Effects of amino acid substitutions at four fully buried sites of the ubiquitin molecule on the thermodynamic parameters (enthalpy, Gibbs energy) of unfolding were evaluated experimentally using differential scanning calorimetry. The same set of substitutions has been incorporated at each of four sites. These substitutions have been designed to perturb packing (van der Waals) interactions, hydration, and/or hydrogen bonding. From the analysis of the thermodynamic parameters for these ubiquitin variants we conclude that: (i) packing of non-polar groups in the protein interior is favorable and is largely defined by a favorable enthalpy of van der Waals interactions. The removal of one methylene group from the protein interior will destabilize a protein by approximately 5 kJ/mol, and will decrease the enthalpy of a protein by 12 kJ/mol. (ii) Burial of polar groups in the non-polar interior of a protein is highly destabilizing, and the degree of destabilization depends on the relative polarity of this group. For example, burial of Thr side-chain in the non-polar interior will be less destabilizing than burial of Asn side-chain. This decrease in stability is defined by a large enthalpy of dehydration of polar groups upon burial. (iii) The destabilizing effect of dehydration of polar groups upon burial can be compensated if these buried polar groups form hydrogen bonding. The enthalpy of this hydrogen bonding will compensate for the unfavorable dehydration energy and as a result the effect will be energetically neutral or even slightly stabilizing. Study holds ProTherm entries: 15501, 15503, 15504, 15505, 15506, 15507, 15508, 15509, 15510, 15511, 15512, 15513, 15514, 15515 Extra Details: Mutation carries two substitutions, K63R and R42E and has a ~13 degree C higher than the other wild type thermostability. protein stability; hydration; packing; amino acid substitutions; differential scanning calorimetry
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:46 p.m.
|Number of data points||28|
|Proteins||E3 ubiquitin-protein ligase RSP5 ; Ubiquitin-binding protein CUE2|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: ddG|
|Libraries||Mutations for sequence A:NDDHESKLSILMDMFPAISKSKLQVHLLENNNDLDLTIGLLLKENDDKS/B:MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG|