Protein Stabilisation by Compatible Solutes: Effect of Mannosylglycerate on Unfolding Thermodynamics and Activity of Ribonuclease A

• Published in: Chembiochem : a European journal of chemical biology Vol. 4; no. 8; pp. 734 - 741
• Main Authors: Faria, Tiago Q, Knapp, Stefan, Ladenstein, Rudolf, Maçanita, António L, Santos, Helena
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 04.08.2003; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Animals; calorimetry; Calorimetry, Differential Scanning; Cattle; compatible solutes; Enzyme Stability - drug effects; Glyceric Acids - pharmacology; Hot Temperature; Hydrogen-Ion Concentration; Mannose - analogs & derivatives; Mannose - pharmacology; mannosylglycerate; Medicin och hälsovetenskap; Protein Folding; protein stabilisation; Rhodothermus - chemistry; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - metabolism; Thermodynamics; Trehalose - pharmacology
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200300574

Differential scanning calorimetry, optical spectroscopy, and activity measurements were used to investigate the effect of mannosylglycerate, a negatively charged osmolyte widely distributed among thermophilic and hyperthermophilic archaea and bacteria, on the thermal unfolding of ribonuclease A (RNase A). For comparison, assays in the presence of trehalose, a canonical solute in mesophiles, and potassium chloride were also carried out. A thermodynamic analysis was performed by using differential scanning calorimetry data. The changes in the heat capacity for unfolding were similar for the different solutes examined. Mannosylglycerate was an efficient thermostabiliser of RNase A and induced an increase of 6 °C mole-¹ in the melting temperature. Moreover, the performance of mannosylglycerate as a stabiliser depended on the net charge of the molecule, with the maximal effect being observed at pH values above 4.5. Analysis of the enthalpic and entropic contributions to unfolding, derived from calorimetric data, revealed that the stabilisation rendered by mannosylglycerate is primarily achieved through a decrease in the unfolding entropy. Also, the number of protons taken up by RNase A upon denaturation in the presence of mannosylglycerate was considerably higher than with other solutes, a result consistent with a more rigid structure of the native protein. Mannosylglycerate (potassium salt) inhibited the activity of RNase A, albeit to a smaller extent than KCl, and acted as an efficient suppressor of aggregation of the denatured protein, thereby having a remarkable beneficial effect on the inactivation of RNase A upon thermal denaturation. The results are discussed in view of the physiological role of this charged compatible solute.