Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins.

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the µM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.

Experimental observation of asymmetrical microwave jets and far-field distribution generated by a dual-material system.

In this paper, we report the experimental and numerical investigation of plane wave diffraction by an all-dielectric dual-material cuboid. Edge diffraction by a cuboid leads to the generation of a narrow, high intensity beam in the near-field region called a photonic jet. We examine the dependence of the jet behavior and orientation on the materials and dimensions of constitutive parts in the microwave frequency domain. The possibility to shift and deviate the resultant microwave jet in the near-field region of such a structure depending on the size of constitutive parts is demonstrated numerically. Experimentally, we observe a shift in the spatial position of the jet. The experimental asymmetric electric field profile observed in the far-field region is attributed to the input of multiple edge waves generated by the dual-material cuboid. The presented results may be scaled at different frequency bands such as optical frequencies for designing nanostructures enabling the focusing and deviation functionality and creation of new optical devices which would satisfy the needs of emerging nanophotonic applications.

Key interactions in the immunoglobulin-like structure of apo-neocarzinostatin: evidence from nuclear magnetic resonance relaxation data and molecular dynamics simulations.

The three-dimensional structure of apo-neocarzinostatin (apo-NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven-stranded antiparallel beta-sandwich, very similar to the immunoglobulin folding domain. We investigated the backbone dynamics of apo-NCS by (13)C-NMR relaxation measurements and molecular dynamics simulation. Model-free parameters determined from the experimental data are compared with a 1.5-nsec molecular simulation of apo-NCS in aqueous solution. This comparison provides an accurate description of both local and collective movements within the protein. This analysis enabled us to correlate dynamic processes with key interactions of this beta-protein. Local motions that could be relevant for the intermolecular association with the ligand are also described.

Role of loops in the folding and stability of yeast phosphoglycerate kinase.

Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstructured sequences (four Gly or four Thr) into the connections between secondary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK. All the mutated proteins can refold efficiently. The effect per residue on stability is larger for the first inserted residue. Insertion in two long betaalpha loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short alphabeta loop (at residue position 89) located on the opposite side of the N-terminal domain. The effect on stability is mainly due to a large increase of the unfolding rate rather than a decrease of the folding rate. This suggests that these connections between secondary structure elements do not play an active role in directing the folding process. Insertion into the short alphabeta loop (position 89) has limited effects on stability and results in the detection of a kinetic phase not previously seen with the wild-type protein, suggesting that insertions in this particular loop do qualitatively affect the folding process without a large effect on folding efficiency. For the two long betaalpha loops (positions 71 and 129) located in the inner surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential scanning calorimetry during thermal unfolding.

Identification and expression of the Pseudomonas syringae pv. aptata hrpZ(Psa) gene which encodes an harpin elicitor.

A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv. syringae hrpZ gene was identified in Pseudomonas syringae pv. aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants. and in E. coli DH10B (pCCP1069) containing the P. syringae pv. aptata hrp gene cluster. PCR with oligonucleotides, based on the hrpZ(Pss) gene and used as primers with the total genomic DNA of P. syringae pv. aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions. The amplicon was cloned into the pGEM-T plasmid vector, amplified in E. coli DH5alpha and sequenced. The sequence showed 95%, 83% and 61% identity with those of hrpZ(Pss), hrpZ(Psg and hrpZ(Pst) genes encoding the harpins of the P. syringae pv. syringae, glycinea and tomato, respectively. The amplicon was cloned into the pMAL expression system. The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography. On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P. syringae pv. aptata harpin.

A "loop entropy reduction" phage-display selection for folded amino acid sequences.

As a step toward selecting folded proteins from libraries of randomized sequences, we have designed a 'loop entropy reduction'-based phage-display method. The basic premise is that insertion of a long disordered sequence into a loop of a host protein will substantially destabilize the host because of the entropic cost of closing a loop in a disordered chain. If the inserted sequence spontaneously folds into a stable structure with the N and C termini close in space, however, this entropic cost is diminished. The host protein function can, therefore, be used to select folded inserted sequences without relying on specific properties of the inserted sequence. This principle is tested using the IgG binding domain of protein L and the lck SH2 domain as host proteins. The results indicate that the loop entropy reduction screen is capable of discriminating folded from unfolded sequences when the proper host protein and insertion point are chosen.

Functionally accepted insertions of proteins within protein domains.

Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural domain. Dihydrofolate reductase and beta-lactamase have been inserted in four different positions of phosphoglycerate kinase. The resultant chimeric proteins are all overexpressed, and the host as well as the inserted partners are functional. Although not explicitly designed, functional coupling between the two fused partners was observed in some of the chimeras. These results show that the tolerance of protein structures to very large structured insertions is more general than previously expected and supports the idea that the natural sequence continuity of a structural domain is not required for the folding process. These results directly suggest a new experimental approach to screen, for example, for folded protein in randomized polypeptide sequences.

Reinvestigation of the proteolytic activity of neocarzinostatin.

Neocarzinostatin (NCS) is the most studied member of a family of chromoproteins secreted by a range of actinomycetes species. It has been proposed that in addition to their antitumoral activity related to the bound chromophores, this group of related proteins could be a secreted proteases superfamily. With the aim of dissecting the molecular basis of the proteolytic activity of NCS, an expression system allowing efficient expression of apo-NCS in Escherichia coli was constructed. The recombinant protein was properly folded and functional. Its histone-specific proteolytic activity was similar to the activity described for the natural protein. Further analyses unambiguously demonstrated that the proteolytic activity could be physically separated from NCS. This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant and natural NCS preparations. The histone degradation test commonly used to monitor proteolytic activity is extremely sensitive and may easily generate false-positive results. These results strongly suggest that the possible proteolytic activity of the proteins of this family should be critically reconsidered.

Retinoic acid, but not arsenic trioxide, degrades the PLZF/RARalpha fusion protein, without inducing terminal differentiation or apoptosis, in a RA-therapy resistant t(11;17)(q23;q21) APL patient.

Primary blasts of a t(11;17)(q23;q21) acute promyelocytic leukaemia (APL) patient were analysed with respect to retinoic acid (RA) and arsenic trioxide (As2O3) sensitivity as well as PLZF/RARalpha status. Although RA induced partial monocytic differentiation ex vivo, but not in vivo, As203 failed to induce apoptosis in culture, contrasting with t(15;17) APL and arguing against the clinical use of As203 in t(11;17)(q23;q21) APL. Prior to cell culture, PLZF/RARalpha was found to exactly co-localize with PML onto PML nuclear bodies. However upon cell culture, it quickly shifted towards microspeckles, its localization found in transfection experiments. Arsenic trioxide, known to induce aggregation of PML nuclear bodies, left the microspeckled PLZF/RARalpha localization completely unaffected. RA treatment led to PLZF/RARalpha degradation. However, this complete PLZF/RARalpha degradation was not accompanied by differentiation or apoptosis, which could suggest a contribution of the reciprocal RARalpha/PLZF fusion product in leukaemogenesis or the existence of irreversible changes induced by the chimera.

Steady state and time-resolved fluorescence study of residual structures in an unfolded form of yeast phosphoglycerate kinase.

A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two tryptophan residues in an unfolded form of yeast phosphoglycerate kinase [Garcia, P., et al. (1995) Biochemistry 34, 397-404]. In this paper, we present a more detailed characterization of these residual structures, through the study of two single tryptophan-containing mutants of yPGK, W333F and W308Y. Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures. On the other hand, the compactness and dynamics of the protein matrix were probed using tryptophan fluorescence quenching by acrylamide at various denaturant concentrations. Taking into consideration the changes in sample viscosity induced by addition of guanidinium chloride made feasible the use of this technique during the denaturation process. These different approaches have shown that the residual structures around tryptophan 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding tryptophan 333. Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy. In the native protein, tryptophan buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas tryptophan that is partially accessible to the solvent, W308, has a lifetime close to 0. 5 ns. The time-resolved tryptophan fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single tryptophan mutants. The significance of minor long lifetime components is discussed for the two tryptophan residues. This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of tryptophan 333 is modified for denaturant concentrations lower than those for tryptophan 308.

Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin.

Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.

Occurrence of transient multimeric species during the refolding of a monomeric protein.

A set of protein fragments from yeast phosphoglycerate kinase were produced by chemical cleavage at a unique cysteinyl residue previously introduced by site-directed mutagenesis. Cross-linking experiments showed that the fragments corresponding to incomplete N-terminal domain form stable oligomeric species. Transient oligomeric species were also observed by both cross-linking and light scattering experiments during the folding process of the whole protein. These transient oligomeric species are formed during the fast folding phase and dissociate during the slow folding phase to produce the monomeric active protein. The multimeric species are not required for the protein to fold correctly. Unexpectedly, the distribution of oligomeric species is not dependent on protein concentration during the folding process. A kinetic competition mechanism is proposed as a possible solution to this paradox. These results provide direct evidence that the polypeptide chain can explore nonnative interactions during the folding process.

Folding and functional complementation of engineered fragments from yeast phosphoglycerate kinase.

A set of protein fragments was produced by site-directed mutagenesis followed by chemical cleavage of phosphoglycerate kinase according to a previously described method [Pecorari et al. (1993) Protein Eng. 6, 313-325]. The cleavage positions were chosen in order to correspond to limits between structural subdomains. These isolated fragments were studied by circular dichroism, folding transitions, and cross-linking analyses. It appears that fragments corresponding to globular subdomains in the protein can recover the expected helix content. However, the cooperativity classically observed in the folding transitions of natural proteins is only observed for fragments larger than a domain. Previous studies have shown that the isolated C-terminal domain is an autonomous folding unit which displays a single cooperatine transition [Missiakas et al. (1990) Biochemistry 29, 8683-8689]. The results presented here show that the presence in a fragment of a sequence overpassing that of the C-terminal domain modifies its folding process. Reassociation experiments suggest that the efficiency of the complementation process is not related to the folding autonomy of the isolated fragments.

Structural and functional properties of mutant Arg203Pro from yeast phosphoglycerate kinase, as a model of phosphoglycerate kinase-Uppsala.

A pathological variant of human phosphoglycerate kinase, phosphoglycerate kinase-Uppsala, associated with chronic nonspherocytic hemolytic anemia has been found to differ from the normal enzyme by substitution of an arginine at position 206 (corresponding to position 203 in yeast) by a proline. In order to understand the structural and functional consequences of this mutation, the corresponding mutant in yeast phosphoglycerate kinase was constructed. The three-dimensional structure of this mutant was resolved at 2.9 A. Although the overall structure is not modified, small local changes were observed. The kinetic parameters of the mutant were not found to be greatly affected, the catalytic constant being lowered by only 10-20%. The most significant difference when compared with the wild-type enzyme is a decrease in stability by about 3 kcal/mol. The physiological implications of this instability are discussed.

Is the continuity of the domains required for the correct folding of a two-domain protein?

The role of domains in protein folding has been widely studied and discussed. Nevertheless, it is not clear whether the continuity of the domains in a protein is an essential requirement in determining the folding pathway. Previous studies have shown that the isolated structural domains of the two-domain monomeric enzyme, yeast phosphoglycerate kinase (yPGK), are able to fold independently into a quasinative structure, but they neither reassociate nor generate a functional enzyme [Minard, P., Hall, L., Betton, J. M., Missiakas, D., & Yon, J. M. (1989) Protein Eng. 3, 55-60; Fairbrother, W. J., Bowen, D., Hall, L., Williams, R. J. P. (1989) Eur. J. Biochem. 184, 617-625; Missiakas, D., Betton, J. M., Minard, P., & Yon, J. M. (1990) Biochemistry 29, 8683-8689]. In the present work, two circularly permuted variants of the yPGK gene were constructed. The natural adjacent chain termini were directly connected and the new extremities were created within the N-domain (at residues 71 and 72) or the C-domain (at residues 291 and 292), respectively. These two proteins were overexpressed and purified. They exhibit 14% and 23% of the wild-type enzyme activity, respectively. The two mutants fold in a compact conformation with slight changes in the secondary and tertiary structure probably related to the presence of a heterogeneous population of molecules. The unfolding studies reveal a large decrease in stability. From the present data it appears that, although the circular permutations induce some perturbations in the structure and stability of the protein, the continuity of the domains is not required for the protein to reach a native-like and functional structure.

Role of the C-terminal helix in the folding and stability of yeast phosphoglycerate kinase.

In order to determine the role of the C-terminal helix in the folding and stability of yeast phosphoglycerate kinase, a mutant deleted of the 12 C-terminal residues (PGK delta 404-415) was constructed. This mutant folds in a conformation very similar to that of the wild-type protein, but exhibits a very low activity (0.1% of that of the wild-type enzyme). The main structural effect of the deletion of the C-terminal helix is an increase in flexibility of the whole protein and a decrease in stability by about 5 kcal/mol. The structural properties of the truncated protein are very similar, at least qualitatively, to those in the isolated domains. The accessibility of the thiol group of Cys 97 is identical to that in the isolated N-domain. The large solvent effect on the tryptophan fluorescence in the native protein at very low concentration of denaturant reveals an increase of flexibility of the C-domain, similar to that observed on the isolated C-domain. NMR measurements show that the pH dependence of His C2H and C4H chemical shifts in the truncated protein perfectly matches those of the isolated domains. The addition of the missing peptide provokes a 40-fold increase in enzyme activity at saturation. A dissociation constant of 80 microM was determined. This peptide, which displays a random structure in solution, folds in a helical structure in the region 405-410 as assessed by TRNOESY. All these results show that the C-terminal part of yeast phosphoglycerate kinase is not necessary for most of the initial folding steps but acts to lock the C-domain on the N-domain, thus ensuring the expression of full enzyme activity. Without this sequence, the protein has the sum of the properties of the two isolated domains.

Transferred nuclear Overhauser effect study of the C-terminal helix of yeast phosphoglycerate kinase: NMR solution structure of the C-terminal bound peptide.

Two-dimensional 1H nuclear magnetic resonance spectroscopy is used to determine the structure of the C-terminal complementary peptide (404-415) bound to a mutant phosphoglycerate kinase (1-403). Conformational changes in the peptide induced by the formation of the peptide-protein complex are followed by transferred nuclear Overhauser effect spectroscopy. Measurement of transferred NOEs and molecular modeling reveal an alpha-helix fold in the 405-409 region. This fold is in good agreement with the corresponding helix XIV of the crystallographic structure of wild-type PGK (Watson et al., 1982). The role of the alpha-helix from the C-terminal peptide in the recovery of catalytic activity in the mutant PGK is discussed.

Evidence for residual structures in an unfolded form of yeast phosphoglycerate kinase.

The unfolding-refolding transition of phosphoglycerate kinase followed by steady-state fluorescence has clearly shown the existence of a hyperfluorescent form [Missiakas et al. (1990) Biochemistry 29, 8683-8689]. In order to determine the contribution of each of the two tryptophans to the fluorescence properties of the enzyme in the equilibrium transition and to characterize the hyperfluorescent form, two single tryptophan mutants in which tryptophans 308 and 333 were replaced by a tyrosine and a phenylalanine, respectively, were constructed. Neither the catalytic nor the physicochemical properties of the enzyme are significantly altered by these mutations. The unfolding-refolding transitions were studied using circular dichroism and tryptophan fluorescence emission. Both tryptophans contribute to the hyperfluorescence observed in the first transition. For guanidine hydrochloride concentrations higher than 0.9 M, it clearly appears that the second transition results from a further unfolding. It is accompanied by a decrease in fluorescence intensity and a 5 mm red shift of the maximum emission wavelength. When the unfolding is induced by urea, the end of the transition corresponds to the hyperfluorescent state. Further addition of guanidine hydrochloride induces complete unfolding. These results suggest the presence of residual microstructures around tryptophan 308 and tryptophan 333 in the hyperfluorescent state. The characterization of these clusters and their contribution as starting structures in the folding process are now under investigation.

Cold denaturation of yeast phosphoglycerate kinase: which domain is more stable?

Under destabilising conditions both heat and cold denaturation of yeast phosphoglycerate kinase (PGK) can be observed. According to previous interpretation of experimental data and theoretical calculations, the C-terminal domain should be more stable than the N-terminal domain at all temperatures. We report on thermal unfolding experiments with PGK and its isolated domains, which give rise to a revision of this view. While the C-terminal domain is indeed the more stable one on heating, it reveals lower stability in the cold. These findings are of importance, because PGK has been frequently used as a model for protein folding and mutual domain interactions.