Long-range surface charge-charge interactions in proteins

Comparison of experimental results with calculations from a theoretical method

Ron Loewenthal, Javier Sancho, Tapani Reinikainen, Alan Fersht

Research output: Contribution to journalArticleScientificpeer-review

71 Citations (Scopus)

Abstract

Long-range coulombic interaction energies between surface-charges in barnase and subtilisin have been determined to provide data for calibrating theoretical methods. The pKa of His18 in barnase can be measured accurately by titrating the fluorescence of Trp94 that is significantly quenched on protonation of His18. The pKa of His64, the active site base of subtilisin, has previously been shown to be measured accurately from the pH dependence of kcat /Km for the hydrolysis of substrates. The titration curves of both histidine residues fit the theoretical equations for the ionization of single groups with great precision; the Hill constants for wild-type and mutant enzymes are all close to 1·0. The coulombic interaction energies of distant charged side-chains with the protonated form of His18 and His64 have been measured from changes in pKa of these residues on mutation of those charged side-chains. The interaction energies between single charges on the surfaces of the proteins at low ionic strength are small, some 0·3-0·5 kcal mol-1 at a distance of 12 Å, and fall gradually with distance to 0·05-0·3 kcal mol-1 at 20 Å. Multiple mutations are frequently additive. Effects are larger in subtilisin than in barnase, possibly related to the degree of solvent exposure of the charge. These data have been used to benchmark the finite-difference method of calculating electrostatic interactions as implemented in the program DelPhi. There is reasonable agreement between the calculated and measured results as a function of both position and ionic strength.

Original languageEnglish
Pages (from-to)574 - 583
Number of pages10
JournalJournal of Molecular Biology
Volume232
Issue number2
DOIs
Publication statusPublished - 1993
MoE publication typeA1 Journal article-refereed

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Subtilisin
Osmolar Concentration
Benchmarking
Mutation
Proteins
Static Electricity
Histidine
Catalytic Domain
Membrane Proteins
Hydrolysis
Fluorescence
Enzymes
Bacillus amyloliquefaciens ribonuclease

Cite this

Loewenthal, Ron ; Sancho, Javier ; Reinikainen, Tapani ; Fersht, Alan. / Long-range surface charge-charge interactions in proteins : Comparison of experimental results with calculations from a theoretical method. In: Journal of Molecular Biology. 1993 ; Vol. 232, No. 2. pp. 574 - 583.
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abstract = "Long-range coulombic interaction energies between surface-charges in barnase and subtilisin have been determined to provide data for calibrating theoretical methods. The pKa of His18 in barnase can be measured accurately by titrating the fluorescence of Trp94 that is significantly quenched on protonation of His18. The pKa of His64, the active site base of subtilisin, has previously been shown to be measured accurately from the pH dependence of kcat /Km for the hydrolysis of substrates. The titration curves of both histidine residues fit the theoretical equations for the ionization of single groups with great precision; the Hill constants for wild-type and mutant enzymes are all close to 1·0. The coulombic interaction energies of distant charged side-chains with the protonated form of His18 and His64 have been measured from changes in pKa of these residues on mutation of those charged side-chains. The interaction energies between single charges on the surfaces of the proteins at low ionic strength are small, some 0·3-0·5 kcal mol-1 at a distance of 12 {\AA}, and fall gradually with distance to 0·05-0·3 kcal mol-1 at 20 {\AA}. Multiple mutations are frequently additive. Effects are larger in subtilisin than in barnase, possibly related to the degree of solvent exposure of the charge. These data have been used to benchmark the finite-difference method of calculating electrostatic interactions as implemented in the program DelPhi. There is reasonable agreement between the calculated and measured results as a function of both position and ionic strength.",
author = "Ron Loewenthal and Javier Sancho and Tapani Reinikainen and Alan Fersht",
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Long-range surface charge-charge interactions in proteins : Comparison of experimental results with calculations from a theoretical method. / Loewenthal, Ron; Sancho, Javier; Reinikainen, Tapani; Fersht, Alan.

In: Journal of Molecular Biology, Vol. 232, No. 2, 1993, p. 574 - 583.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Long-range surface charge-charge interactions in proteins

T2 - Comparison of experimental results with calculations from a theoretical method

AU - Loewenthal, Ron

AU - Sancho, Javier

AU - Reinikainen, Tapani

AU - Fersht, Alan

N1 - Project code: BIO8018

PY - 1993

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N2 - Long-range coulombic interaction energies between surface-charges in barnase and subtilisin have been determined to provide data for calibrating theoretical methods. The pKa of His18 in barnase can be measured accurately by titrating the fluorescence of Trp94 that is significantly quenched on protonation of His18. The pKa of His64, the active site base of subtilisin, has previously been shown to be measured accurately from the pH dependence of kcat /Km for the hydrolysis of substrates. The titration curves of both histidine residues fit the theoretical equations for the ionization of single groups with great precision; the Hill constants for wild-type and mutant enzymes are all close to 1·0. The coulombic interaction energies of distant charged side-chains with the protonated form of His18 and His64 have been measured from changes in pKa of these residues on mutation of those charged side-chains. The interaction energies between single charges on the surfaces of the proteins at low ionic strength are small, some 0·3-0·5 kcal mol-1 at a distance of 12 Å, and fall gradually with distance to 0·05-0·3 kcal mol-1 at 20 Å. Multiple mutations are frequently additive. Effects are larger in subtilisin than in barnase, possibly related to the degree of solvent exposure of the charge. These data have been used to benchmark the finite-difference method of calculating electrostatic interactions as implemented in the program DelPhi. There is reasonable agreement between the calculated and measured results as a function of both position and ionic strength.

AB - Long-range coulombic interaction energies between surface-charges in barnase and subtilisin have been determined to provide data for calibrating theoretical methods. The pKa of His18 in barnase can be measured accurately by titrating the fluorescence of Trp94 that is significantly quenched on protonation of His18. The pKa of His64, the active site base of subtilisin, has previously been shown to be measured accurately from the pH dependence of kcat /Km for the hydrolysis of substrates. The titration curves of both histidine residues fit the theoretical equations for the ionization of single groups with great precision; the Hill constants for wild-type and mutant enzymes are all close to 1·0. The coulombic interaction energies of distant charged side-chains with the protonated form of His18 and His64 have been measured from changes in pKa of these residues on mutation of those charged side-chains. The interaction energies between single charges on the surfaces of the proteins at low ionic strength are small, some 0·3-0·5 kcal mol-1 at a distance of 12 Å, and fall gradually with distance to 0·05-0·3 kcal mol-1 at 20 Å. Multiple mutations are frequently additive. Effects are larger in subtilisin than in barnase, possibly related to the degree of solvent exposure of the charge. These data have been used to benchmark the finite-difference method of calculating electrostatic interactions as implemented in the program DelPhi. There is reasonable agreement between the calculated and measured results as a function of both position and ionic strength.

U2 - 10.1006/jmbi.1993.1412

DO - 10.1006/jmbi.1993.1412

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