Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes

Kazumi Kimura, Tomoyuki Numata, Yoshimitsu Kakuta, Makoto Kimura

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Abstract

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr101, Phe 102, Ala105, and Phe190 resulted in a significant decrease in themostability; the Tm values were 47-58°C compared to that for the wild type (64°C). Mutations of Pro 125, Gly127, Gly144, and Val165 caused a moderate decrease in thermostability (Tm: 60-62°C). In contrast, mutations of Asp107 and Gly173 did little effect on thermostability. The contribution of Tyr101, Phe102, Pro125, and Gly127 to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr101, Phe102, Ala 105, Pro125, Gly127, Gly144, Leu162, Val165, and Phe190 conserved in the RNase T2 family play an important role in the stability of the proteins.

Original languageEnglish
Pages (from-to)1748-1757
Number of pages10
JournalBioscience, Biotechnology and Biochemistry
Volume68
Issue number8
DOIs
Publication statusPublished - Aug 1 2004

Fingerprint

ribonuclease T(2)
Protein Stability
Amino Acids
Enzymes
Momordica charantia
Protein folding
Mutation
Proteins
Protein Folding
Seed
Digestion
Seeds
Peptide Hydrolases
Substitution reactions
Hot Temperature
Experiments

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

Cite this

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abstract = "The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr101, Phe 102, Ala105, and Phe190 resulted in a significant decrease in themostability; the Tm values were 47-58°C compared to that for the wild type (64°C). Mutations of Pro 125, Gly127, Gly144, and Val165 caused a moderate decrease in thermostability (Tm: 60-62°C). In contrast, mutations of Asp107 and Gly173 did little effect on thermostability. The contribution of Tyr101, Phe102, Pro125, and Gly127 to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr101, Phe102, Ala 105, Pro125, Gly127, Gly144, Leu162, Val165, and Phe190 conserved in the RNase T2 family play an important role in the stability of the proteins.",
author = "Kazumi Kimura and Tomoyuki Numata and Yoshimitsu Kakuta and Makoto Kimura",
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AU - Kimura, Kazumi

AU - Numata, Tomoyuki

AU - Kakuta, Yoshimitsu

AU - Kimura, Makoto

PY - 2004/8/1

Y1 - 2004/8/1

N2 - The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr101, Phe 102, Ala105, and Phe190 resulted in a significant decrease in themostability; the Tm values were 47-58°C compared to that for the wild type (64°C). Mutations of Pro 125, Gly127, Gly144, and Val165 caused a moderate decrease in thermostability (Tm: 60-62°C). In contrast, mutations of Asp107 and Gly173 did little effect on thermostability. The contribution of Tyr101, Phe102, Pro125, and Gly127 to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr101, Phe102, Ala 105, Pro125, Gly127, Gly144, Leu162, Val165, and Phe190 conserved in the RNase T2 family play an important role in the stability of the proteins.

AB - The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr101, Phe 102, Ala105, and Phe190 resulted in a significant decrease in themostability; the Tm values were 47-58°C compared to that for the wild type (64°C). Mutations of Pro 125, Gly127, Gly144, and Val165 caused a moderate decrease in thermostability (Tm: 60-62°C). In contrast, mutations of Asp107 and Gly173 did little effect on thermostability. The contribution of Tyr101, Phe102, Pro125, and Gly127 to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr101, Phe102, Ala 105, Pro125, Gly127, Gly144, Leu162, Val165, and Phe190 conserved in the RNase T2 family play an important role in the stability of the proteins.

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