The folding pathway of an engineered circularly permuted PDZ domain

To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.     

Demonstration by burst-phase analysis of a robust folding intermediate in the FF domain

The role of intermediates in the folding reaction of single-domainproteins is a controversial issue. It was previously shown bydifferent methods that an on-pathway intermediate is populatedin the presence of sodium sulphate during the folding of theFF domain from HYPA/FBP11. Here we demonstrate using analysisof the amplitudes of kinetic traces that this burst-phase foldingintermediate is present at different salt concentration andat various pH, and is also found in roughly 30 site-directedmutants. The intermediate appears robust to changing conditionsand thus fulfils an important criterion for a productive molecularspecies on the folding reaction pathway.     

Kinetic folding mechanism of PDZ2 from PTP-BL

PDZ domains represent a large family of protein-interaction modules associated with a variety of unrelated proteins with different functions. We report a complete characterization of the kinetic folding mechanism of a fluorescent variant of PDZ2 from PTP-BL, investigated under a variety of different experimental conditions. For this purpose, we engineered a fluorescent variant of this protein Y43W (called pseudo-wild-type, pWT43). The results suggest the presence of a high-energy intermediate in the folding of PDZ2, as revealed by a pronounced non-linear dependence of the unfolding rate constant on denaturant concentration. Such an intermediate may or may not be detectable depending on the experimental conditions, giving rise to apparent two-state folding under stabilizing conditions (e.g. in the presence of sodium sulfate). Interestingly, even under these conditions, three-state folding can be restored by selectively destabilizing the native-like rate-limiting barrier by one specific mutation (V44A). Finally, we show that data taken on pWT43 under different experimental conditions (e.g. different pH values from 2.1 to 8.0 or in the presence of a stabilizing salt) and also data on a site-directed conservative mutant can be rationalized in terms of a simple reaction scheme involving a single set of intermediates and transition states.     

Fluorescence resonance energy transfer analysis of the folding pathway of Engrailed Homeodomain

The Engrailed Homeodomain folds on the microsecond time scale via an intermediate that is experimentally well characterised using structural Engrailed-Homeodomain mimics. Here, we analysed directly the changes in distance between key residues during the kinetics of unfolding and at equilibrium using fluorescence resonance energy transfer (FRET). Trp was the donor and 5-(((acetylamino)ethyl)amino) naphthalene-1-sulphate, the acceptor, substituted in positions that caused little change in stability. Distances calculated for the native state were in good agreement with those derived from the NMR structure. The distances between the N- and C-termini of Helix I and of Helix III increased, then decreased and finally increased again with increasing GdmCl concentration on equilibrium denaturation. This behaviour implied that there was a folding intermediate on the folding pathway and that this intermediate was populated at low concentrations of GdmCl concentration ( approximately 1 M). We analysed the changes in distance during temperature-jump relaxation kinetics, using a qualitative and very conservative procedure that drew conclusions only when changes in fluorescence of mutants containing either the donor or the acceptor alone would not obscure the change in the FRET signal when both donor and acceptor were present. The distance changes obtained under equilibrium and kinetic measurements were self-consistent and also consistent with the known high-resolution structures of the mimics of the folding intermediates. We showed that for analysing distances in disordered ensembles, it is important to use FRET probes with a critical distance close to the average separation in the ensemble. Otherwise, average distances could be over or underestimated.     

Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9

Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10 degrees C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.     

Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains

A single polypeptide chain containing two dihydrofolate reductase( D M ) sequences from Escherichia coli was constructed to determineif a repeat sequence fusion protein could be expressed in anactive form. The possibility that intersequence interactionscould play a significant role for this enzyme is suggested bythe results of Hall and Frieden (1989, Proc. NatlAcad. Sri.USA, 86, 3060-3064) who observed a substantial decrease in theyield of active enzyme when folded hi the presence of a largeC-terminal fragment. The fusion protein [DHFR(Cysl52Ghi)-De-DHFR(MetlGln)] was efficiently expressed in E.coli cells and hasan activity which is twice that of the wild-type enzyme in thestandard assay. The Michaelis constants of the fusion proteinfor the substrate, dihydrofolate and the cofactor, NADPH, areessentially unchanged from those of the wild-type protein. Theureainduced in vitro unfolding reaction of the fusion proteinat low concentrations was found to be fully reversible and followa three state model, suggesting that the two domains unfoldindependently. At higher protein concentrations the unfoldingtransition broadened and shifted to a higher urea concentration.Size-exclusion chromatography results are consistent with theformation of aggregates at the higher protein concentration,even in the absence of denaturant.     

Recombinant C-terminal domain of pancreatic lipase retains full ability to bind colipase

The organization of the pancreatic lipase in two well defineddomains has been correlated to a specific function for eachdomain, catalytic activity for the N-terminal domain and colipasebinding for the C-terminal domain. In order to see if such anorganization implies that the two domains can behave as separateentities, we expressed the N- and C-terminal domains in insectcells. The recombinant proteins secreted in the cell supernatantspresent the expected molecular properties. However, whereasthe C-terminal domain retains its function of colipase binding,the N-terminal domain appears to be unable to ensure catalysis.The lack of activity of the recombinant N-terminal domain couldresult either from a (partially) incorrect folding or from anincapacity to function by itself. These results suggest that,although both are structurally well defined, the two domainsof the pancreatic lipase behave differently when they are expressedas separate entities.     

Engineering allosteric protein switches by domain insertion

Domain insertion is proving to be an effective way to construct hybrid proteins exhibiting switch-like behavior. In this strategy, two existing domains, the first exhibiting a signal recognition function and the second containing the function to be modulated, are fused such that the recognition of the signal by the first domain is transmitted to the second domain, thereby modulating its activity. Recent directed evolution experiments indicate that the structural space comprised of the recombination of unrelated protein domains may be rich in switching behavior, particularly when the circular permutation of domains is also employed. This bodes well for potential basic science, sensing and therapeutic applications of molecular switches.     

Repertoires of aggregation-resistant human antibody domains

We recently described a method for the generation of a large human domain antibody repertoire involving combinatorial assembly of CDR building blocks from a smaller repertoire comprising a high frequency of aggregation-resistant antibody domains. Here we show that the frequency of aggregation-resistant domains in the combinatorial repertoire remained high. Furthermore, one of the antigen-binding domains selected from the combinatorial repertoire retained its binding properties through 25 cycles of thermal denaturation, suggesting that antibody domains can be created that rival the heat-resistance of thermophilic proteins such as Taq polymerase.     

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.     

Tissue-type plasminogen activator variants with domain duplications and rearrangements

The interactions between tPA domains that are important forcatalysis are poorly understood. We have probed the functionof interdomain interactions by generating tPA variants in whichdomains are duplicated or rearranged. The proteins were expressedin a transient mammalian expression system and tested in vitrofor their ability to activate plasminogen, induce fibrinolysisand bind to a forming fibrin clot. Duplication of the heavychain domains of tPA produced enzymatically active tPA variants,many of which demonstrated similar in vitro amidolytic and fibrinolyticactivity and similar fibrin affinity to the parent molecule.Zymographic analysis of the domain duplication tPA variantsshowed one major active species for each variant. Selectionof the residues duplicated and the interdomain spacing werefound to be critical considerations in the design of tPA variantswith duplicated domains. We also rearranged the domains of tPAsuch that kringle 1 replaced the second kringle domain and viceversa. An analysis of these variants indicates that the firstkringle domain can confer fibrin affinity to a tPA variant andfunction in place of kringle 2. Therefore, in wild-type tPA,the functions of kringle 1 and kringle 2 must be dependent partiallyon their orientation within the heavy chain of the protein.The functional autonomy of the heavy and light chains of tPAis demonstrated by the activity of a tPA variant in which theorder of the heavy and light chains was reversed.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminalextracellular domains of human CD4 antigen, a cell surface glycoprotein,is reported. This region of CD4, particularly the first domain,has been identified as containing the binding region for theenvelope gp120 protein of the human immuno-deficiency virus.The model was predicted based on the sequence homology of eachdomain with the variable light chain of immunoglobulins. Theframework ß-sheet regions were taken from the crystalcoordinates of REI. For one region in the first domain of CD4there was an ambiguity in the alignment with REI and two alternatemodels are presented. Loops connecting the framework were modeledfrom fragments selected from a database of main chain coordinatesfrom all known protein structures. Residues identified as involvedin binding gp120 have been located in several other studieswithin the first domain of CD4. Epitopes from eight monoclonalantibodies have been mapped onto residues in both domains. Competitionof these antibodies with each other and with gp120 can be interpretedfrom the structural model.     

An investigation of protein subunit and domain interfaces

Protein structures were collected from the Brookhaven Databaseof tertiary architectures that displayed oligomeric association(24 molecules) or whose polypeptide folding revealed domains(34 proteins). The subunit and domain interfaces for these proteinswere respectively examined from the following aspects: percentagewater-accessible surface area buried by the respective associations,surface compositions and physical characteristics of the residuesinvolved in the subunit and domain contacts, secondary structuralstate of the interface amino acids, preferred polar and non-polarinteractions, spatial distribution of polar and non-polar residueson the interface surface, same residue interactions in the oligomeric:contacts, and overall cross-section and shape of the contactsurfaces. A general, consistent picture emerged for both thedomain and subunit interfaces.     

Effective renaturation of reduced lysozyme by gentle removal of urea

To increase the folding yield of concentrated reduced lysozyme,we developed a renaturation method by means of dialysis fromconcentrated urea with redox agents. After lysozyme was incubatedin the reducing buffer (8 M urea solution) with oxidized glutathione,renaturation of reduced lysozyme was started by dialysis againstthe dialyzing buffer containing 8 M urea with redox agents.The urea concentration of the dialyzing bottle was graduallydiluted with dialyzing buffer without urea at a flow rate of0.1 ml/min by high pressure pump. Using this systematic dialysis,a concentration as high as 5 mg/ml of reduced lysozyme couldbe renaturated in 80% yield, while the folding yield was <5%even at a concentration of 1 mg/ml using a conventional rapiddilution method [Goldberg et al. (1991) Biochemistry, 30, 2790–2797].Therefore, it was concluded that gentle removal of urea fromdenatured proteins, dissolved in concentrated urea solution,by means of dialysis should be useful to renature denaturedproteins effectively.     

Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression

Three different forms of dihydrofolate reductase (DHFR) fromEscherichia coli with amino acid replacements Thr35 Asp, Asn37 Ser and Arg57 His, and one form containing all three of thesechanges were obtained by oligonucleotide-directed mutagenesis.These amino acids are on the surface of the protein and twoof them (Thr35 and Arg57) are invariant for known sequencesof DHFR. Conversion of Asn37 Ser has no effect on the functionalactivity or the protein level in the cells. The Thr35 Asp replacementleads to a sharp decrease in the protein level, while the additionof a DHFR inhibitor, trimethoprim (Tmp), to the growth mediumincreases the level of DHFR in the ceus. There is a very smallquantity of DHFR with all three amino acid changes. The additionof Tmp to the growth medium also leads to an increase in themutant protein levels. The mutant with the Arg57 His replacementrenders the cells sensitive to Tmp, but the level of DHFR isthe same as for the wild-type protein. It is suggested thatthe invariant Thr35 is important for the stable conformationof DHFR whereas Arg57 is essential for protein activity. Variousstructural and functional aspects of these results are discussed.     

Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1{beta} point mutant modified in a surface loop

Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (T_agg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97—Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the T_m of the global transition. This hypothesisis supported by the location of residue 97 in the long 86–99loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolatedehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolatesynthetase was engineered to contain a prokaryotic ribosomebinding site and was expressed under the bacteriophage T7 RNApolymerase promoter in Escherichia coli. Site-directed mutagenesiswas used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase(D/C) domain as amino acid residues 1–301, and the synthetase(Syn) domain as residues 304–935. Both domains formedactive enzymes thereby demonstrating their ability to fold independently.The full-length enzyme, D/C and Syn domains were expressed atlevels 4-, 55- and 3-fold higher than the specific activitiesfound in liver. Additional mutagenesis and independent expressionof domains further defined the interdomain region to includeamino acids 292–310. The D/C domain was purified to homogeneityby a single affinity chromatographic step, and the full-lengthprotein in a twostep procedure. The kinetic properties of theD/C domain appear unaltered from those of the trifunctionalenzyme.     

Effect of additives on the renaturation of reduced lysozyme in the presence of 4 M urea

Reduced lysozyme was renatured by sulfhydryl-disuffide interchangereactions at pH 8.0 in the presence of 4 M urea, with or withoutadditives at 40°C. In the absence of additives, the finalfolding yield of reduced lysozyme was 40%. In the presence ofsarcosine, glycerol, ammonium sulfate, N-acetyl glucosamineand glucose, its folding yields increased in all cases. In particular,yields increased up to 90% in the presence of 4 M sarcosine.On the other hand, the melting temperatures of lysozyme withor without additives in 0.02 M citrate buffer (pH 6.0) wereevaluated using differential scanning calorimetry. In the absenceof additive, the melting temperature of lysozyme was 73.8°C.In the presence of additives, all melting temperatures werehigher than that of lysozyme in the absence of additives. Moreover,there was a good correlation on addition of additives betweenan increase in the folding yield of reduced lysozyme with 4M urea and an increase in the melting temperature without 4M urea. Therefore, we conclude that additives, which stabilizenative lysozyme, are effective at increasing the folding yieldof reduced lysozyme in 4 M urea.     

Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis

Biochemical, genetic and primary sequence analyses of the Erwiniachrysanthemi endoglucanase EGZ allowed us to identify two functionaldomains and to locate their boundaries. The catalytic domainextends from residue 1 to 288, while a domain required for EGZto bind to microcrystalline cellulose lies from residues 324to 385. Each domain was found capable of functioning in theabsence of the other. A region rich in Pro, Thr and Ser residueslinks both domains and appeared to be susceptible to proteolyticattack. Based upon predictions derived from a method developedto compare sequences sharing a low level of similarity, e.g.hydrophobic cluster analysis (HCA), we analysed the importanceof either residue His98 or Glul33 in EGZ catalytic activity.Two EGZ-derived proteins were engineered in which either His98or Glul33 amino acid was converted to an Ala residue. Characterizationof the purified proteins showed that no enzymatic activity couldbe detected, by using carboxymethylcellulose (CMC) or paranitrophenyl-cellobioside(pNPC) as substrates, while both mutated proteins retained thecapacity to bind to microcrystalline cellulose. These studies,which to date constitute the first experimental testing of HCA-derivedpredictions, allowed us to identify two particular amino acidsinvolved in cellulolytic activity. By taking into account datafrom chemical modification studies of other cellulases, we speculatethat the His98 residue is involved in the folding of the catalyticdomain while the Glul33 residue intervenes directly in the ß,1–4 glycosidic bond cleavage.