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How Site‐Directed Mutagenesis Alters the Solubility and Stability of RNase Sa
Author(s) -
Schmalzer Katherine Michelle,
Trevino Saul,
Pace Nick,
Scholtz J. Martin
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a501
Subject(s) - solubility , asparagine , serine , amino acid , rnase p , mutant , biochemistry , threonine , mutagenesis , chemistry , mutation , escherichia coli , enzyme , organic chemistry , rna , gene
The study of protein solubility has been a recurring research topic in the field of biochemistry and medicine for the last several years. Protein solubility research has allowed insight into the characteristics of human diseases including Alzheimer’s and Type II diabetes mellitus. The goals and aims of this experiment were to determine how alterations in the amino acid sequence of Ribonuclease (RNase) Sa affected the solubility of the protein. Additionally, double mutants were constructed to determine if the mutations have an additive effect. Recombinant proteins were synthesized using Escherichia coli and mutations were made to specific amino acids to obtain substantial alterations in the solubility. The data was used with accessible surface area calculations to rationalize the effects of the mutations on the solubility. The thermodynamic stability of these variants was also measured and compared to wild type. Solubility results suggest serine and threonine contribute much more favorably than asparagine. Therefore, future mutations will be made to Thr76 in order to create an amino acid solubility scale. Stability of the double mutants was additive and the overall stability of RNase Sa was not altered for the single or double mutants. Funding for this research was from grant DBI‐013924 from the National Science Foundation (NSF).