Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein

In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837–841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [³H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors.     

Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through Hydrophobic Cluster Analysis and molecular modelling

A 21 kDa protein purified from bovine brain cytosol was previouslydescribed as a hydrophobic ngand binding protein; however, itsaccurate biological function remained still uncertain. In orderto get further information about its potential biological role,an extended prediction of its secondary and three dimensionalstructures was undertaken. We describe here a process whichpermitted us to discover a structural homology between the 21kDa protein and the N-domain of yeast phosphoglycerate kinase(PGK). This process is based on comparing the 21 kDa proteinwith all the proteins presenting a slight homology, by usingthe Hydrophobic Cluster Analysis (HCA) method. According tothe observed similarity between the N-domaln of yeast PGK andthe 21 kDa protein, we built a model which was shown to possessa potential binding site for nucleotides. Moreover, the modelobtained presents three-dimensional (3D) structure similaritywith adenylate kinase. These results suggest two main hypotheses:(0 the 21 kDa protein may belong to the kinase family; (ii)the binding of a nucleotide could imply a modification of the3D structure of the 21 kDa protein that can promote the transferof hydrophobic ligands to the plasma membrane. Meanwhile, verificationof these hypotheses has been in part performed experimentally:the 21 kDa protein binds MgATP as well as, to a lesser extent,phosphoglycerate.     

Physicochemical factors for discriminating between soluble and membrane proteins: hydrophobicity of helical segments and protein length

The average hydrophobicity of a polypeptide segment is consideredto be the most important factor in the formation of transmembranehelices, and the partitioning of the most hydrophobic (MH) segmentinto the alternative nonpolar environment, a membrane or hydrophobiccore of a globular protein may determine the type of proteinproduced. In order to elucidate the importance of the MH segmentin determining which of the two types of protein results froma given amino acid sequence, we statistically studied the characteristicsof MH helices, longer than 19 residues in length, in 97 membraneproteins whose three-dimensional structure or topology is known,as well as 397 soluble proteins selected from the Protein DataBank. The average hydrophobicity of MH helices in membrane proteinshad a characteristic relationship with the length of the protein.All MH helices in membrane proteins that were longer than 500residues had a hydrophobicity greater than 1.75 (Kyte and Doolittlescale), while the MH helices in membrane proteins smaller than100 residues could be as hydrophilic as 0.1. The possibilityof developing a method to discriminate membrane proteins fromsoluble ones, based on the effect of size on the type of proteinproduced, is discussed.     

Homology modelling of transferrin-binding protein A from Neisseria meningitidis

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded beta-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligand-binding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed.     

Mammalian cell transient expression of tissue factor for the production of antigen

We describe a mammalian cell expression system used to rapidlyproduce microgram quantities of a membrane protein used as animraunogen. A fusion protein expression vector was constructedwhich contained the signal sequence and 27 amino acids of theHerpes simplex virus glycoprotein D (gD), followed by a factorVIII (fVIII) thrombin cleavage site and the mature tissue factor(TF) sequence. This fusion protein was transiently expressedand then purified using an antibody to gD. The purified fusionprotein, gDTF, was incubated with thrombin to remove the gD-fVIIImoiety and the resulting rTF served as antigen for the generationof TF-specific antibodies. The antibodies produced were thenused for a comparison of the turnover rates of the constitutivelyand transiently produced fusion protein. In addition, sensitivityto glycosidases indicated that the transiently and constitutivelyproduced recombinant proteins do not contain identical carbohydratestructures.     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

Generation of GPI-linked CCL5 based chemokine receptor antagonists for the suppression of acute vascular damage during allograft transplantation

Limiting the acute vascular damage associated with leukocyte infiltration is a central issue in solid organ transplantation. The family of chemotactic cytokines (chemokines) helps to regulate leukocyte recruitment. Systemic treatment with the chemokine ligand-5 (CCL5) based antagonist Met-RANTES has previously shown to suppress acute damage to transplanted kidneys by blocking effector cell recruitment. To address problems associated with systemic long-term administration of chemokine receptor antagonists, a chemokine based reagent was designed to be integrated into endothelial surfaces of the organ just before transplantation. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells, are efficiently incorporated into their cell surface membranes. A series of modifications were introduced into the CCL5 protein to generate a functional antagonist. These included the addition of an N-terminal methionine group, a mutation to render the protein a dimer and a GPI signal sequence for surface expression. The resultant protein was stably expressed in CHO cells, GPI anchorage was confirmed and the protein purified by FPLC. Exogenously administered Met-CCL5(dimer)-GPI was efficiently inserted into the membrane of microvascular endothelial cells. The reagent is being tested in murine models of renal transplantation. The effect on subsequent immune induced damage will be assessed.     

Designing amino acid sequences to fold with good hydrophobic cores

We present two methods for designing amino acid sequences ofproteins that will fold to have good hydrophobic cores. Giventhe coordinates of the desired target protein or polymer structure,the methods generate sequences of hydrophobic (H) and polar(P) monomers that are intended to fold to these structures.One method designs hydrophobic inside, polar outside; the otherminimizes an energy function in a sequence evolution process.The sequences generated by these methods agree at the levelof 60–80% of the sequence positions in 20 proteins inthe Protein Data Bank. A major challenge in protein design isto create sequences that can fold uniquely, i.e. to a singleconformation rather than to many. While an earlier lattice-basedsequence evolution method was shown not to design unique folders,our method generates unique folders in lattice model tests.These methods may also be useful in designing other types offoldable polymer not based on amino acids     

Detection of secondary structure elements in proteins by hydrophobic cluster analysis

Hydrophobic cluster analysis (HCA) is a protein sequence comparisonmethod based on -helical representations of the sequences wherethe size, shape and orientation of the clusters of hydrophobicresidues are primarily compared. The effectiveness of HCA hasbeen suggested to originate from its potential ability to focuson the residues forming the hydrophobic core of globular proteins.We have addressed the robustness of the bidimensional representationused for HCA in its ability to detect the regular secondarystructure elements of proteins. Various parameters have beenstudied such as those governing cluster size and limits, thehydrophobic residues constituting the clusters as well as thepotential shift of the cluster positions with respect to theposition of the regular secondary structure elements. The followingresults have been found to support the -helical bidimensionalrepresentation used in HCA: (i) there is a positive correlation(clearly above background noise) between the hydrophobic clustersand the regular secondary structure elements in proteins; (ii)the hydrophobic clusters are centred on the regular secondarystructure elements; (iii) the pitch of the helical representationwhich gives the best correspondence is that of an -helix. Thecorrespondence between hydrophobic clusters and regular secondarystructure elements suggests a way to implement variable gappenalties during the automatic alignment of protein sequences.     

Cloning and expression in E.coli of a synthetic gene for the bacteriocidal protein caltrin/seminalplasmin

A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasminwas constructed and expressed in Escherichia coli as a fusionwith ß-galactosidase. The gene was designed with arecognition site for the plasma protease, Factor X_a, coded forimmediately prior to the N-terminus of caltrin. The ß-galactosidase-caltrinfusion protein was cleaved with Factor X_a to give caltrin, whichwas identified by its size on SDS-PAGE, its ability to reactwith an antiserum raised to the N-terminal nonapeptide of caltrinand its N-terminal amino acid sequence. After partial purification,synthetic caltrin was found to be active in an assay involvinginhibition of growth of E.coli.     

Distinctive properties of signal sequences from bacterial lipoproteins

We have compared a number of attributes (hydrophobicity, aminoacid size, charge and secondary structure propensities) of signalsequences from bacterial lipoproteins with the same attributesof signal peptides from other prokaryotic proteins (non-lipoproteins).Lipoprotein leader sequences tend to be shorter, more hydrophobicand bulky, and they have stronger conformational preferences,the most conspicuous being a predicted ß-turn comprisingpositions 2 or 3 of the mature protein. Another distinctivefeature is a maximum in the local energy profile between positions–1 and +2. With one exception (ß-lactamase III),the lipoproteins do not have Pro in their signal peptides, andthey tend to have fewer Ser and Thr but more Gly than non-lipoproteins.Lipoproteins also lack a net negative charge in the N-terminalregions of the mature proteins. The signal peptides of the bacteriocinplasmid-coded lysis proteins appear to be unique in that theyhave all the ascribed features of lipoprotein signals; thesecharacteristics can be used to guide signal peptide mutagenesisexperiments and to construct new secretion vehicles.     

Emulsifying performance of modular beta-sandwich proteins: the hydrophobic moment and conformational stability

Our understanding of protein emulsifying properties is largely based on analysis of emulsifiers found in milk and seed. The 9th-10th type III fibronectin domain pair retains full biological activity following emulsification-encapsulation into polyester microspheres, for controlled delivery, but the conformational criteria determining emulsification efficiency (EE) are unknown. Here, we have generated a series of mutants of this beta-sandwich protein, changing the hydrophobic moment and conformational stability, to investigate the structure-emulsification relationship. Predictive modelling of the hydrophobic moment of beta-strands and mutations known to increase conformational stability were used to generate the series. The proteins were tested for their emulsion stability and EE for oil-in-water mixtures. We show that the stabilization of emulsions by beta-sandwich proteins is best predicted by conformational stability during equilibrium denaturation in ionic surfactant. In contrast, the EE of these proteins is inversely related to an increase in their surface hydrophobicity following unfolding in surfactant. We also describe a novel beta-sandwich emulsifier with strong EE. The requirement for interdomain flexibility to achieve maximum emulsion stability and EE is also shown. This work increases our understanding of the mechanisms involved in protein emulsification and will be of use to the microencapsulation of proteins into polyester microspheres via emulsion-extraction protocols.     

Membrane proteins: from sequence to structure

The prediction of protein structure from sequence has been along-standing goal of molecular biology. Integral membrane proteins,once abhorred by protein chemists and crystallographers becauseof their insolubility and stubborn refusal to yield good crystals,now appear to hold great promises for efficient structure predictionand engineering. This is mainly due to the constraints on permissiblestructures imposed by the lipid environment, and to the apparentuncoupling between an initial membrane targeting and insertionprocess which determines the overall topological arrangementof the transmembrane segments and a subsequent –condensation—of these segments into a unique folded state. Recent work suggeststhat the membrane insertion process is controlled by simplesequence elements composed of different combinations of longhydrophobic regions and flanking charged residues. In this reviewwe sketch the most unportant structural rules relating aminoacid sequence to membrane insertion to fully folded molecule,and their use for prediction and protein-engineering purposes.     

Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence

Statistical analysis of 12 unstable and 32 stable proteins revealedthat there are certain dipeptides, the occurrence of which issignificantly different in the unstable proteins compared withthose in the stable ones. Based on the impact of these dipeptideson the unstable proteins over the stable ones, a weight valueof instability is assigned to each of the dipeptides. For agiven protein the summation of these weight values normalizedto the length of its sequence helps to distinguish between unstableand stable proteins. Results suggest that the in vivo instabilityof proteins is possibly determined by the order of certain aminoacids in its sequence. An attempt is made to correlate metabolicstability of proteins with features of their primary sequencewhere weight values of instability for a protein of known sequencecould thus be used as an index for predicting its stabilitycharacteristics.     

Properties of N-terminal tails in G-protein coupled receptors: a statistical study

The extramembraneous segments in a large collection of G-proteincoupled receptors (GPCRs) have been analysed in terms of aminoacid composition and length. It is shown that this family ofmulti-spanning integral membrane proteins conforms well to the‘positive inside’ rule. Further, the extracellularN-terminal tails of GPCRs lacking a cleavable signal peptideare shown to be considerably shorter and to have a reduced contentof positively charged amino acids compared with the N-terminaltails of GPCRs endowed with a signal peptide. This suggeststhat extracellular N-terminal tails of eukaryotic plasma membraneproteins may be translocated by different mechanisms dependingon whether or not they are preceded by a signal peptide.     

Non-polar nuclei in fungal microbial RNases

An application of a previously proposed method for the analysisof the non-polar structure of proteins is presented. A detailedanalysis of the composition and properties of non-polar nucleiand microclusters of fungal microbial ribonucleases has beenperformed on the basis of the 3-D structures of RNase T1 andrelated proteins. Three hydrophobic nuclei were found in thesestructures. It has been shown that all residues in non-polarnuclei have high homology ({small tilde}89%). Residues in thenuclei are practically fully buried in the interior of a molecule.Detailed analysis of non-polar nuclei properties shows thatthese nuclei determine the hydrophobic core of a protein andthe location and role of each residue in the non-polar interiorof proteins. In addition it was found that there are variableresidues not only on the surface of a protein but on the surfaceof the nuclei inside the protein and between the nuclei andthat there is a consistent region in all proteins, the hydrophobic-nuclei. An evaluation of the stability of non-polar nuclei,the conservation of their compositions and their positions inthe protein globule, allows one to assume that these three nucleiplay an important functional role in the stability and foldingof molecules of RNases and possibly can be considered as independentstructural elements of 3-D structures of these proteins.     

Secondary structure prediction of the H5 pore of potassium channels

The ‘H5’ segment located between the putative fifthand sixth transmembrane helices is the most highly conservedregion in voltage-gated potassium channels and it is believedto constitute a major part of the ion conduction path (pore).Here we present a two-step procedure, comprising secondary structureprediction and hydrophobic moment profiling, to predict thestructure of this important region. Combined results from theapplication of the procedure to the H5 region of four voltage-gatedand five other K⁺ channel sequences lead to the prediction ofa ß-strand-turn-(3-strand structure for H5. The reasonsfor the application of these soluble protein methods to partsof membrane proteins are: (i) that pore-lining residues areaccessible to water and (ii) that a large enough database ofhighresolution membrane protein structures does not yet existThe results are compared with experimental results, in particularspectroscopic studies of two peptides based on the H5 sequenceof SHAKER potassium channel. The procedure developed here maybe applicable to wateraccessible regions of other membrane proteins.     

Development of pswudoenergy potentials for assessing protein 3-D-1-D compatibility and detecting weak homologies

Recent approaches to the 3-D-l-D compatibility problem ave triedto predict protein 3-D structure from sequence. One of the criticalfactors in this issue is the evaluation of fitness between agiven 3-D structure and any sequence mounted on it. We havedeveloped an evaluation function composed of four terms, sidechain packing, hydration, hydrogen bonding and local conformationpotentials, which were empirically derived from 101 proteinsof known structure. The efficiency of the evaluation functionwas tested hi two ways. In the first test, the sequence of proteinA is mounted (without gaps) on the structure of protein B whichis greater in size than A. For 81 proteins examined, the nativestructure was always detected. In the second test, a standardsequence homology search is performed against the entire database,followed by an assessment of the alignment with its proposedstructure, using the empirical evaluation function. When thistest was applied to the 101 proteins, our evaluation functionsuccessfully discriminated truly homologous sequence pairs fromnon-homologous proteins even when the sequence similaritieswere very weak. This approach was found to have clear advantagesover conventional sequence search methods.     

Hydrophobic regions on protein surfaces: definition based on hydration shell structure and a quick method for their computation

The hydrophobic part of the solvent-accessible surface of atypical monomeric globular protein consists of a single, largeinterconnected region formed from faces of apolar atoms andconstituting –60% of the solvent-accessible surface area.Therefore, the direct delineation of the hydrophobic surfacepatches on an atom-wise basis is impossible. Experimental dataindicate that, in a two-state hydration model, a protein canbe considered to be unified with its first hydration shell inits interaction with bulk water. We show that, if the surfacearea occupied by water molecules bound at polar protein atomsas generated by AUTOSOL is removed, only about two-thirds ofthe hydrophobic part of the protein surface remains accessibleto bulk solvent. Moreover, the organization of the hydrophobicpart of the solvent-accessible surface experiences a drasticchange, such that the single interconnected hydrophobic regiondisintegrates into many smaller patches, i.e. the physical definitionof a hydrophobic surface region as unoccupied by first hydrationshell water molecules can distinguish between hydrophobic surfaceclusters and small interconnecting channels. It is these remaininghydrophobic surface pieces that probably play an important rolein intraand intermolecular recognition processes such as ligandbinding, protein folding and protein–protein associationin solution conditions. These observations have led to the developmentof an accurate and quick analytical technique for the automaticdetermination of hydrophobic surface patches of proteins. Thistechnique is not aggravated by the limiting assumptions of themethods for generating explicit water hydration positions. Formationof the hydrophobic surface regions owing to the structure ofthe first hydration shell can be computationally simulated bya small radial increment in solvent-accessible polar atoms,followed by calculation of the remaining exposed hydrophobicpatches. We demonstrate that a radial increase of 0.35–0.50Å resembles the effect of tightly bound water on the organizationof the hydrophobic part of the solvent-accessible surface.