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deltaV88
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deletion mutant
G68A
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substitution in the NS2B sequence, decreased activity compared with that of the wild-type enzyme
G81A
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substitution in the NS2B sequence, decreased activity compared with that of the wild-type enzyme
L73A
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substitution in the NS2B sequence, decreased activity compared with that of the wild-type enzyme
Q77A
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substitution in the NS2B sequence, mutation has greater effects on substrate binding than on the reaction rate
V88D
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reduced activity compared with that of the wild-type
V88K
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reduced activity compared with that of the wild-type
W60A
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substitution in the NS2B sequence, decreased activity compared with that of the wild-type enzyme
D129A
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mutant shows 39fold reduction in catalytic efficacy compared to wild-type
D129E
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no significant loss of activity
D129L
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no significant loss of activity
D129R
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no significant loss of activity
D129S
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no significant loss of activity
G133A
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mutant shows 10fold reduced catalytic activity
G148A
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mutant exhibits impaired autolytic activity
I165A
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mutant shows a drastic decrease in catalytic efficacy and a drastic increase in Km
K26A
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mutant exhibits impaired autolytic activity
L115A
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mutant shows activity comparable to wild-type
N152A
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mutant shows no a complete inactivation but a 60fold reduced catalytic activity
S163A
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mutant shows 12fold reduction in catalytic efficacy compared to wild-type
T134A
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mutant shows 2.2fold reduction in catalytic efficacy compared to wild-type
A125C/V162C
site-directed mutagenesis, the mutant shows 32% reduction in activity due to the cysteine substitution. Linked A125C/V162C, in which both mutations are within the protease domain, does not migrate differently without DTT. The unlinked either trap does not form intermolecular disulfide bonds
H51A
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site-directed mutagenesis, the mutation of NS3 protein impairs the charge interaction and the pH dependence of the conformational changes. It stabilizes the open conformation, while the addition of BPTI still converts NS2B-NS3p from open to closed conformation
H51A/S75C
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complex mutations
I73C/P106C
site-directed mutagenesis, the linked open trap mutant does not form intermolecular disulfide bonds appreciably, as evidenced by the lack of higher-molecular weight bands. Unlinked I73C/P106C forms a small amount of NS2BS-SNS3pro, which shows retarded migration as NS2B and NS3pro electrophorese as a single disulfide-bonded complex
K117A
site-directed mutagenesis
K117C
site-directed mutagenesis
K117R
site-directed mutagenesis
K117R/T122R
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mutant is analyzed by 15N-HSQC spectra with and without inhibitor 4-guanidino-benzoic acid-4-nitrophenyl ester
K15A
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autocleavage detected by SDS-PAGE is supressed
P176G
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mutation in the 11-amino acid linker region (169-179): a 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis are observed
S75C/K117C
site-directed mutagenesis, linked S75C/K117C is induced to form the NS2BS-SNS3pro closed conformation in the presence of active site inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that the mutation forms a disulfide bond when the enzyme is resting in the active conformation. The S75C/K117C pair is close together in the closed conformation but far apart in the open conformation and should therefore trap this variant in the closed conformation upon intramolecular disulfide bond formation
A125C/V162C
Dengue virus type 2 Thailand/16681/1984
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site-directed mutagenesis, the mutant shows 32% reduction in activity due to the cysteine substitution. Linked A125C/V162C, in which both mutations are within the protease domain, does not migrate differently without DTT. The unlinked either trap does not form intermolecular disulfide bonds
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I73C/P106C
Dengue virus type 2 Thailand/16681/1984
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site-directed mutagenesis, the linked open trap mutant does not form intermolecular disulfide bonds appreciably, as evidenced by the lack of higher-molecular weight bands. Unlinked I73C/P106C forms a small amount of NS2BS-SNS3pro, which shows retarded migration as NS2B and NS3pro electrophorese as a single disulfide-bonded complex
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K117A
Dengue virus type 2 Thailand/16681/1984
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site-directed mutagenesis
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S75C/K117C
Dengue virus type 2 Thailand/16681/1984
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site-directed mutagenesis, linked S75C/K117C is induced to form the NS2BS-SNS3pro closed conformation in the presence of active site inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that the mutation forms a disulfide bond when the enzyme is resting in the active conformation. The S75C/K117C pair is close together in the closed conformation but far apart in the open conformation and should therefore trap this variant in the closed conformation upon intramolecular disulfide bond formation
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C1123A
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results in dramatic reductions in both auto-cleavage of the NS2/3 precursor and NS3 protease activities to levels that are close to the background of the assay
C1125A
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results in dramatic reductions in both auto-cleavage of the NS2/3 precursor and NS3 protease activities to levels that are close to the background of the assay
C1171A
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results in dramatic reductions in both auto-cleavage of the NS2/3 precursor and NS3 protease activities to levels that are close to the background of the assay
C1185A
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has no effect on either auto-cleavage of the NS2/3 precursor and NS3 protease activities
C184A
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mutation in the NS2 active site, is defective in NS2-3 processing. When Hepatitis C virus polyproteins with NS2 containing a C184A mutation are co-expressed, NS2 and NS3 cleavage products are detected
C922A
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reduces NS2/3 auto-cleavage, but has no significant effect on NS3 protease activity
H1175A
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reduces NS3 protease activity. Auto-cleavage activity is indistinguishable from wild-type
H143A
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mutation in the NS2 active site, is defective in NS2-3 processing. When Hepatitis C virus polyproteins with NS2 containing a H143A mutation are co-expressed, NS2 and NS3 cleavage products are detected
H143A/C184A
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mixing wild-type Flag-NS2-3 with double-mutant HA-NS2-3(H143A/C184A) or vice versa yields only cleaved wild-type NS2, whereas the double-mutant polypeptide remains unprocessed because neither of the composite active sites is functional, when a wild-type and a double-mutant NS2-3 dimerize
H952A
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totally abolishes auto-cleavage of the NS2/3 precursor, whereas NS3 protease activity is not abolished
L1026P/A1027P
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totally abolishes auto-cleavage of the NS2/3 precursor, whereas NS3 protease activity is not abolished
S1165A
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completely abrogates NS3 protease activity, but has no effect on NS2/3 auto-cleavage
D42G
has no dramatic effect on either the catalytic activity (50% of wild-type) or self-proteolysis of NS2B-NS3pro. Aprotinin efficiently inhibits proteolytic activity
G460L
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no autolytic cleavage, but is an efficient enzyme against the artificial substrate CFP-LQYTKRGGVLWD-YFP
P131K/T132P
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decrease in kcat, which is partially compensated by an improvement in the substrate binding. Shift of the cleavage preferences toward that of the Dengue virus proteinase
D42G
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has no dramatic effect on either the catalytic activity (50% of wild-type) or self-proteolysis of NS2B-NS3pro. Aprotinin efficiently inhibits proteolytic activity
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G460L
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no autolytic cleavage, but is an efficient enzyme against the artificial substrate CFP-LQYTKRGGVLWD-YFP
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R76L
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the mutant displays reduced catalytic efficiency compared to the wild type enzyme
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S135A
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no autolytic cleavage, is enzymatically inactive
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T52V
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the mutant displays increased catalytic efficiency compared to the wild type enzyme
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S138A
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is inactive towards Bz-Nle-Lys-Arg-Arg-7-amino-4-methylcoumarin
C143S
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site-directed mutagenesis, the NS2B mutant cannot form the disulfide bond between C143 residues of neighboring NS3
S135A
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inactive
S75C
site-directed mutagenesis
S75C
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site-directed mutagenesis, the mutation of NS2B protein
G22S
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no activity
G22S
resistant to autoproteolysis at the flexible linker region, retains 3% residual activity
H51A
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no activity
H51A
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devoid of any proteolytic activity and incapable of autolytically cleaving either the NS2B sequence or the NS3pro-hel boundary. Resistant to the proteolysis in trans by the external, highly active NS2B-NS3pro construct
K48A
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autolysis-resistant
K48A
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is a single-chain protein and fully resistant to autolysis at the linker region, exhibits high stability and functional activity, cleaves pyroglutamic acid-Arg-Thr-Lys-Arg-7-amino-4-methylcoumarin
K48A
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mutation of the C-terminal amino acid residue of the NS2B sequence, inactivates the autolytic cleavage site. Does not have any significant effect on the catalytic activity. Readily cleaved in cis by the integral NS2B-NS3pro activity
K48A
resistant to autoproteolysis at the flexible linker region. Aprotinin efficiently inhibits proteolytic activity
K48A
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the mutant displays slightly increased catalytic efficiency compared to the wild type enzyme
R76L
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kinetic parameters are similar to those of the wild-type. Shift of the cleavage preferences toward that of the Dengue virus proteinase
R76L
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the mutant displays reduced catalytic efficiency compared to the wild type enzyme
S135A
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inactive mutant
S135A
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no autolytic cleavage, is enzymatically inactive
T52V
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exhibits a significant loss of kcat, which is compensated by an improvement in substrate binding. Does not cause any significant differences in the cleavage subsite specificity. Cleavage preferences of the T52V mutant construct are similar to those of the original West Nile virus proteinase
T52V
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the mutant displays increased catalytic efficiency compared to the wild type enzyme
G22S
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no activity
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G22S
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resistant to autoproteolysis at the flexible linker region, retains 3% residual activity
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H51A
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devoid of any proteolytic activity and incapable of autolytically cleaving either the NS2B sequence or the NS3pro-hel boundary. Resistant to the proteolysis in trans by the external, highly active NS2B-NS3pro construct
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K48A
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mutation of the C-terminal amino acid residue of the NS2B sequence, inactivates the autolytic cleavage site. Does not have any significant effect on the catalytic activity. Readily cleaved in cis by the integral NS2B-NS3pro activity
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K48A
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the mutant displays slightly increased catalytic efficiency compared to the wild type enzyme
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K48A
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resistant to autoproteolysis at the flexible linker region. Aprotinin efficiently inhibits proteolytic activity
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R95*A/R29G
construction of a complex containing residues 45 to 95 of the NS2B cofactor linked via a Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly (G4SG4) linker to the NS3 protease to avoid auto-proteolysis and create the linked protease, the last arginine residue of the NS2B cofactor before the G4SG4 linker is mutated to alanine (R95*A), and arginine 29 of the NS3 protease domain is mutated to glycine (R29G)
R95*A/R29G
Zika virus Puerto Rico isolate
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construction of a complex containing residues 45 to 95 of the NS2B cofactor linked via a Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly (G4SG4) linker to the NS3 protease to avoid auto-proteolysis and create the linked protease, the last arginine residue of the NS2B cofactor before the G4SG4 linker is mutated to alanine (R95*A), and arginine 29 of the NS3 protease domain is mutated to glycine (R29G)
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additional information
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mutation at G153 results in inactivation
additional information
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random mutagenesis study. Among 103 clones, 58 represent functionally inactive mutants. All tested alanine-substituted mutants exhibit impaired autolytic activity
additional information
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separate expression of dengue virus NS3 protease and its NS2B cofactor domain and efficient co-refolding to form a stable complex, method evaluation, overview. This straightforward and robust method allows for separate isotope labeling of the two proteins, facilitating analysis by NMR spectroscopy, detailed overview. Unlinked NS2B-NS3pro behaves better in NMR spectroscopy than linked NS2B-NS3pro, which has resulted in the backbone resonance assignment of the unlinked NS2B-NS3 complex bound to a peptidic boronic acid inhibitor
additional information
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16 possible mutants bearing and an exchanged Ile to Val are analyzed by 15N-HSQC spectra with and without inhibitor 4-guanidino-benzoic acid-4-nitrophenyl ester
additional information
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7 Ser to Ala mutants are analyzed by 15N-HSQC spectra with and without inhibitor 4-guanidino-benzoic acid-4-nitrophenyl ester
additional information
multiple types of protein constructs have been used for NS2B-NS3pro expression. In one type, a Gly4SerGly4 linker joins NS2B to NS3pro, reported to improve expression and purification, while another type requires co-expression of the unlinked NS2B and NS3 regions. The catalytic activity is slightly different between the linked and unlinked protein constructs, with the unlinked form showing activity approximately 0-5fold greater than that of the linked form, likely due to constraints introduced by the synthetic linker
additional information
Dengue virus type 2 Thailand/16681/1984
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multiple types of protein constructs have been used for NS2B-NS3pro expression. In one type, a Gly4SerGly4 linker joins NS2B to NS3pro, reported to improve expression and purification, while another type requires co-expression of the unlinked NS2B and NS3 regions. The catalytic activity is slightly different between the linked and unlinked protein constructs, with the unlinked form showing activity approximately 0-5fold greater than that of the linked form, likely due to constraints introduced by the synthetic linker
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additional information
mutation in the 11-amino acid linker region (169-179): a Gly residue before Pro174 is introduced in DENV4 NS2B18NS3: The mutant protein having a glycine insertion in its linker region adopts another conformation where the protease domain has not rotated by an angle of 161° relative to the helicase domain
additional information
D(32)DD(34) substitution for AAA inactivates NS2B-NS3pro, retains 1% residual activity
additional information
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D(32)DD(34) substitution for AAA inactivates NS2B-NS3pro, retains 1% residual activity
additional information
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D(32)DD(34) substitution for AAA inactivates NS2B-NS3pro, retains 1% residual activity
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additional information
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mutation studies at the residues His94, Asp118 and Ser176 reveal that Asp118-His94 bond play an important role in the structural stability of NS2B-NS3 complex
additional information
construction of eZiPro that contains the NS2B cofactor region (residues 45-96) linked with NS3 protein (residues 1-177) through the last five residues of NS2B (K126TGKR130), and the gZiPro construct, which is similar except that the G4SG4 linker replaced K126TGKR130 of NS2B. Protease activity analysis of gZiPro, eZiPro and bZiPro
additional information
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construction of eZiPro that contains the NS2B cofactor region (residues 45-96) linked with NS3 protein (residues 1-177) through the last five residues of NS2B (K126TGKR130), and the gZiPro construct, which is similar except that the G4SG4 linker replaced K126TGKR130 of NS2B. Protease activity analysis of gZiPro, eZiPro and bZiPro
additional information
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construction of the NS2B cofactor region linked to the NS3 protease domain via a glycine-rich flexible linker (Gly4-Ser-Gly4 linker), structural dynamics of this conventional Zika protease (gZiPro) using NMR spectroscopy, overview. Although the glycine-rich linker in gZiPro does not alter the overall folding of the protease in solution, gZiPro is not homogenous in ion exchange chromatography. Compared to the unlinked protease construct, the artificial linker affects the chemical environment of many residues including H51 in the catalytic triad. Direct comparison of ZIKV protease constructs with and without an artificial linker, namely gZiPro (with the Gly4-Ser-Gly4 linker), bZiPro (unlinked NS2B NS3protease), and eZiPro (NS2B-NS3 junction sequence KTGKR as the linker). Solution NMR spectrum of recombinant gZiPro enzyme without inhibitor, overview. The inhibitor-bound gZiPro-AcKR-H complex adopts the closed conformation. Effect of the linker on the chemical environment of residues, dynamics of the linked protease, overview