It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect. Study holds ProTherm entries: 6820, 6821, 6822, 6823, 6824, 6825, 6826, 14370, 14371 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:32 p.m.
|Number of data points||18|
|Proteins||Guanyl-specific ribonuclease Sa ; RIBONUCLEASE ; Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Experimental Assay: ddG_H2O pH:2.5, buffers:glycine: 30 mM ; Experimental Assay: Cm pH:2.5, buffers:glycine: 30 mM ; Experimental Assay: m pH:2.5, buffers:glycine: 30 mM ; Experimental Assay: ddG_H2O buffers:MOPS: 30 mM, pH:7.0 ; Experimental Assay: Cm buffers:MOPS: 30 mM, pH:7.0 ; Experimental Assay: m buffers:MOPS: 30 mM, pH:7.0|
|Libraries||Mutations for sequence ACDYTCGSNCYSSSDVSTAQAAGYQLHEDGETVGSNSYPHKYNNYEGFDFSVSSPYYEWPILSSGDVYSGGSPGADRVVFNENNQLAGVITHTGASGNNFVECT ; Mutations for sequence DVSGTVCLSALPPEATDTLNLIASDGPFPYSQDGVVFQNRESVLPTQSYGYYHEYTVITPGARTRGTRRIITGEATQEDYYTGDHYATFSLIDQTC|