Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface

Thermal inactivation of oligomeric enzymes is most often irreversible and is frequently accompanied by precipitation. We have engineered two symmetry related disulfide bridges (155-188′ and 188-155′) across the subunit interface of Lactobacillus casei thymidylate synthase, at sites chosen on the basis of an algorithm for the introduction of stereochemically unstrained bridges into proteins. In this communication, we demonstrate a remarkable enhancement in the thermal stability of the covalently cross-linked double disulfide containing dimeric enzyme. The mutant enzyme remains soluble and retains secondary structure even at 90°C, in contrast to the wild-type enzyme which precipitates at 52°C. Furthermore, the mutant enzyme has a temperature optimum of 55°C and possesses appreciable enzymatic activity at 65°C. Cooling restores complete activity, in the mutant protein, demonstrating reversible thermal unfolding. The results suggest that inter-subunit crosslinks can impart appreciable thermal stability in multimeric enzymes

Sub-Zero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen Deuterium Exchange Mass Spectrometry

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of deuterium label (back-exchange) during the course of the analysis. A method to limit loss of label during the separation stage of the analysis using sub-zero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30°C with negligible loss of deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of HDX MS in terms of mixture complexity, and the magnitude with which changes in deuteration level can be quantified

Covalent reinforcement of a fragile region in the dimeric enzyme thymidylate synthase stabilizes the protein against chaotrope-induced unfolding

Urea and guanidinium chloride induced unfolding of thymidylate synthase, a dimeric enzyme, and engineered interface mutants have been monitored by circular dichroism, fluorescence, and size-exclusion chromatography. Equilibrium unfolding studies show biphasic transitions, with a plateau between 3.5 and 5 M urea, when monitored by far-UV CD and fluorescence energy transfer employing an (aminoethylamino)naphthalenesulfonyl (AEDANS) label at the active site residue, Cys198. AEDANS was also specifically incorporated at position Cys155 in the mutant protein T155C. Direct excitation of this extrinsic fluorophore in the wild type protein (labeled at Cys198) and mutant T155C (labeled at Cys155) showed remarkable differences in the unfolding profiles. C155 AEDANS has a transition centered at 3.5 M urea, which is in contrast to Cys 198 AEDANS (5.5 M urea). Unfolding studies monitored by following intrinsic fluorescence of Trp residues which are located in a small structural domain suggest that this region of the protein is intrinsically fragile. The stable equilibrium intermediate is identified to be an ensemble of partially unfolded aggregated species by gel filtration studies. The chaotrope-induced denaturation of TS appears to proceed through a partially unfolded intermediate that is stabilized by aggregation. Dissociation and loss of structure occur concomitantly at high denaturant concentrations. Introduction of two symmetrically positioned disulfide bridges across the dimer interface in the triple mutant T155C/E188C/C244T (TSMox) stabilized the protein against denaturant-induced unfolding. Aggregate formation was completely abolished in the mutant TSMox, which also enhanced the overall structural stability of the protein. Structural reinforcement of the fragile interface in thymidylate synthase results in dramatic stabilization toward chaotrope-induced unfolding

Covalent Reinforcement of a Fragile Region in the Dimeric Enzyme Thymidylate Synthase Stabilizes the Protein against Chaotrope-Induced Unfolding

Urea and guanidinium chloride induced unfolding of thymidylate synthase, a dimeric enzyme, and engineered interface mutants have been monitored by circular dichroism, fluorescence, and sizeexclusion chromatography. Equilibrium unfolding studies show biphasic transitions, with a plateau between 3.5 and 5 M urea, when monitored by far-UV CD and fluorescence energy transfer employing an (aminoethylamino)naphthalenesulfonyl (AEDANS) label at the active site residue, Cys198. AEDANS was also specifically incorporated at position Cys155 in the mutant protein T155C. Direct excitation of this extrinsic fluorophore in the wild type protein (labeled at Cys198) and mutant T155C (labeled at Cys155) showed remarkable differences in the unfolding profiles. C155 AEDANS has a transition centered at 3.5 M urea, which is in contrast to Cys 198 AEDANS (5.5 M urea). Unfolding studies monitored by following intrinsic fluorescence of Trp residues which are located in a small structural domain suggest that this region of the protein is intrinsically fragile. The stable equilibrium intermediate is identified to be an ensemble of partially unfolded aggregated species by gel filtration studies. The chaotrope-induced denaturation of TS appears to proceed through a partially unfolded intermediate that is stabilized by aggregation. Dissociation and loss of structure occur concomitantly at high denaturant concentrations. Introduction of two symmetrically positioned disulfide bridges across the dimer interface in the triple mutant T155C/E188C/C244T (TSMox) stabilized the protein against denaturant-induced unfolding. Aggregate formation was completely abolished in the mutant TSMox, which also enhanced the overall structural stability of the protein. Structural reinforcement of the fragile interface in thymidylate synthase results in dramatic stabilization toward chaotrope-induced unfolding

Subzero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as −30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified

Experimental and Computational Assessment of Conditionally Essential Genes in Escherichia coli

Genome-wide gene essentiality data sets are becoming available for Escherichia coli, but these data sets have yet to be analyzed in the context of a genome scale model. Here, we present an integrative model-driven analysis of the Keio E. coli mutant collection screened in this study on glycerol-supplemented minimal medium. Out of 3,888 single-deletion mutants tested, 119 mutants were unable to grow on glycerol minimal medium. These conditionally essential genes were then evaluated using a genome scale metabolic and transcriptional-regulatory model of E. coli, and it was found that the model made the correct prediction in ∼91% of the cases. The discrepancies between model predictions and experimental results were analyzed in detail to indicate where model improvements could be made or where the current literature lacks an explanation for the observed phenotypes. The identified set of essential genes and their model-based analysis indicates that our current understanding of the roles these essential genes play is relatively clear and complete. Furthermore, by analyzing the data set in terms of metabolic subsystems across multiple genomes, we can project which metabolic pathways are likely to play equally important roles in other organisms. Overall, this work establishes a paradigm that will drive model enhancement while simultaneously generating hypotheses that will ultimately lead to a better understanding of the organism