Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.

Mosley, Pamela L

Daniels, Kyle G

Oas, Terrence G

United States

2015-09-09T15:48:21Z

2015-09-08

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.

http://www.ncbi.nlm.nih.gov/pubmed/26267651

1520-4995

https://hdl.handle.net/10161/10596

eng

American Chemical Society (ACS)

Biochemistry

10.1021/acs.biochem.5b00138

Bacillus subtilis

Histidine

Hydrogen-Ion Concentration

Nuclear Magnetic Resonance, Biomolecular

Protein Structure, Secondary

Protein Structure, Tertiary

Ribonuclease P

Static Electricity

Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.

Journal article

Oas, Terrence G|0000-0002-3067-2743

http://www.ncbi.nlm.nih.gov/pubmed/26267651

5379

5388

35

Basic Science Departments

Biochemistry

Duke

Duke Cancer Institute

Institutes and Centers

School of Medicine

Published

54