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At least 307 records · Page 17Linked to original sources
PubMed2004

Natural protein engineering: a uniquely salt-tolerant, but not halophilic, alpha-type carbonic anhydrase from algae proliferating in low- to hyper-saline environments.

Dunaliella salina is a unicellular green alga thriving in environments ranging from fresh water to hyper-saline lakes, such as the Dead Sea. An unusual, internally duplicated, 60 kDa alpha-type carbonic anhydrase (dCA I), located on the surface of this alga, is expected to function over a broad range of salinities. It would therefore differ from other carbonic anhydrases that already lose activity at low salinities and also from halophilic proteins that require high salinities for conformational stability. Enzymatic analyses indeed indicated that dCA I retained activity at salt concentrations ranging from low salt to at least 1.5 M NaCl or KCl for CO(2) hydration, 2.0 M NaCl for esterase activity and 0.5 M for bicarbonate dehydration. Although measurements at higher salinities were constrained by the interference of salt in the respective assayed reactions, activity was noticeable even at 4.0 M NaCl. Comparisons of the internally duplicated dCA I to single-domain derivatives indicated that inter-domain interactions played a decisive role in the stability, activity, salt tolerance and pH responses of dCA I. Hence dCA I is a uniquely salt- tolerant protein, retaining an active conformation over a large range of salinities and, as a Zn metalloenzyme, largely immune to the specific inhibitory effects of anions. Its unique features make dCA I a useful model to understand the physico-chemical basis of halotolerance and protein-salt interactions in general.

Amino Acid Sequence↗
PubMed2004

Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation.

It is controversial whether fast-folding proteins can form productive on-pathway intermediates that are more stable than the denatured state because noncovalent intermediates are usually evanescent. Here, we apply the classical criteria for the existence of intermediates: namely, the intermediates form and react rapidly enough to be on pathway and they can be isolated and characterized. The folding of the 71-residue, mainly alpha-helical FF domain from human HYPA/FBP11 fulfills these classical criteria, as was found for Im7. The FF domain folds in two phases, one on the micros and the other on the ms time scale. An engineered mutant folds only to a partly folded state, with some 20-40% of the native helical content. The kinetic properties of the mutant are identical to those found for the fast phase of the wild-type protein, and it is likely that the mutant folds just to the intermediate state. A full kinetic analysis of the folding of wild-type protein, using the amplitudes of its native and denatured states and the observed values for the mutant, rules out an off-pathway scheme but fits an on-pathway scheme, with a low energy intermediate that is modeled by the mutant. The experimental proof benchmarks a molecular dynamics method that identifies an obligatory intermediate observed in multiple simulations. The conformational space defining this intermediate is visited several times in the simulations, leading to high populations consistent with the presence of a low energy intermediate.

Kinetics↗
PubMed2005

Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor.

Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.

Chromatography, Affinity↗
PubMed2002

Interatomic potentials and solvation parameters from protein engineering data for buried residues.

Van der Waals (vdW) interaction energies between different atom types, energies of hydrogen bonds (H-bonds), and atomic solvation parameters (ASPs) have been derived from the published thermodynamic stabilities of 106 mutants with available crystal structures by use of an originally designed model for the calculation of free-energy differences. The set of mutants included substitutions of uncharged, inflexible, water-inaccessible residues in alpha-helices and beta-sheets of T4, human, and hen lysozymes and HI ribonuclease. The determined energies of vdW interactions and H-bonds were smaller than in molecular mechanics and followed the "like dissolves like" rule, as expected in condensed media but not in vacuum. The depths of modified Lennard-Jones potentials were -0.34, -0.12, and -0.06 kcal/mole for similar atom types (polar-polar, aromatic-aromatic, and aliphatic-aliphatic interactions, respectively) and -0.10, -0.08, -0.06, -0.02, and nearly 0 kcal/mole for different types (sulfur-polar, sulfur-aromatic, sulfur-aliphatic, aliphatic-aromatic, and carbon-polar, respectively), whereas the depths of H-bond potentials were -1.5 to -1.8 kcal/mole. The obtained solvation parameters, that is, transfer energies from water to the protein interior, were 19, 7, -1, -21, and -66 cal/moleA(2) for aliphatic carbon, aromatic carbon, sulfur, nitrogen, and oxygen, respectively, which is close to the cyclohexane scale for aliphatic and aromatic groups but intermediate between octanol and cyclohexane for others. An analysis of additional replacements at the water-protein interface indicates that vdW interactions between protein atoms are reduced when they occur across water.

Amino Acid Substitution↗
PubMed1999

Different equilibrium stability behavior of ScFv fragments: identification, classification, and improvement by protein engineering.

A classification of scFv fragments concerning their unfolding/refolding equilibria is proposed. It is based on the analysis of different mutants of the levan-binding A48 scFv fragment and the HER-2 binding 4D5 scFv fragment as well as a "hybrid" scFv carrying the VL domain of 4D5 and the VH domain of an A48 mutant. The denaturant-induced unfolding curves of the corresponding scFv fragments were measured and, if necessary for the classification, compared with the denaturation of the isolated domains. Depending on the relative intrinsic stabilities of the domains and the stability of the interface, the different scFv fragments were grouped into different classes. We also demonstrate with several examples how such a classification can be used to improve the stability of a given scFv fragment, by concentrating engineering efforts on the "weak part" of the particular molecule, which may either be the intrinsic stability of VL, of VH, or the stability of the interface. One of the scFv fragments obtained by this kind of approach is extremely stable, starting denaturation only at about 7 M urea. We believe that such extremely stable frameworks may be very suitable recipients in CDR grafting experiments. In addition, the thermodynamic equilibrium stabilities of seven related A48 scFv mutants covering a broad range of stabilities in urea unfolding were shown to be well correlated with thermal aggregation properties measured by light scattering and analytical gel filtration.

Chromatography, Gel↗
PubMed1998

A new method to characterize hydrophobic organization of proteins: application to rational protein engineering of barnase.

We present a new algorithm for characterization of protein spatial structure basing on the molecular hydrophobicity potential approach. The method is illustrated by the analysis of three-dimensional structure of barnase and barnase-barstar complex. Current approach enables identification of amino acid residues situated in unfavorable environment (these residues may be "active" for binding), and to map quantitatively hydrophobic, hydrophilic and unfavorable hydrophobic-hydrophilic intra- and inter-molecular contacts involving backbone and side-chain segments of amino acid residues. Calculation of individual contributions of amino acid residues to such contacts permits identification of structurally-important residues. The contact plots obtained with molecular hydrophobicity potential calculations, provide easy rules to choose sites for mutations, which can increase a strength of intra- or inter-molecular hydrophobic interactions. The unfavorable hydrophobic-hydrophilic contact can be mutated to favorable hydrophobic, and already existing weak hydrophobic contact can be strengthen by increasing hydrophobicity of residues in contact. Basing on the analysis of the contact plots, we suggest several mutations of barnase which are supposed to increase intramolecular hydrophobic interactions, and thus might lead to increased stability of the protein. Part of these mutations was studied previously experimentally, and indeed stabilized barnase. The other of predicted mutations were not studied experimentally yet. Several new mutations of barnase and barstar are also proposed to enhance the hydrophobic interactions on their binding interface.

Algorithms↗
PubMed2003

Temperature adaptation of proteins: engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase.

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic alpha-amylase) with or without an extra disulfide bond specific to warm-blooded animals. Both multiple mutants display large modifications of stability and activity arising from synergic effects in comparison with single mutations. Newly introduced weak interactions and the disulfide bond confer mesophilic-like stability parameters, as shown by increases in the melting point t(m), in the calorimetric enthalpy DeltaH(cal) and in protection against heat inactivation, as well as by decreases in cooperativity and reversibility of unfolding. In addition, both kinetic and thermodynamic activation parameters of the catalyzed reaction are shifted close to the values of the porcine enzyme. This study confirms the central role of weak interactions in regulating the balance between stability and activity of an enzyme in order to adapt to the environmental temperature.

Animals↗
PubMed1997

Structural constraints in protein engineering--the coenzyme specificity of Escherichia coli isocitrate dehydrogenase.

In a previous study we reported on the successful inversion of coenzyme specificity in isocitrate dehydrogenase (IDH) from NADP to NAD [Chen, R., Greer, A. & Dean, A. M. (1995) A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity, Proc. Natl Acad. Sci. USA 92, 11666-11670]. Here, we explore alternative means to generate NAD dependence in the NADP-dependent scaffold of Escherichia coli IDH. The results reveal that engineering a preference for NAD is constrained by the architecture of the IDH coenzyme binding pocket and confirms that the substituted Asp344 in the engineered enzyme is the major determinant of coenzyme specificity. Mutations in the 316-325 loop, which forms part of the coenzyme binding site, reduce activity through transmission of long-range conformational changes into the active site some 14 A distant. Conformational changes seen upon substituting Cys332-->Tyr are not directly involved with improving activity. Replacements at Cys201 reveal that subtle changes in the packing of hydrophobic residues (Met and Ile versus Leu) can elicit markedly different responses. We caution against using sequence alignments as the sole guide for mutagenesis and show how a combination of rational design of active-site residues based on X-ray structures and random substitutions at surrounding residues provides an efficient means to improve enzyme preference and catalytic efficiency towards novel substrates.

Amino Acid Sequence↗
PubMed2005

Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B.

The sensitivity of acetylcholinesterase (AChE) biosensors for insecticide detection could be increased substantially by engineering AChE B of Nippostrongylus brasiliensis. The introduction of 10 single and 4 double mutations into the AChE peptide chain led to an increase in sensitivity to 10 of the 11 insecticides tested. The combination of three mutants with the wild-type enzyme in a multienzyme biosensor array enabled the detection of 11 out of the 14 most important organophosphates and carbamates at concentrations below 10 microg/kg, the maximum residue limit of infant food. The detection limit for pirimiphos methyl could be reduced from 10 microg/L to a value as low as 1 ng/L (3.5 x 10(-)(12) mol/L). The newly created biosensors exhibited an extraordinary high storage stability. There was no loss of sensitivity of N. brasiliensis AChE B, immobilized on screen-printed, disposable electrodes, even after 17-month storage at room temperature.

Acetylcholinesterase↗
PubMed2005

Protein engineering of interferon alphas.

Interferon (IFN)-alphas constitute a family of proteins exhibiting high degree of homology in primary, secondary, and tertiary structure and display a high level of species specificity in their biological properties. However, small structural differences in these proteins may be responsible for a significant variety of biological actions. Understanding the structure and function of human IFN-alpha is very important. Recombinant techniques are important tools for the production and modification of IFN proteins. The first IFN hybrid, IFN-alpha1/alpha2 was constructed using recombinant technology in 1981. Subsequently, a number of IFN hybrids and mutants have been constructed, expressed and characterized. These hybrids and mutants have resulted in novel IFNs that either combine different biological properties from the parental proteins or have significantly different biological activity. Therefore, IFN hybrids and mutants have provided a powerful tool for studying the structure and function of these molecules. Also, these engineered IFNs may have important new therapeutic applications and may provide greater sights into understanding of the clinical activities of these molecules.

Amino Acid Sequence↗
PubMed2006

Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events.

Antiangiogenic therapy is nowadays one of the most active fields in cancer research. The first strategies, aimed at inhibiting tumor vascularization, included upregulation of endogenous inhibitors and blocking of the signals delivered by angiogenic factors. But interaction between endothelial cells and their surrounding extracellular matrix also plays a critical role in the modulation of the angiogenic process. This study introduces a new concept to enhance the efficacy of antibody-based antiangiogenic cancer therapy strategies, taking advantage of a key molecular event occurring in the tumor context: the proteolysis of collagen XVIII, which releases the endogenous angiogenesis inhibitor endostatin. By fusing the collagen XVIII NC1 domain to an antiangiogenic single-chain antibody, a multispecific agent was generated, which was efficiently processed by tumor-associated proteinases to produce monomeric endostatin and fully functional trimeric antibody fragments. It was demonstrated that the combined production in the tumor area of complementary antiangiogenic agents from a single molecular entity secreted by gene-modified cells resulted in enhanced antitumor effects. These results indicate that tailoring recombinant antibodies with extracellular matrix-derived scaffolds is an effective approach to convert tumor progression associated processes into molecular clues for improving antibody-based therapies.

Angiogenesis Inhibitors↗
PubMed2000

Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase. A protein engineering study of bacteriophage T4 NrdD.

Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX(2)C-CX(2)C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.

Bacterial Proteins↗
PubMed1996

Protein engineering of venom toxins by synthetic approach and NMR dynamic simulation: status of basic amino acid residues in waglerin I.

The tertiary structure of waglerin I has been determined by NMR and dynamic simulated annealing [Chuang et al., Biochim. Biophys. Acta 1292, 145-155 (1996)]. It is believed that the peptide basicity of waglerin may play an important role for its activity due to its high content of basic amino acids. In order to investigate the active site of the toxin, seven analogues of waglerin, [Ala3]-waglerin, [Ala7]-waglerin, [Ala10]-waglerin, [Ala14]-waglerin, [Ala18]-waglerin, [Ala20]-waglerin and [Ala22]-waglerin have been synthesized chemically by single replacement of basic amino acid residues one by one with Ala. By correlation of structures for each analogue with LD50 toxicity bioassays, it is found that the [Ala10]-waglerin exhibits no toxicity and the active site of the native toxin seems to reside in the proximity of the disulfide loop, which is spatially close to His10. Furthermore, the closer is the disulfide loop to the basic amino acid in waglerin, the more influential is the basic amino acid on the toxicity of waglerin. Based on the tertiary structure of waglerin, the structures of all synthetic analogues were derived based on computer-simulated modelling. By the pair-wise structural comparison, the disulfide loop in [Ala10]-waglerin analogue is found to be twisted as compared to the native form, in agreement with the lack of toxicity for this synthetic analogue.

Amino Acid Sequence↗
PubMed2004

Effects of protein engineering of canola procruciferin on its physicochemical and functional properties.

The primary structure of Brassica napus procruciferin 2/3a was engineered to elucidate structure-function relationships and to improve the functionality of cruciferin. The following mutants were constructed: (1) C287T, (2) DeltaII, variable region II was deleted; (3) C287T/DeltaII, mutation involving (1) and (2); (4) DeltaIV + A1aIV; and (5) DeltaIV + A3IV, variable region IV was replaced with variable region IV containing many charged residues from soybean glycinin A1aB1b and A3B4 subunits. Differential scanning calorimetry analysis revealed that the A1aIV region has a more favorable interaction with the procruciferin molecule than does A3IV as well as the original regions. On the basis of heat-induced precipitation analysis, it was concluded that replacement of the free cysteine residue with threonine (C287T) and insertion of charged regions (DeltaIV + A1aIV and DeltaIV + A3IV) could lead procruciferin to form soluble aggregates after heating. Low solubility was observed in mutants DeltaIV + A3IV, DeltaII, and C287T/DeltaII, especially between pH 4 and 6 at mu = 0.08, but not in DeltaIV + A1aIV, indicating that the number of acidic amino acid residues and the high number of glutamine residues are important factors for solubility at mu = 0.08. None of the mutants showed any improvements in emulsifying ability, indicating that destabilization and addition of the hydrophilic region are not effective for emulsification. The insertion of the A1aIV region in procruciferin made the molecule more susceptible to alpha-chymotrypsin.

Allergens↗
PubMed1999

Mapping the pro-region of carboxypeptidase B by protein engineering. Cloning, overexpression, and mutagenesis of the porcine proenzyme.

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 310 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.

Amino Acid Sequence↗