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Journal of Biochemistry Advance Access originally published online on March 2, 2009
Journal of Biochemistry 2009 145(6):751-762; doi:10.1093/jb/mvp034
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Sequence Perturbation Analysis: Addressing Amino Acid Indices to Elucidate the C-Terminal Role of Escherichia Coli Dihydrofolate Reductase

Hisashi Takahashi{dagger}, Akiko Yokota{dagger}, Tatsuyuki Takenawa and Masahiro Iwakura*

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

*To whom correspondence should be addressed. Tel: +81-29-861-6179, Fax: +81-29-856-4055, E-mail: masa-iwakura{at}aist.go.jp

Received January 19, 2009; Accepted February 19, 2009


  Abstract

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.

Key Words: amino acid indices, C-terminal role, dihydrofolate reductase, mutational sensitivity, sequence perturbation analysis

Abbreviations: CD, circular dichroism; DHF, dihydrofolate; DHFR, dihydrofolate reductase; MRE, mean residue ellipticity; MTX, methotrexate; SVD, singular value decomposition; THF, tetrahydrofolate; TMP, trimethoprim; UV, ultraviolet

{dagger}The first two authors are equal contributors to this work.


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