Literature summary for 3.1.31.1 extracted from

Baran, K.L.; Chimenti, M.S.; Schlessman, J.L.; Fitch, C.A.; Herbst, K.J.; Garcia-Moreno, B.E.
Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease (2008), J. Mol. Biol., 379, 1045-1062.

Cloned(Commentary)

Cloned (Comment)	Organism
gene insert cloned into pET24a+plasmid, transformation of BL21(DE3) Escherichia coli cells for expression	Staphylococcus sp.

Crystallization (Commentary)

Crystallization (Comment)	Organism
E75A mutant: 4°C, hanging-drop, precipitating solution 37% (v/v) 2-methyl-2,4-pentanediol and 25 mM potassium phosphate buffer at pH 6.0, protein concentration 9.9 mg/ml before mixing with equal volume of precipitating solution. E75Q: 4°C, hanging-drop, precipitating solution 38% (v/v) 2-methyl-2,4-pentanediol and 25 mM potassium phosphate buffer at pH 6.0, starting from 14.2 mg/ml protein. Side chain of His121 is unaffected by elimination of Glu75, histidine moves closer to Glu101 in the structure with E75A. Both crystal structures suggest that the network of polar or ionizable groups connected through hydrogen bonding or charge-charge interactions is a rigid unit, incapable of reorganizing even when strongly stabilizing interactions between Glu75, His124, and Tyr93 are disrupted	Staphylococcus sp.

Crystallization (Comment)

Organism

E75A mutant: 4°C, hanging-drop, precipitating solution 37% (v/v) 2-methyl-2,4-pentanediol and 25 mM potassium phosphate buffer at pH 6.0, protein concentration 9.9 mg/ml before mixing with equal volume of precipitating solution. E75Q: 4°C, hanging-drop, precipitating solution 38% (v/v) 2-methyl-2,4-pentanediol and 25 mM potassium phosphate buffer at pH 6.0, starting from 14.2 mg/ml protein. Side chain of His121 is unaffected by elimination of Glu75, histidine moves closer to Glu101 in the structure with E75A. Both crystal structures suggest that the network of polar or ionizable groups connected through hydrogen bonding or charge-charge interactions is a rigid unit, incapable of reorganizing even when strongly stabilizing interactions between Glu75, His124, and Tyr93 are disrupted

Staphylococcus sp.

Protein Variants

Protein Variants	Comment	Organism
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)	Staphylococcus sp.
D21N/T33V/T41I/S59A/P117G/A128A	hyperstable engineered form of staphylococcal nuclease (SNase)	Staphylococcus sp.
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	Staphylococcus sp.
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	Staphylococcus sp.
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)	Staphylococcus sp.
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)	Staphylococcus sp.
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	Staphylococcus sp.
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)	Staphylococcus sp.
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)	Staphylococcus sp.
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)	Staphylococcus sp.
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)	Staphylococcus sp.
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	Staphylococcus sp.
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	Staphylococcus sp.
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	Staphylococcus sp.

General Stability

General Stability	Organism
examination of acid-induced denaturation (monitored by intrinsic fluorescence of Trp140) measuring deltaG0H2O: D21N/T33V/T41I/S59A/P117G/A128A mutant: 9.5 kcal/mol, K9A mutant: 6.5 kcal/mol, Y91A mutant: 4.5 kcal/mol, Y91F mutant: 6.7 kcal/mol, Y93A mutant: 3.6 kcal/mol, Y93F mutant: 7.5 kcal/mol, E101A mutant: 8.4 kcal/mol, K127A mutant: 8.9 kcal/mol, A128S mutant: 8.2 kcal/mol. Examination of denaturation (monitored by intrinsic fluorescence of Trp140) with guanidinium chloride measuring the pH at the midpoint of the acid-induced unfolding: D21N/T33V/T41I/S59A/P117G/A128A mutant: 3.05, K9A mutant: 3.16, E73A mutant: 3.21, E75A mutant: 3.28, E75Q mutant: 3.27, D77A mutant: 3.31, Y91A mutant: 3.83, Y93A mutant: 4.10, E101A mutant: 3.14, K127A mutant: 2.94, A128S mutant: 3.20	Staphylococcus sp.

Organism

examination of acid-induced denaturation (monitored by intrinsic fluorescence of Trp140) measuring deltaG0H2O: D21N/T33V/T41I/S59A/P117G/A128A mutant: 9.5 kcal/mol, K9A mutant: 6.5 kcal/mol, Y91A mutant: 4.5 kcal/mol, Y91F mutant: 6.7 kcal/mol, Y93A mutant: 3.6 kcal/mol, Y93F mutant: 7.5 kcal/mol, E101A mutant: 8.4 kcal/mol, K127A mutant: 8.9 kcal/mol, A128S mutant: 8.2 kcal/mol. Examination of denaturation (monitored by intrinsic fluorescence of Trp140) with guanidinium chloride measuring the pH at the midpoint of the acid-induced unfolding: D21N/T33V/T41I/S59A/P117G/A128A mutant: 3.05, K9A mutant: 3.16, E73A mutant: 3.21, E75A mutant: 3.28, E75Q mutant: 3.27, D77A mutant: 3.31, Y91A mutant: 3.83, Y93A mutant: 4.10, E101A mutant: 3.14, K127A mutant: 2.94, A128S mutant: 3.20

Staphylococcus sp.

Metals/Ions

Metals/Ions	Comment	Organism	Structure
additional information	examination of salt sensitivity of pka-values (chemical shifts analyzed by NMR while titrating) His8, His46, His121 and His124. Tested variants D21N/T33V/T41I/S59A/P117G/A128A mutant, E75A mutant, E75Q mutant, and E101A mutant in 0.01 M, 0.10 M, and 1.0 M KCl	Staphylococcus sp.

Organism

Organism	UniProt	Comment	Textmining
Staphylococcus sp.	-	-	-

Synonyms

Synonyms	Comment	Organism
SNase	-	Staphylococcus sp.
staphylococcal nuclease	-	Staphylococcus sp.

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	examination of denaturation (monitored by intrinsic fluorescence of Trp140) due to heat (pH 7): D21N/T33V/T41I/S59A/P117G/A128A mutant(Tm = 72°C), K9A mutant (Tm = 71°C), E73A mutant (Tm = 67°C), E75A mutant (Tm = 70°C), E75Q mutant (Tm = 70°C), D77A mutant (Tm = 67°C), Y91A mutant (Tm = 58°C), Y93A mutant (Tm = 52°C), E101A mutant (Tm = 71°C), K127A mutant (Tm = 75°C), A128S mutant (Tm = 71°C)	Staphylococcus sp.