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Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.
Abstract
The pKa values of ionizable groups in proteins report the free energy of site-specific
proton binding and provide a direct means of studying pH-dependent stability. We measured
histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and
sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate
nuclear magnetic resonance-monitored titration approach that utilizes internal reference
compounds and a parametric fitting method. The three histidines in the sulfate-bound
folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01
units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide.
In the unliganded and unfolded protein, the pKa values are depressed relative to that
of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively.
Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose
apparent pKa value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than
in either U or F. The depressed pKa values we observe are consistent with repulsive
interactions between protonated histidine side chains and the net positive charge
of the protein. However, the pKa differences between F and U are small for all three
histidines, and they have little ionic strength dependence in F. Taken together, these
observations suggest that unfavorable electrostatics alone do not account for the
fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple
factors encoded in the P protein sequence account for its IUP property, which may
play an important role in its function.
Type
Journal articleSubject
Bacillus subtilisHistidine
Hydrogen-Ion Concentration
Nuclear Magnetic Resonance, Biomolecular
Protein Structure, Secondary
Protein Structure, Tertiary
Ribonuclease P
Static Electricity
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https://hdl.handle.net/10161/10596Published Version (Please cite this version)
10.1021/acs.biochem.5b00138Publication Info
Mosley, Pamela L; Daniels, Kyle G; & Oas, Terrence G (2015). Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by
Nuclear Magnetic Resonance-Based Histidine pKa Measurements. Biochemistry, 54(35). pp. 5379-5388. 10.1021/acs.biochem.5b00138. Retrieved from https://hdl.handle.net/10161/10596.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Terrence Gilbert Oas
Professor of Biochemistry
Our laboratory is primarily interested in the mechanisms of protein folding. We use
nuclear magnetic resonance (NMR) and other types of spectroscopy to study the solution
structure, stability and folding reactions of small protein models. These include
monomeric λ repressor, the B domain of protein A (BdpA) and various regulator
of G-protein signalling (RGS) domains. Our biophysical studies are used to inform
our investigations of the role of folding mechanism in the function of pro

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