritima GAPDH to revealthe structural basis of thermostability. Three known GAPDH structureswere used as reference proteins. First, the rough model of onesubunit was constructed using the identified structurally conservedand variable regions of the reference proteins. The holoenzymewas assembled from four subunits and the NAD molecules. Thestructure was refined by energy minimization and molecular dynamicssimulated annealing. No errors were detected in the refinedmodel using the 3-D profile method. The model was compared withthe structure of Bacillus stearothermophilus GAPDH to identifystructural details underlying the increased thermostability.In all, 12 extra ion pairs per subunit were found at the proteinsurface. This seems to be the most important factor responsiblefor thermostability. Differences in the non-specific interactions,including hydration effects, were also found. Minor changeswere detected in the secondary structure. The model predictsthat a slight increase in a-helical propensities and helix-dipoleinteractions also contribute to increased stability, but toa lesser degree.