TY - JOUR
T1 - Structural origins of high apparent dielectric constants experienced by ionizable groups in the hydrophobic core of a protein
AU - García-Moreno, Bertrand
AU - Chimenti, Michael S.
AU - Castañeda, Carlos A.
AU - Majumdar, Ananya
N1 - Funding Information:
This work was supported by a grant from the National Institutes of Health ( GM-065197 to B.G.M.E.). M.S.C. received additional support from the Chapman Charitable Trust. C.A.C. was supported by a Burroughs-Wellcome Fellowship. The authors wish to thank Dr. Juliette Lecomte for helpful discussions and Dr. Rossitza Gitti for measuring the preliminary spectra of the Δ+PHS/V66K variant. All NMR experiments were performed at the Biomolecular NMR Center at the Johns Hopkins University.
PY - 2011/1/14
Y1 - 2011/1/14
N2 - The side chains of Lys66, Asp66, and Glu66 in staphylococcal nuclease are fully buried and surrounded mainly by hydrophobic matter, except for internal water molecules associated with carboxylic oxygen atoms. These ionizable side chains titrate with pKa values of 5.7, 8.8, and 8.9, respectively. To reproduce these pKa values with continuum electrostatics calculations, we treated the protein with high dielectric constants. We have examined the structural origins of these high apparent dielectric constants by using NMR spectroscopy to characterize the structural response to the ionization of these internal side chains. Substitution of Val66 with Lys66 and Asp66 led to increased conformational fluctuations of the microenvironments surrounding these groups, even under pH conditions where Lys66 and Asp66 are neutral. When Lys66, Asp66, and Glu66 are charged, the proteins remain almost fully folded, but resonances for a few backbone amides adjacent to the internal ionizable residues are broadened. This suggests that the ionization of the internal groups promotes a local increase in dynamics on the intermediate timescale, consistent with either partial unfolding or increased backbone fluctuations of helix 1 near residue 66, or, less likely, with increased fluctuations of the charged side chains at position 66. These experiments confirm that the high apparent dielectric constants reported by internal Lys66, Asp66, and Glu66 reflect localized changes in conformational fluctuations without incurring detectable global structural reorganization. To improve structure-based pKa calculations in proteins, we will need to learn how to treat this coupling between ionization of internal groups and local changes in conformational fluctuations explicitly.
AB - The side chains of Lys66, Asp66, and Glu66 in staphylococcal nuclease are fully buried and surrounded mainly by hydrophobic matter, except for internal water molecules associated with carboxylic oxygen atoms. These ionizable side chains titrate with pKa values of 5.7, 8.8, and 8.9, respectively. To reproduce these pKa values with continuum electrostatics calculations, we treated the protein with high dielectric constants. We have examined the structural origins of these high apparent dielectric constants by using NMR spectroscopy to characterize the structural response to the ionization of these internal side chains. Substitution of Val66 with Lys66 and Asp66 led to increased conformational fluctuations of the microenvironments surrounding these groups, even under pH conditions where Lys66 and Asp66 are neutral. When Lys66, Asp66, and Glu66 are charged, the proteins remain almost fully folded, but resonances for a few backbone amides adjacent to the internal ionizable residues are broadened. This suggests that the ionization of the internal groups promotes a local increase in dynamics on the intermediate timescale, consistent with either partial unfolding or increased backbone fluctuations of helix 1 near residue 66, or, less likely, with increased fluctuations of the charged side chains at position 66. These experiments confirm that the high apparent dielectric constants reported by internal Lys66, Asp66, and Glu66 reflect localized changes in conformational fluctuations without incurring detectable global structural reorganization. To improve structure-based pKa calculations in proteins, we will need to learn how to treat this coupling between ionization of internal groups and local changes in conformational fluctuations explicitly.
KW - NMR
KW - conformational reorganization
KW - dielectric constant
KW - electrostatics
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U2 - 10.1016/j.jmb.2010.10.001
DO - 10.1016/j.jmb.2010.10.001
M3 - Article
C2 - 21059359
AN - SCOPUS:78650906637
SN - 0022-2836
VL - 405
SP - 361
EP - 377
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -