Electrostatic Effects in a Network of Polar and Ionizable Groups in Staphylococcal Nuclease Kelli L. Baran 1 , Michael S. Chimenti 1 , Jamie L. Schlessman 2 , Carolyn A. Fitch 1 , Katie J. Herbst 1 and Bertrand E. Garcia-Moreno 1 ⁎ 1 Department of Biophysics, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA 2 Department of Chemistry, U. S. Naval Academy, 572 Holloway Rd., Annapolis, MD 21402, USA Received 12 November 2007; received in revised form 27 February 2008; accepted 9 April 2008 Available online 16 April 2008 His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tauto- meric state and the pK a values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK a values of 5.2 and 5.6, respectively. In the variants, where the net- work was disrupted, the pK a values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK a was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N ε2 state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb inter- actions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK a calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK a values are governed by short-range polar and Coulomb interactions. © 2008 Elsevier Ltd. All rights reserved. Edited by B. Honig Keywords: pK a values; histidine; NMR spectroscopy; continuum electro- statics; networks Introduction Electrostatic energy is useful for correlating the structure and function of proteins. To this end, improved understanding of the molecular determi- nants of electrostatic effects in proteins and im- proved computational methods for structure-based calculation of pK a values and electrostatic energies are necessary. One problem that has not received much attention from the experimental perspective concerns the properties of ionizable groups that are members of networks of polar and ionizable groups. In these environments, the pK a values of ionizable groups can be influenced by short-range Cou- lomb interactions, hydrogen bonding and inductive effects, charge delocalization, tautomerism and side- chain flexibility. These factors are poorly understood and can be difficult to treat computationally. Clusters of ionizable and polar groups have been studied ex- perimentally in complex systems such as photosyn- thetic reaction centers, 1,2 but not in a small protein where detailed structural and equilibrium thermo- dynamic analysis is possible. The goal of this study was to examine these short-range effects experimen- tally and systematically in a model protein to obtain *Corresponding author. E-mail address: bertrand@jhu.edu. Abbreviations used: MD, molecular dynamics; SNase, staphylococcal nuclease; NVIAGA, hyperstable form of SNase; GdmCl, guanidinium chloride. doi:10.1016/j.jmb.2008.04.021 J. Mol. Biol. (2008) 379, 1045–1062 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.