Experimental and theoretical study of electrostatic effects on the isoelectric pH and the pKa of the catalytic residue His-102 of the recombinant ribonuclease fromBacillus amyloliquefaciens (barnase)

Bastyns, Katrin; Froeyen, Matheus; Dı́az, José Fernando; Volckaert, Guido; Engelborghs, Yves

doi:10.1002/(sici)1097-0134(199603)24:3<370::aid-prot10>3.0.co;2-j

Cited by 10 publications

(2 citation statements)

References 27 publications

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“…This analysis provides apparent titration behavior for the ionizable groups involved in the cleavage reaction. This approach of comparing pK a prediction results with kinetic data has literature precedent [20, 43, 44]. For a couple of related metallonucleases, one limb of the profile has been assigned to the attacking water molecule [8, 37].…”

Section: Resultsmentioning

confidence: 99%

Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

Xie

Briggs

Dupureur

2010

Journal of Inorganic Biochemistry

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Metallonucleases conduct metal dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pK a 's for active site moieties very close to this species, and measurement of the pK a of a specific water molecule is difficult to access experimentally. Here we describe a computational approach for exploring the local electrostatic influences on the water-derived nucleophile in metallonucleases featuring the common PD…(D/E)xK motif. We utilized UHBD to predict the pK a 's of active site groups, including that of a water molecule positioned to act as a nucleophile. The pK a of a Mg(II)-ligated water molecule hydrogen bonded to the conserved Lys70 in a Mg(II)-PvuII enzyme complex was calculated to be 6.5. The metal and the charge on the Lys group were removed in separate experiments; both resulted in the elevation of the pK a of this water molecule, consistent with contributions from both moieties to lowering this pK a . This behavior is preserved among other PD…(D/E)xK metallonucleases. pK a 's extracted from the pH dependence of the single turnover rate constant are compared to previous experimental data and the above predicted pK a 's.

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Section: Resultsmentioning

confidence: 99%

Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

Xie

Briggs

Dupureur

2010

Journal of Inorganic Biochemistry

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“…We conclude from the comparisons with eGFP (t = 2.7 ns) [39] and with GFP-S65T (t = 2.9 ns) [40] that the emission stems from the B-state, which is the predominant anionic state of GFP. [28,41] Additionally, histidine, in spite of being a known fluorescence quencher and here in the intermediate vicinity of the chromophore, [42] does not quench fluorescence of the anionic GFP chromophore. Obviously, the chromophore and the histidine do not adopt a suitable geometry for efficient electron transfer.…”

Section: In Memoriam Stephan Gerharzmentioning

confidence: 94%

Replacement of Highly Conserved E222 by the Photostable Non‐photoconvertible Histidine in GFP

et al. 2014

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The widely used green fluorescent protein (GFP) decarboxylates upon irradiation; this involves removal of the acidic function of the glutamic acid at position 222, thereby resulting in the irreversible photoconversion of GFP. To suppress this phenomenon, the photostable, non-photoconvertible histidine was introduced at position 222 in GFP. The variant E222H shows negligible photodynamic processes and high expression yield. In addition, the stable and bright fluorescence over a wide pH range makes the E222H protein an alternative for GFP in fluorescence imaging and spectroscopy. Other fluorescent proteins are predicted to benefit from replacement of the catalytic glutamic acid by histidine.

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Barnase: Fluorescence Analysis of A Three Tryptophan Protein

Engelborghs

Fersht

Topics in Fluorescence Spectroscopy

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Experimental and theoretical study of electrostatic effects on the isoelectric pH and the pKa of the catalytic residue His-102 of the recombinant ribonuclease fromBacillus amyloliquefaciens (barnase)

Cited by 10 publications

References 27 publications

Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

Replacement of Highly Conserved E222 by the Photostable Non‐photoconvertible Histidine in GFP

Barnase: Fluorescence Analysis of A Three Tryptophan Protein

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