Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4428 Extra Details: osmolytes; two-state unfolding transitions;,thermodynamic parameters; sarcosine
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:25 p.m.
|Number of data points||1|
|Assays/Quantities/Protocols||Experimental Assay: Tm|
|Libraries||Mutations for sequence MAMSSLWWTAILLLALTVSMCYGVPTYQDFLRTHVDFPKTSFPNIAAYCNVMMVRRGINVHGRCKSLNTFVHTDPRNLNTLCINQPNRALRTTQQQLPVTDCKLIRSHPTCSYTGNQFNHRVRVGCWGGLPVHLDGTFP|