Sequence perturbation analysis: addressing amino acid indices to elucidate the C-terminal role of Escherichia coli dihydrofolate reductase.
Because amino acid residues intrinsically possess many factors participating in protein structures and functions, to determine main (or unique) factors at a specific site in a protein sequence should be of great help for understanding how a protein obtains its structure and function. In this study, we proposed a means of sequence perturbation analysis to address the above concerns involving comprehensive AA indices. We constructed all 19 possible single mutant proteins as to the three sites in the C-terminal of Escherichia coli dihydrofolate reductase (DHFR), and measured the activity and thermal stability of each of all the single mutant proteins. The significantly perturbed properties with each systematic single mutation at each mutational site were examined in terms of the linear correlation with each AA index. As a result, at each of Arg158 and Arg159 of DHFR, the AA index for the isoelectric points of amino acids showed strong correlation with the transition temperature of thermal denatuation, suggesting that the electrostatic interaction is the main factor influencing the C-terminal role of the DHFR. The feasibility and general versatility of our sequence perturbation analysis were also examined by application to other sites of DHFR.
Takahashi
Hisashi
H
Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Yokota
Akiko
A
Takenawa
Tatsuyuki
T
Iwakura
Masahiro
M
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
D029968:Escherichia coli Proteins; D013762:Tetrahydrofolate Dehydrogenase