Protein stabilisation by compatible solutes: effect of mannosylglycerate on unfolding thermodynamics and activity of ribonuclease A.
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 degrees 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.
Faria
Tiago Q
TQ
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal.
Knapp
Stefan
S
Ladenstein
Rudolf
R
Maçanita
António L
AL
Santos
Helena
H
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D004795:Enzyme Stability / Q000187:drug effects*; D005988:Glyceric Acids / Q000494:pharmacology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008358:Mannose / Q000031:analogs & derivatives* / Q000494:pharmacology*; D017510:Protein Folding; D045245:Rhodothermus / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014199:Trehalose / Q000494:pharmacology
D005988:Glyceric Acids; C402688:mannosylglycerate; D014199:Trehalose; D012259:Ribonuclease, Pancreatic; D008358:Mannose