Chemical shifts in proteins: a shielding trajectory analysis of the fluorine nuclear magnetic resonance spectrum of the Escherichia coli galactose binding protein using a multipole shielding polarizability-local reaction field-molecular dynamics approach

Pearson, John G.; Oldfield, Eric; Lee, Frederick S.; Warshel, Arieh

doi:10.1021/ja00068a049

Cited by 87 publications

(103 citation statements)

References 6 publications

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“…However, the largest shifts coincided with cases that likely involved the making or breaking of hydrogen bonds to the probe, as hypothesized on structural grounds (46,61), which would convolute the observed effects (28). In the second case, the chemical shift dispersion for 19 F-labeled amino acids incorporated at multiple locations within galactose binding protein were calibrated using a computational model to suggest that site-to-site variations in the projection of the electrostatic field on the C─F bond vector as large as 40 MV∕cm occur (20). However, conflicting computational arguments have been made that short-range chemical interactions (i.e., van der Waals interactions) between fluorine atoms and surrounding groups can dominate the chemical shift dispersion rather than local electrostatics (19,62,63).…”

Section: Discussionmentioning

confidence: 99%

“…These studies have inferred electrostatic potentials based on free energy measurements [e.g., pK a shifts of ionizable residues (9-11), reactivity differences among cysteines (12,13), equilibrium binding constants of charged ligands (14), and redox potential shifts (15)] or spectroscopic variations in probes incorporated into or bound to proteins [e.g., fluorescent dyes (16), 19 F and 13 C chemical shifts (17)(18)(19)(20)(21), and EPR-measured g-factor perturbations of nitroxides (22)]. These studies have spurred the ongoing development of electrostatic calculation methodology (6,7) and contributed greatly to our general recognition that the solvation environment within proteins is very different from water.…”

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confidence: 99%

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Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Fafarman¹,

Sigala²,

Schwans³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

216

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Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV∕cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK a prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK a near 7 was included. UV-absorbance and 13 C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis. E xtensive structural studies of enzymes have revealed that biological catalysis occurs within sequestered active site crevices that solvate reacting substrates with an anisotropic and chemically complex constellation of charged, polar, and hydrophobic groups. But beyond visualization of the chemical composition and architecture of active sites permitted by the rich library of available protein structures, our understanding of the electrostatic nature and properties of this highly heterogeneous environment and its role in molecular recognition and catalysis is largely based on simulations. Computations using three-dimensional structures routinely provide electrostatic potentials, as in Fig. 1A. These potentials have been used to identify and characterize protein-protein and protein-ligand binding sites, and interaction energies derived from these calculations have suggested key contributions from the electrostatic environment to enzymatic rate enhancement and specificity (1-5). However, even the most sophisticated computational methods have multiple approximations and limitations (6-8) and, most importantly, have not been rigorously evaluated through cycles of nontrivial computational predictions and experimental tests. Incisive and quantitative experimental measures o...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Fafarman¹,

Sigala²,

Schwans³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

216

View full text Add to dashboard Cite

show abstract

“…It is known that, because of the large polarizability of C-F bonds, 19 F chemical shifts are susceptible to weak long-range electronic interactions. [28] Apparently, this is even more so for the B-F bond, presumably because of the increased electronegativity difference between the two atoms. As far as we are aware, splitting of the fluorine resonances has not been reported previously.…”

Section: F Nmr Spectroscopymentioning

confidence: 99%

Redox‐Controlled Fluorescence Modulation in a BODIPY‐Quinone Dyad

et al. 2008

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The synthesis and properties of two closely related boron dipyrromethene (BODIPY) derived dyads, incorporating redoxactive quinone units appended at the meso position, are described. For one dyad, the quinone unit is attached directly to the BODIPY core, whereas a phenylene spacer separates the two units in the second compound. Each of the quinone units is readily converted, by both chemical and electrochemical means, to the corresponding hydroquinone derivative. The strong fluorescence normally associated with the BODIPY unit is efficiently quenched in both dyads in their quinone forms. This is attributed to deactivation of the first excited singlet state by a way of an intramolecular electron-

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“…Based on a Stark tuning rate of 0.7 cm −1 /(MV/cm), this peak shift indicates that the net electric field experienced along the -CN bond axis has increased by 0.6 MV/cm, a minor change compared to reports of electric field changes in proteins of 10 MV/cm or higher upon mutation. [20][21][22][23] This minor change suggested, most simply, a minimal contribution to local field from replacing active site disordered waters with hydrophobic steroid rings, a conclusion we directly tested as described below.…”

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confidence: 95%

Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site?

et al. 2007

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We report the site-specific incorporation of a thiocyanate vibrational probe into the active site oxyanion hole of ketosteroid isomerase (KSI) to test the effect of hydrophobic steroid binding and solvent exclusion on the local electrostatic environment at this position. While binding of an uncharged ground state steroid analog shifts the observed -CN vibrational frequency by +0.4 cm −1 relative to unliganded KSI, binding of an intermediate steroid analog containing localized negative charge results in a +2.8 cm −1 shift. Based on a Stark tuning rate of 0.7 cm −1 /(MV/cm), this shift indicates a fivefold larger change in the projection of the local electric field along the -CN bond in the presence of the charged ligand. Binding of a single ring phenolate with oxyanion charge localization equivalent to the intermediate steroid analog but lacking distal hydrocarbon rings results in an identical -CN peak shift. We conclude that solvent exclusion and replacement by hydrophobic steroid rings negligibly alter the electrostatic environment within the KSI oxyanion hole. Development of localized negative charge analogous to that of the dienolate intermediate during steroid isomerization dramatically increases the magnitude of the local electric field. This increase reflects field contributions from the localized negative charge itself as well as possible increased ordering of active site dipoles in response to charge localization.The thousands of enzyme structures solved to date have consistently revealed that biological catalysis occurs within sequestered pockets containing complex interdigitations of polar and hydrophobic groups and from which water molecules are displaced upon substrate binding. 1 This chemical complexity has sparked considerable controversy regarding the electrostatic nature of active sites and the role of substrate binding and solvent exclusion in shaping active site electrostatics. [2][3][4][5][6][7] We report herein the site-specific incorporation of a thiocyanate vibrational probe 8 into the active site of Pseudomonas putida ketosteroid isomerase (KSI) to directly and quantitatively test the effect of steroid binding and concomitant solvent exclusion on the local electrostatic environment.KSI catalyzes a double-bond migration reaction in steroid substrates that involves formation of a dienolate intermediate within an active site oxyanion hole composed of Y16, protonated D103, and a preponderance of hydrophobic residues (Scheme 1A and Figure S1). Numerous physical changes occur upon steroid binding that might alter the local electrostatic environment within the KSI active site. In the free enzyme an ordered water molecule is sboxer@stanford.edu, herschla@stanford.edu. Supporting Information Available: Experimental methods and Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 September 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Auth...

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Chemical shifts in proteins: a shielding trajectory analysis of the fluorine nuclear magnetic resonance spectrum of the Escherichia coli galactose binding protein using a multipole shielding polarizability-local reaction field-molecular dynamics approach

Cited by 87 publications

References 6 publications

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Redox‐Controlled Fluorescence Modulation in a BODIPY‐Quinone Dyad

Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site?

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