Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic n-terminal mutations
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Standard
Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic n-terminal mutations. / Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter Waaben; Christensen, Hans Erik Mølager; Kožíšek, Milan; Nagata, Kyosuke; Czene, Anikó; Gyurcsik, Béla.
I: Protein Science, Bind 23, Nr. 8, 2014, s. 1113-1122.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic n-terminal mutations
AU - Németh, Eszter
AU - Körtvélyesi, Tamás
AU - Thulstrup, Peter Waaben
AU - Christensen, Hans Erik Mølager
AU - Kožíšek, Milan
AU - Nagata, Kyosuke
AU - Czene, Anikó
AU - Gyurcsik, Béla
PY - 2014
Y1 - 2014
N2 - The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7=KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7 >> KGNK>KGNG ∼ GGNK> GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.
AB - The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7=KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7 >> KGNK>KGNG ∼ GGNK> GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.
KW - DNA cleavage
KW - Flow linear dichroism
KW - Isothermal calorimetry
KW - Positively charged N-terminal residues
U2 - 10.1002/pro.2497
DO - 10.1002/pro.2497
M3 - Journal article
C2 - 24895333
AN - SCOPUS:84904734052
VL - 23
SP - 1113
EP - 1122
JO - Protein Science
JF - Protein Science
SN - 0961-8368
IS - 8
ER -
ID: 130978368