A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor

Koone, Jordan C.; Dashnaw, Chad M.; Gonzalez, Mayte; Shaw, Bryan F.

doi:10.1002/pro.4384

Cited by 3 publications

(5 citation statements)

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“…Measuring the net charge (Z) of a folded, solvated protein (at pH ≠ pI) is analytically challenging. ,, Only a fraction of proteins (<40 by our estimate) have had their values of net charge directly measured in the folded, solvated state at pH ≠ pI. Isoelectric points represent only one value of Z (i.e., Z= 0) at one pH (i.e., pH = pI) and do not express the magnitude of charge at pH ≠ pI. , In this study, “protein charge ladders” of trunc Tt Rp were synthesized and analyzed with capillary zone electrophoresis (CE) to measure changes in the net charge of trunc Tt Rp upon reduction of Fe(III) to Fe(II). − ,− …”

Section: Resultsmentioning

confidence: 95%

“…Tryptic fragments of acetylated protein were generated as previously described. 37 Prior to proteolysis, trunc Tt Rp was reduced with dithiothreitol (DTT, 2 mM) at room temperature for 1 h. Trypsin Gold (Promega, WI, USA) was added at a molar ratio of 1:20 trypsin to trunc Tt Rp and incubated at 37 °C for 24 h in 50 mM Tris-HCl buffer (pH 8.8). Following digestion this sample was sequenced with LC-MS/MS (LTQ LX/Orbitrap Q Exactive Focus Thermo Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…H/D exchange was performed as previously described. 37 Briefly, isotopic exchange was initiated by diluting 100 μL of protein into 900 μL of 99.9% D 2 O (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA). H/D exchange was conducted at 22 °C and pH read = 7.4.…”

Section: Methodsmentioning

confidence: 99%

“…Amide Hydrogen/Deuterium Exchange LC-ESI-MS. H/D exchange was performed as previously described. 37 Briefly, isotopic exchange was initiated by diluting 100 μL of protein into 900 μL of 99.9% D 2 O (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA). H/D exchange was conducted at 22 °C and pH read = 7.4.…”

Section: Recombinant Expression and Purification Of The Truncated Rie...mentioning

confidence: 99%

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Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer

Koone,

Simmang,

Saenger

et al. 2023

J. Am. Chem. Soc.

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The degree to which redox-driven proton pumps regulate net charge during electron transfer (ΔZ ET ) remains undetermined due to difficulties in measuring the net charge of solvated proteins. Values of ΔZ ET can reflect reorganization energies or redox potentials associated with ET and can be used to distinguish ET from proton(s)-coupled electron transfer (PCET). Here, we synthesized protein "charge ladders" of a Rieske [2Fe−2S] subunit from Thermus thermophilus (truncTtRp) and made 120 electrostatic measurements of ΔZ ET across pH. Across pH 5−10, truncTtRp is suspected of transitioning from ET to PCET, and then to two proton-coupled ET (2PCET). Upon reduction, we found that truncTtRp became more negative at pH 6.0 by one unit (ΔZ ET = −1.01 ± 0.14), consistent with single ET; was isoelectric at pH 8.8 (ΔZ ET = −0.01 ± 0.45), consistent with PCET; and became more positive at pH 10.6 (ΔZ ET = +1.37 ± 0.60), consistent with 2PCET. These ΔZ ET values are attributed to protonation of H154 and H134. Across pH, redox potentials of TtRp (measured previously) correlated with protonation energies of H154 and H134 and ΔZ ET for truncTtRp, supporting a discrete proton pumping mechanism for Rieske proteins at the Fe-coordinating histidines.

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Section: Resultsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Recombinant Expression and Purification Of The Truncated Rie...mentioning

confidence: 99%

See 2 more Smart Citations

Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer

Koone,

Simmang,

Saenger

et al. 2023

J. Am. Chem. Soc.

Self Cite

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show abstract

“…However, once the protein is partially unfolded, the positive charge of di-Zn (II) is polarized, and more positive charges will interact with the O Glu atoms in the bridging glutamic acid and N His atoms. The polarization of the positive charge might increase the coordination bond strength, as well as their mechanical and kinetic stability (Cheng et al, 2015;Koone et al, 2022). This may be a self-protection mechanism developed by multimetal-containing enzymes, where the charge in the metal center can be redistributed when the enzyme partially unfolds due to the interference caused by shearing force or reaction substrate moving.…”

Section: δLc (Nm)mentioning

confidence: 99%

Quantification of carboxylate‐bridged di‐zinc site stability in protein due ferri by single‐molecule force spectroscopy

Wang

Zhao

et al. 2023

Protein Science

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Carboxylate-bridged diiron proteins belong to a protein family involved in different physiological processes. These proteins share the conservative EXXH motif, which provides the carboxylate bridge and is critical for metal binding. Here, we choose de novo-designed single-chain due ferri protein (DFsc), a four-helical protein with two EXXH motifs as a model protein, to study the stability of the carboxylate-bridged di-metal binding site. The mechanical and kinetic properties of the di-Zn site in DFsc were obtained by atomic force microscopy-based single-molecule force spectroscopy. Zn-DFsc showed a considerable rupture force of $200 pN, while the apo-protein is mechanically labile. In addition, multiple rupture pathways were observed with different probabilities, indicating the importance of the EXXH-based carboxylatebridged metal site. These results demonstrate carboxylate-bridged di-metal site

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Harnessing Water Competition to Drive Enzyme Crosstalk

Meziadi,

Bloquert,

Greschner

et al. 2024

Biomacromolecules

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In nature, enzymatic pathways often involve compartmentalization effects that can modify the intrinsic activity and specificity of the different enzymes involved. Consequently, extensive research has focused on replicating and studying the compartmentalization effects on individual enzymes and on multistep enzyme "cascade" reactions. This study explores the influence of compartmentalization achieved using molecular crowding on the glucose oxidase/horseradish peroxidase (GOx/HRP) cascade reaction. The crowder tested is methoxy poly-(ethylene glycol) (mPEG) that can, depending on conditions, promote GOx and HRP coassociation at the nanoscale and extend their contact time. Low-molecular-weight mPEG (0.35 kDa), but not mPEG of higher molecular weights (5 or 20 kDa), significantly enhanced the cascade reaction where up to a 20-fold increase in the rate of the cascade reaction was observed under some conditions. The combined analyses emphasize the particularity of low-molecular-weight mPEG and point toward mPEG-induced coassociation of HRP and GOx, producing nearest crowded neighbor effects of HRP on GOx, and vice versa. These altered the nanoscale environments of these enzymes, which influenced substrate affinity. Using mPEG to promote protein coassociation is simple and does not chemically modify the proteins studied. This approach could be of interest for more broadly characterizing nearest crowded neighbor effects (i.e., protein−protein interactions) for multiprotein systems (i.e., more than just two), thus making it an interesting tool for studying very complex systems, such as those found in nature.

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A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor

Cited by 3 publications

References 98 publications

Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer

Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer

Quantification of carboxylate‐bridged di‐zinc site stability in protein due ferri by single‐molecule force spectroscopy

Harnessing Water Competition to Drive Enzyme Crosstalk

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