Literature summary for 3.4.21.B57 extracted from

Takeuchi, Y.; Tanaka, S.; Matsumura, H.; Koga, Y.; Takano, K.; Kanaya, S.
Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon (2009), Biochemistry, 48, 10637-10643.

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Crystallization (Commentary)

Crystallization (Comment)	Organism
DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) is crystallized using sitting-drop vapor-diffusion method at 4°C. DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) is crystallized using hanging-drop method at 20°C. The structures of DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) are identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca2+-binding loop is destabilized. These proteins are slightly more stable than Pro-S324A. These results suggest that the Ca2+-binding loop is required for folding of Tk-subtilisin but does not seriously contribute to the stabilization of Tk-subtilisin in a native structure. The counting of amino acids refers to the enzyme protein without the signal peptide (amino acid 1-24) and the propeptide (amino acid 25-106)	Thermococcus kodakarensis

Crystallization (Comment)

Organism

DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) is crystallized using sitting-drop vapor-diffusion method at 4°C. DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) is crystallized using hanging-drop method at 20°C. The structures of DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) are identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca2+-binding loop is destabilized. These proteins are slightly more stable than Pro-S324A. These results suggest that the Ca2+-binding loop is required for folding of Tk-subtilisin but does not seriously contribute to the stabilization of Tk-subtilisin in a native structure. The counting of amino acids refers to the enzyme protein without the signal peptide (amino acid 1-24) and the propeptide (amino acid 25-106)

Thermococcus kodakarensis

Protein Variants

Protein Variants	Comment	Organism
additional information	to analyze the role of the Ca2+-binding loop, three mutant proteins, Deltaloop-Tk-subtilisin (Ca2+-binding loop is removed), DeltaCa2-Pro-S324A (Ca2+-binding site Ca2 is removed), and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed), are constructed. The structures of DeltaCa2-Pro-S324A (Ca2+-binding site Ca 2 is removed) and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed) are identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca2+-binding loop is destabilized. These proteins are slightly more stable than Pro-S324A	Thermococcus kodakarensis

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	the enzyme requires Ca2+ for folding and assumes a molten globule-like structure in the absence of Ca2+ even in the presence of Tk-propeptide. Tk-subtilisin contains seven Ca2+-binding sites. Four of them are located within a long loop, which mostly consists of a unique insertion sequence of this protein	Thermococcus kodakarensis

Organism

Organism	UniProt	Comment	Textmining
Thermococcus kodakarensis	P58502	sequence including singnal peptide (amino acid 1-24) and propeptide (amino acid 25-106)	-

Purification (Commentary)

Purification (Comment)	Organism
mutant proteins, Deltaloop-Tk-subtilisin (Ca2+-binding loop is removed), DeltaCa2-Pro-S324A (Ca2+-binding site Ca2 is removed), and DeltaCa3-Pro-S324A (Ca2+-binding site Ca3 is removed), are purified in a Ca2+-bound form	Thermococcus kodakarensis

Synonyms

Synonyms	Comment	Organism
Tk-subtilisin	-	Thermococcus kodakarensis