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D790E
by site-directed mutagenesis, proteolysis of Tudor staphylococcal nuclease does not occur either when the P1 position of the DAVD motif is mutated or after treatment with the pan-caspase inhibitor zVAD-fluoromethylketone, expression of mutant under normal conditions enhances cell proliferation in both cancer (HeLa) and non-cancer (HEK-293) cells compared with mock- and wild-type Tudor staphylococcal nuclease-transfected samples, under camptothecin-induced apoptosis, expression of Tudor staphylococcal nuclease mutant results in a 35% increment in viable HeLa cells, suggesting that caspase-mediated proteolysis of enzyme is important for the progression of apoptosis
DELTA140-149
-
deletion of the 10 C-terminal residues, mutant proteins are in a non-native or disordered state under physiological conditions, folding is induced by addition of an inhibitor or substrate
F34A
-
site-directed mutagenesis
G79S/H124LC80-C116
-
effects on the stability and conformation of the folded protein
H124LC77-C118
-
effects on the stability and conformation of the folded protein
H124LC79-C118
-
effects on the stability and conformation of the folded protein
H124LC80-C116
-
effects on the stability and conformation of the folded protein
I92A
-
site-directed mutagenesis, the mutant shows similar global stability like the wild-type enzyme
INS33A34
-
insertion of an alanine between residues 33 and 34, mutant proteins are in a non-native or disordered state under physiological conditions, folding is induced by addition of an inhibitor or substrate
K45C
-
insertion of a cysteine to enable labeling with thiol reactive ligands, e.g. 5,5'-dithiobis-2-nitrobenzoic acid, CD-spectra of wild type enzyme, mutant and mutant with 5,5'-dithiobis-2-nitrobenzoic acid label indicate, that the protein have very similar secondary structures
L103A
-
site-directed mutagenesis, the mutant shows similar global stability like the wild-type enzyme
L125A
-
site-directed mutagenesis, the mutant shows similar global stability like the wild-type enzyme
L25A
-
site-directed mutagenesis
L36A
-
site-directed mutagenesis
L38A
-
site-directed mutagenesis
P117G,/H124L/S128A
-
site-directed mutagenesis, a highly stable triple mutant
P117G/H124L/S128A
-
site-directed mutagenesis
P11A/P31A/P42A/P47T/P56A/P117G
proline free mutant, conformationally different from wild type protein, 1.4% of wild type activity
P47G/P117G/H124L/W140H
-
tryptophan-free mutant used for the insertion of a unique tryptophan at positions 15, 27, 61, 76, 91, 102, and 121, mutant enzymes used to study the enzyme folding kinetics, variants are destabilized but maintain the ability to refold in the native-like structure
T62C
-
designed for the insertion of a cysteine reactive label
T62P
highly destabilized variant of enzyme, exists in the unfolded state over a wide pH-range, can be fully refolded to the native folding by addition of osmolytes
V23A
-
site-directed mutagenesis
V66A
-
site-directed mutagenesis, the mutant shows similar global stability like the wild-type enzyme
V66F/P117G,/H124L/S128A
-
site-directed mutagenesis of the highly stable triple mutant P117G,/H124L/S128A, thermodynamic stability during guanidine hydrochloride denaturation of mutants is compared
V66G/P117G,/H124L/S128A
-
site-directed mutagenesis of the highly stable triple mutant P117G,/H124L/S128A, thermodynamic stability during guanidine hydrochloride denaturation of mutants is compared
V66N/P117G,/H124L/S128A
-
site-directed mutagenesis of the highly stable triple mutant P117G,/H124L/S128A, thermodynamic stability during guanidine hydrochloride denaturation of mutants is compared
V66Q/P117G,/H124L/S128A
-
site-directed mutagenesis of the highly stable triple mutant P117G,/H124L/S128A, thermodynamic stability during guanidine hydrochloride denaturation of mutants is compared
V66S/P117G,/H124L/S128A
-
site-directed mutagenesis of the highly stable triple mutant P117G,/H124L/S128A, thermodynamic stability during guanidine hydrochloride denaturation of mutants is compared
V66T/P117G,/H124L/S128A
-
site-directed mutagenesis of the highly stable triple mutant P117G,/H124L/S128A, thermodynamic stability during guanidine hydrochloride denaturation of mutants is compared
V66Y/P117G,/H124L/S128A
-
site-directed mutagenesis of the highly stable triple mutant P117G,/H124L/S128A, thermodynamic stability during guanidine hydrochloride denaturation of mutants is compared
V74A
-
site-directed mutagenesis
A128S
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to study inductive effects and longer-range interactions between elements of the network, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy)
A1C/Q149C
-
SNase double mutant, N- and C-terminal residues replaced by cysteine, constructed from the plasmid (pMT7-SN) of wild-type SNase using the Kunkel method, can form disulfide bond
A90S
-
pH-value, at which the acid-denaturation is half completed is 4.19, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 1.0 for the mutant enzyme compared to 1.8 for wild-type enzyme
D143G
-
Tm value for mutant enzyme is 50.53°C, compared to 50.98°C for wild-type enzyme
D146G
-
Tm value for mutant enzyme is 50.99°C, compared to 50.98°C for wild-type enzyme
D19G
-
Tm value for mutant enzyme is 52.06°C, compared to 50.98°C for wild-type enzyme
D21G
-
Tm value for mutant enzyme is 53.74°C, compared to 50.98°C for wild-type enzyme
D21N/T33V/T41I/S59A/P117G/A128A
-
hyperstable engineered form of staphylococcal nuclease (SNase)
D40G
-
Tm value for mutant enzyme is 50.44°C, compared to 50.98°C for wild-type enzyme
D77A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to study inductive effects and longer-range interactions between elements of the network, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy) , determination of tautomeric states of His121 and His124, pH near pI
D83G
-
Tm value for mutant enzyme is 37.21°C, compared to 50.98°C for wild-type enzyme
D95G
-
Tm value for mutant enzyme is 37.38°C, compared to 50.98°C for wild-type enzyme
DELTA1-139
-
mutant lacks tertiary structure, fluorescence of the mutant is much lower than that of the wild-type enzyme
DELTA1-141
-
intact tertiary conformation, melting point is nearly identical to wild-type enzyme
E101A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine shortrange effects on His124, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy)
E101G
-
Tm value for mutant enzyme is 43.04°C, compared to 50.98°C for wild-type enzyme
E10G
-
Tm value for mutant enzyme is 43.8°C, compared to 50.98°C for wild-type enzyme
E122G
-
Tm value for mutant enzyme is 44.12°C, compared to 50.98°C for wild-type enzyme
E129G
-
Tm value for mutant enzyme is 34.59°C, compared to 50.98°C for wild-type enzyme
E135G
-
Tm value for mutant enzyme is 44.54°C, compared to 50.98°C for wild-type enzyme
E135Q
-
charge neutralization
E142G
-
Tm value for mutant enzyme is 49.41°C, compared to 50.98°C for wild-type enzyme
E43G
-
Tm value for mutant enzyme is 54.99°C, compared to 50.98°C for wild-type enzyme
E52G
-
Tm value for mutant enzyme is 52.1°C, compared to 50.98°C for wild-type enzyme
E57G
-
Tm value for mutant enzyme is 46.6°C, compared to 50.98°C for wild-type enzyme
E67G
-
Tm value for mutant enzyme is 46.53°C, compared to 50.98°C for wild-type enzyme
E73A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to study inductive effects and longer-range interactions between elements of the network, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy)
E73G/D77G
-
loss of thermal stabilty of 47% relative to the wild-type protein
E73G/E75G
-
loss of thermal stabilty of 59% relative to the wild-type protein
E73G/E75G/D77G
-
loss of thermal stabilty of 65% relative to the wild-type protein
E75A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine short-range (up to 6.4 Angstrom) Coulomb and hydrogen bonding effects on His121, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy), determination of tautomeric states of His121 and His124, pH near pI
E75G
-
Tm value for mutant enzyme is 36.99°C, compared to 50.98°C for wild-type enzyme
E75G/D77G
-
loss of thermal stabilty of 58% relative to the wild-type protein
F34W/W140F
-
characterized by far and near UV CD, gel filtration, ANS-binding fluorescence, enzymatic parameters are similar to those of the wild type, similar substrate affinity to the wild type enzyme
F61W/W140A
-
the mutant shows reduced activity with higher Michaelis-Menten constants, Km, and lower maximum reaction rate compared to the wild-type enzyme, the mutant also shows a more rapid loss of secondary and tertiary structure by Gdn-HCl unfolding than the wild-type enzyme
F76W/W140H
-
mutation causes decrease in thermal stability
G20A
-
2% of wild-type activity. Km(Ca) is almost 20fold higher than the wild-type enzyme. Denaturation midpoint for the mutant enzyme is 1.5 M urea compared to 2.0 M urea for the wild-type enzyme
G20I
-
0.21% of wild-type activity. Km(Ca) is almost 50fold higher than the wild-type enzyme. Denaturation midpoint for mutant enzyme is 0.82 M urea compared to 2.0 M urea for the wild-type enzyme
G20V
-
0.45% of wild-type activity. Km(Ca) is almost 20fold higher than the wild-type enzyme. Denaturation midpoint for the mutynt enzyme is 1.1 M urea compared to 2.0 M urea for the wild-type enzyme
G50F/V51N/P117G/H124L/S128A/DELTA44-49
-
hyperstable, acid-resistant mutant form of enzyme known as delta+PHS
G79S
-
pH-value, at which the acid-denaturation is half completed is 4.39, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 1.4 for the mutant enzyme compared to 1.8 for wild-type enzyme
G88V
-
pH-value, at which the acid-denaturation is half completed is 3.57, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 3.0 for the mutant enzyme compared to 1.8 for wild-type enzyme
H124L
-
pH-value, at which the acid-denaturation is half completed is 2.98, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 2.8 for the mutant enzyme compared to 1.8 for wild-type enzyme
H124Q
-
charge neutralization
I92E
-
wild-type enzyme exhibits a broad range of pH-independence from pH 4.5 to pH 9, mutant enzyme exhibits pronounced pH-dependence with a maximal stability at pH 4.9
I92K
-
wild-type enzyme exhibits a broad range of pH-independence from pH 4.5 to pH 9.0, mutant enzyme exhibits pronounced pH-dependence with a maximal stability at pH 9.8
K127A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine shortrange effects on His124, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy)
K127Q
-
charge neutralization
K133A
-
intact tertiary conformation, melting point is 4.6°C lower than that of the wild-type enzyme
K133Q
-
charge neutralization
K28C/H124C
-
generated by site-directed mutagenesis, native and non-native conformations are observed, and the non-native conformation expands with increasing guanidinium hydrochloride concentrations, the non-native chains of the derivative exhibits different changes of persistence length at higher guanidinium hydrochloride concentrations, suggesting a subdomain-specific collapse of the denatured state of SNase, this local chain specific collapse is likely to play a role in modulating the formation of early intermediate during protein folding
K28C/K97C
-
generated by site-directed mutagenesis, native and non-native conformations are observed, and the non-native conformation expands with increasing guanidinium hydrochloride concentrations, the non-native chains of the derivative exhibits different changes of persistence length at higher guanidinium hydrochloride concentrations, suggesting a subdomain-specific collapse of the denatured state of SNase, this local chain specific collapse is likely to play a role in modulating the formation of early intermediate during protein folding
K28Q
-
charge neutralization
K48Q
-
charge neutralization
K63Q
-
charge neutralization
K64Q
-
charge neutralization
K70Q
-
charge neutralization
K78Q
-
charge neutralization
K84Q
-
charge neutralization
K97Q
-
charge neutralization
K9A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to study inductive effects and longer-range interactions between elements of the network, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy)
L25A
-
pH-value, at which the acid-denaturation is half completed is 4.15, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 1.2 for the mutant enzyme compared to 1.8 for wild-type enzyme
S28C
-
this mutant contains a five-amino acid type I beta-turn from concanavalin A in place of residues 27-30 of SNase
V66D/P117G/H124L/S128A
-
production by site-directed mutagenesis, pka value shifts to 7.79 and, after chemical denaturation, to 8.05
V66D/P117G/H124L/S128A/G50F/V51N/DELTA44-49
-
production by site-directed mutagenesis, pka value shifts to 8.95 and, after chemical denaturation, to 8.73
V66E/P117G/H124L/S128A
-
production by site-directed mutagenesis, pka value shifts to 8.80 and, after chemical denaturation, to 8.99
V66E/P117G/H124L/S128A/G50F/V51N/DELTA44-49
-
production by site-directed mutagenesis, pka value shifts to 9.07 and, after chemical denaturation, to 8.80
V66K/P117G/H124L/S128A
-
production by site-directed mutagenesis, pka value shifts to 6.35
V66K/P117G/H124L/S128A/G50F/V51N/DELTA44-49
-
production by site-directed mutagenesis, pka value shifts to 5.63 and, after chemical denaturation, to 5.83
V66L
-
pH-value, at which the acid-denaturation is half completed is 3.36, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 2.6 for the mutant enzyme compared to 1.8 for wild-type enzyme
V66L/G79S/G88V
-
pH-value, at which the acid-denaturation is half completed is 3.67, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 1.1 for the mutant enzyme compared to 1.8 for wild-type enzyme
V66L/G88V
-
pH-value, at which the acid-denaturation is half completed is 3.42, compared to pH 3.76 for wild-type enzyme. The apparent number of protons which trigger the denaturation and are taken up by the protein upon denaturation is 1.6 for the mutant enzyme compared to 1.8 for wild-type enzyme
W140C
-
DNA hydrolysis activity is 75% of wild-type activity
W140D
-
DNA hydrolysis activity is 65% of wild-type activity
W140E
-
DNA hydrolysis activity is 65% of wild-type activity
W140G
-
DNA hydrolysis activity is 75% of wild-type activity
W140I
-
DNA hydrolysis activity is 70% of wild-type activity
W140K
-
DNA hydrolysis activity is 70% of wild-type activity
W140M
-
DNA hydrolysis activity is 70% of wild-type activity
W140N
-
DNA hydrolysis activity is 75% of wild-type activity
W140P
-
DNA hydrolysis activity is 55% of wild-type activity
W140Q
-
DNA hydrolysis activity is 75% of wild-type activity
W140R
-
DNA hydrolysis activity is 75% of wild-type activity
W140S
-
DNA hydrolysis activity is 75% of wild-type activity
W140T
-
DNA hydrolysis activity is 75% of wild-type activity
W140V
-
DNA hydrolysis activity is 75% of wild-type activity
Y54C/I139C
-
production by site-directed mutagenesis, the oxidized form assumes a more compact denatured structure under acidic conditions than the wild type, the kinetic measurements reveal that the refolding reactions of both the reduced and oxidized forms of mutant are similar to those of the wild type protein
Y54C/I139C/DELTA140-149
-
production by site-directed mutagenesis, under physiological conditions, the reduced form appears to assume a denatured structure, in contrast, the oxidized form forms a native-like structure
Y91A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine short-range (up to 6.4 Angstrom) Coulomb and hydrogen bonding effects on His121, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy)
Y91F
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine short-range (up to 6.4 Angstrom) Coulomb and hydrogen bonding effects on His121, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy) , determination of tautomeric states of His121 and His124, pH near pI
Y93A
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine short-range (up to 6.4 Angstrom) Coulomb and hydrogen bonding effects on His121, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy) , determination of tautomeric states of His121 and His124, pH near pI
Y93F
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine short-range (up to 6.4 Angstrom) Coulomb and hydrogen bonding effects on His121, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy) , determination of tautomeric states of His121 and His124, pH near pI
Y93W/W140A
-
the mutant shows reduced activity with higher Michaelis-Menten constants, Km, and lower maximum reaction rate compared to the wild-type enzyme, the mutant also shows a more rapid loss of secondary and tertiary structure by Gdn-HCl unfolding than the wild-type enzyme
D77G
-
loss of catalytic efficiency of 16% and thermal stabilty of 26% relative to the wild-type protein
D77G
-
Tm value for mutant enzyme is 44.14°C, compared to 50.98°C for wild-type enzyme
D77N
-
charge reversal
D77N
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to study inductive effects and longer-range interactions between elements of the network, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy) , determination of tautomeric states of His121 and His124, pH near pI
E73G
-
loss of catalytic efficiency of 24% and thermal stabilty of 22% relative to the wild-type protein
E73G
-
Tm value for mutant enzyme is 37°C, compared to 50.98°C for wild-type enzyme
E75Q
-
charge reversal
E75Q
-
single point mutation in D21N/T33V/T41I/S59A/P117G/A128A, designed to examine short-range (up to 6.4 Angstrom) Coulomb and hydrogen bonding effects on His121, pKa values of histidines (His8, His46, His121, His124) are obtained by analysis of the pH titration monitored through the 1 H chemical shifts of the C(epsilon) H resonance of each histidine (NMR spectroscopy)
V66K
-
production by site-directed mutagenesis, pka value shifts to 6.38 after chemical denaturation
V66K
-
the substitution affects the H/D exchange properties of the protein globally, even when Lys66 is neutral. The conformational dynamics of the protein probed by H/D exchange indicate that, while both the global fluctuation and the local fluctuation are increased by the substitution, the global fluctuations are enhanced by protonation of Lys-66
W140A
-
DNA hydrolysis activity is 70% of wild-type activity
W140A
-
mutant lacks tertiary structure, fluorescence of the mutant is much lower than that of the wild-type enzyme
W140A
-
the mutant shows reduced activity with higher Michaelis-Menten constants, Km, and lower maximum reaction rate compared to the wild-type enzyme, the mutant also shows a more rapid loss of secondary and tertiary structure by Gdn-HCl unfolding than the wild-type enzyme
W140F
-
mutation causes decrease in thermal stability
W140F
-
native-like structure and native-like activity under physiological conditions
W140H
-
mutation causes decrease in thermal stability
W140H
-
native-like structure and native-like activity under physiological conditions
W140L
-
DNA hydrolysis activity is 75% of wild-type activity
W140L
-
mutation causes decrease in thermal stability
W140Y
-
mutation causes decrease in thermal stability
W140Y
-
native-like structure and native-like activity under physiological conditions
additional information
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enzyme inactivation by siRNA expression in enzyme-expressing HeLa cells
additional information
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knockdown of SND1 in QGY-7703 cells inhibits establishment of xenografts in nude mice
additional information
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suppression in QGY-7703 cells by si RNA, SND1-17-AT1Rsi
additional information
knockdown of the enzyme by dsRNA
additional information
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knockdown of the enzyme by dsRNA
additional information
construction of nuc1 and nuc1/nuc2 deletion mutant strains
additional information
construction of nuc1 and nuc1/nuc2 deletion mutant strains
additional information
four chimeric His-tagged fusion proteins are constructed by splicing together: 1. the N-terminal Nuc secretion signal or NucB leader to the Nuc2 C-terminal active domain, or 2. the N-terminal Nuc2 membrane anchor to the NucA and NucB C-terminal active domains, construction of nuc2 and nuc1/nuc2 deletion mutant strains
additional information
four chimeric His-tagged fusion proteins are constructed by splicing together: 1. the N-terminal Nuc secretion signal or NucB leader to the Nuc2 C-terminal active domain, or 2. the N-terminal Nuc2 membrane anchor to the NucA and NucB C-terminal active domains, construction of nuc2 and nuc1/nuc2 deletion mutant strains
additional information
generation of a nuc1/nuc2 double deletion mutant
additional information
generation of a nuc1/nuc2 double deletion mutant
additional information
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generation of a nuc1/nuc2 double deletion mutant
additional information
-
generation of six single mutations were made in a highly stable triple mutant of nucleasemost nuclease, the mutants do not denature by a three-state mechanism, modeling, overview
additional information
-
ten cavity-containing variants of the highly stable form of the enzyme known as DELTA+PHS SNase are described, the DELTA+ PHS reference protein bears stabilizing substitutions in the C-terminal helix (G50F, V51N, P117G, H124L, and S128A), and a deletion of the mobile X loop (residues 44-49), which is part of the active site. Variants with substitutions in the C-terminal domain and the interface between alpha and beta subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. In contrast, cavity creation in the beta-barrel subdomain leads to minimal perturbation of the structure of the folded state
additional information
-
the enzyme is fused in a chimeric protein to artificial zinc-finger protein, which inhibits virus DNA replication in planta and in 293H cells by blocking binding of a viral replication protein to its replication origin. The resulting hybrid nuclease AZP-SNase cleaves its target DNA plasmid efficiently and sequence-specifically in vitro, and expressed in cells, it inhibits human papillomavirus HPV-18 DNA replication cleaving an HPV-18 ori plasmid around its binding site, overview
additional information
-
four chimeric His-tagged fusion proteins are constructed by splicing together: 1. the N-terminal Nuc secretion signal or NucB leader to the Nuc2 C-terminal active domain, or 2. the N-terminal Nuc2 membrane anchor to the NucA and NucB C-terminal active domains, construction of nuc2 and nuc1/nuc2 deletion mutant strains
-
additional information
-
construction of nuc1 and nuc1/nuc2 deletion mutant strains
-
additional information
-
generation of a nuc1/nuc2 double deletion mutant
-
additional information
-
almost all mutations are destabilizing, the average loss of stability for all of the charge neutralization substitutions is 0.5 kcal/mol and the average loss of stability for all of the charge reversal substitutions is 1.0 kcal/mol
additional information
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generation by site-directed mutagenesis of C-terminal truncated SNases, i.e. SNase137, SNase139, SNase140, and SNase141 containing residues 1-137, 1-139, 1-140, and 1-141, respectively. The mutants show reduced activities compared to the wild-type enzyme. Determination of the secondary structures of the four SNase fragments, overview