ABSTRACT
A plot of the Gibbs free energy of unfolding vs. temperature is calculated for baker's yeast phosphoglycerate kinase in 150 mM sodium phosphate (pH=7.0) from a combination of reversible differential scanning calorimetry measurements and isothermal guanidine hydrochloride titrations. The stability curve reveals the existence of two stable, folded conformers with an abrupt conformational transition occurring at 24 degrees C. The transition state thermodynamics for the low- to high-temperature conformational change are calculated from slow-scan-rate differential scanning calorimetry measurements where it is found that the free energy barrier for the conversion is 90 kJ/mol and the transition state possesses a significant unfolding quality. This analysis also confirms a nondenaturational conformational transition at 24 degrees C. The data therefore suggest that X-ray structures obtained from crystals grown below this temperature may differ considerably from the physiological structure and that the two conformers are not readily interconverted.
Subject(s)
Phosphoglycerate Kinase/chemistry , Saccharomyces cerevisiae/enzymology , Calorimetry, Differential Scanning , Enzyme Stability , Guanidine , Protein Conformation , Protein Denaturation , Protein Folding , Saccharomyces cerevisiae Proteins/chemistry , Temperature , ThermodynamicsABSTRACT
The stability curve - a plot of the Gibbs free energy of unfolding versus temperature - is calculated for bovine erythrocyte carbonic anhydrase in 150 mM sodium phosphate (pH = 7.0) from a combination of reversible differential scanning calorimetry measurements and isothermal guanidine hydrochloride titrations. The enzyme possesses two stable folded conformers with the conformational transition occurring at ~30 degrees C. The methodology yields a stability curve for the complete unfolding of the enzyme below this temperature but only the partial unfolding, to the molten globule state, above it. The transition state thermodynamics for the low- to physiological-temperature conformational change are calculated from slow-scan-rate differential scanning calorimetry measurements where it is found that the free energy barrier for the conversion is 90 kJ/mole and the transition state possesses a substantial unfolding quality. The data therefore suggest that the x-ray structure may differ considerably from the physiological structure and that the two conformers are not readily interconverted.
Subject(s)
Carbonic Anhydrases/chemistry , Carbonic Anhydrases/metabolism , Temperature , Animals , Calorimetry, Differential Scanning , Cattle , Guanidine/pharmacology , Kinetics , Protein Conformation/drug effects , Protein Denaturation/drug effects , Protein Folding , Thermodynamics , TitrimetryABSTRACT
Wide-complex tachycardia (WCT) is defined as a rhythm disturbance with a rate greater than 100 beats/min and a QRS complex duration of 0.12 seconds or more in the adult patient; in the pediatric patient, both rate and QRS complex width are age related. In evaluating this type of tachycardia, there are 2 broad categories usually discussed in the medical literature: ventricular and supraventricular with aberrant intraventricular conduction. There are several other important causes of a WCT encountered in clinical practice, which are less often discussed; these tachycardias often require specific therapies differing from the standard approach to WCT. These tachycardias are diverse; as such, the pathophysiology behind each form of WCT includes toxic, metabolic, and conduction system dysfunction mechanisms.
