In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, DeltaC(p), that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn. As a control, Ala was introduced at the same three positions. We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln. As a control, Asn was introduced at Gln41 as well. The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry. The effect of the amino acid substitutions on the structure was also evaluated by comparing the (1)H-(15)N HSQC spectra of the ubiquitin variants. It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding. However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding. The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants. The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding. These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign. However, the observed changes in DeltaC(p) are several times larger than those predicted, based on the changes in water accessible surface area upon substitution. Study holds ProTherm entries: 11393, 11394, 11395, 11396, 11397, 11398, 11399, 11400, 11401, 11402, 11403 Extra Details: a 6xHis-tag was added to the C-terminus of the molecule heat capacity changes; hydration; amino acid substitutions;,differential scanning calorimetry; NMR
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:42 p.m.
|Number of data points||12|
|Proteins||E3 ubiquitin-protein ligase RSP5 ; Ubiquitin-binding protein CUE2|
|Assays/Quantities/Protocols||Experimental Assay: dCp temp:25.0 C ; Experimental Assay: dCp ; Experimental Assay: Tm|
|Libraries||Mutations for sequence A:NDDHESKLSILMDMFPAISKSKLQVHLLENNNDLDLTIGLLLKENDDKS/B:MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG|