Local Backbone Flexibility as a Determinant of the Apparent pKa

Sep 27, 2017 - Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly ano...
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Local Backbone Flexibility as a Determinant of the Apparent pKa Values of Buried Ionizable Groups in Proteins Meredith T. Peck,† Gabriel Ortega,‡,§ Javier N. De Luca-Johnson,∥ Jamie L. Schlessman,∥ Aaron C. Robinson,† and Bertrand García-Moreno E*,† †

Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, United States Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park Ed. 800, 48160 Derio, Spain § Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, United States ∥ Chemistry Department, U.S. Naval Academy, Annapolis, Maryland 21402, United States ‡

S Supporting Information *

ABSTRACT: Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly anomalous pKa values that reflect the incompatibility between charges and dehydrated environments. A systematic study of pKa values of buried ionizable groups in staphylococcal nuclease (SNase) suggests that these pK a values are determined in part by conformational reorganization of the protein. Lys-66 is one of the most deeply buried residues in SNase. We show that its apparent pKa of 5.7 reflects the average of the pKa values of Lys-66 in different conformational states of the protein. In the fully folded state, Lys-66 is deeply buried in the hydrophobic core of SNase and must titrate with a pKa of ≪5.7. In other states, the side chain of Lys-66 is fully solvent-exposed and has a normal pKa of ≈10.4. We show that the pKa of Lys-66 can be shifted from 5.7 toward a more normal value of 7.1 via the insertion of flanking Gly residues at positions 64 and 67 to promote an “open” conformation of SNase. Crystal structures and nuclear magnetic resonance spectroscopy show that in these Gly-containing variants Lys-66 can access bulk water as a consequence of overwinding of the C-terminal end of helix 1. These data illustrate that the apparent pKa values of buried groups in proteins are governed in part by the difference in free energy between different conformational states of the protein and by differences in the pKa values of the buried groups in the different conformations.

T

physiological range. This may explain why in many pH sensitive proteins such as hemoglobin2 and the hemagglutinin protein of the influenza virus10 histidine residues act as the pH-sensing moieties. However, the pKa of an ionizable moiety is exquisitely sensitive to its microenvironment, and examples of proteins that rely on other ionizable groups (e.g., Asp, Glu, and Lys) with highly anomalous pKa values shifted into the physiological range are not uncommon.5,11,12 A detailed understanding of how proteins have evolved to respond to changes in pH requires an understanding at the molecular level of the determinants of pKa values of ionizable groups. Early attempts to examine this problem with structurebased electrostatics calculations were stymied by the use of static structures that required use of artificially large and physically meaningless protein dielectric constants (εp) to reproduce experimentally measured pKa values.13−15 The measured εp for dry protein powders is in the range of 2− 4,16,17 similar to the dielectric constants of waxes and oils. These low dielectric constants are consistent with extreme

ight regulation of pH is a feature common and essential to all living organisms. Small differences or changes in pH are often used to drive important biological processes by modulating the function of proteins through ligand binding and/or release,1,2 protein (dis)assembly,3 or conformational change.4,5 Furthermore, dysregulation of cellular pH has now been implicated in a number of diseases, including cancer6 and some neurodegenerative disorders.7,8 Given the essential role that pH plays as an important biological signal, it is not surprising that evolution has selected for many proteins to be poised to respond to small changes in pH. For protein regulation by pH to occur, and for proteins to be able to act as pH sensors that can undergo a conformational transition in response to a small change in pH, they must contain one or more ionizable residues that titrate over the range of pH where sensing is to occur. In fact, as noted by Tanford, for a protein to act as a pH sensor, an ionizable group must experience different, conformation-specific pKa values;9 that is, the proton affinity of an ionizable group must differ depending on the conformational state of the protein such that a change in the pH of the system will bias which conformational state is preferred. Of the naturally occurring amino acids, only histidine has an intrinsic pKa (∼6.5) in the © XXXX American Chemical Society

Received: July 18, 2017 Revised: September 9, 2017

A

DOI: 10.1021/acs.biochem.7b00678 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry Table 1. Thermodynamic Parameters from Denaturation Experiments protein

ΔG°a at pH 7 (kcal/mol)

Δ+PHS K64A K64G E67A E67G K64G/E67G V66Kf K64A/V66K K64G/V66K V66K/E67A V66K/E67G K64G/V66K/E67G

11.9 ± 0.1 11.6 ± 0.1 11.0 ± 0.1 10.8 ± 0.1 10.8 ± 0.1 9.9 ± 0.1 − − − − − −

e

ΔG°a at pH ≥10 (kcal/mol) − 8.9 8.5 9.2 8.7 7.6 5.2 5.6 4.8 5.4 4.9 3.6

± ± ± ± ± ± ± ± ± ± ±

ΔΔGmut (kcal/mol)b − −0.3 −0.9 −1.1 −1.1 −2.0 − −3.3 −3.5 −3.8 −3.8 −3.4

0.2 0.2 0.2 0.2 0.3 0.1 0.2 0.2 0.4 0.2 0.3

pKNa c − − − − − − 5.7 5.7 6.3 6.6 6.4 7.1

± ± ± ± ± ±

0.3 0.2 0.3 0.2 0.2 0.2

pHmidd 2.06 − − − − − − 3.90 3.99 3.93 4.00 3.99

± 0.01

± ± ± ± ±

0.01 0.01 0.01 0.01 0.01

ΔG° is the thermodynamic stability measured with chemical denaturation monitored with Trp fluorescence. bΔΔGmut = ΔΔGvar − ΔΔGref. For Lys-66 variants, ΔΔGmut corresponds to the energy required to substitute the group when Lys is neutral (i.e., at pH 10.4). cΔpKNa values were determined by fitting the pH dependence of ΔG°H2O to eq 1. dpH titrations fit with a two- or three-state model as described in ref 27. The value reported corresponds to the major unfolding transition. eData from ref 56. fData from ref 27. a

for critical assessment of the ability of computational methods to identify the different conformations that govern the pKa value of a buried ionizable group, and to correctly assign the relative statistical weights required to predict accurate pKa values.

levels of dehydration and with suppression of dipolar relaxation processes. However, calculations with low εp and static structures fail to reproduce electrostatic properties of proteins. The need for high dielectric constants (i.e., εp > 4) to reproduce experimental pKa values with static structures was correctly regarded as a failure of methods to account for protein dynamics.13,14,18 Nevertheless, even as new computational methods have been developed to account for both side chain and backbone dynamics, accurate prediction of pKa values remains challenging.18−21 Problems with conformational sampling have received the most attention as it is widely accepted that accurate modeling of a pH-driven conformational change requires knowledge of the conformation-specific, or microscopic, pKa values of the ionizable group(s) driving the conformational change.22−26 From these microscopic pKa values and knowledge of the population of each conformational state, a phenomenological or apparent pKa may be determined, corresponding to the pH at which conformational switching occurs. In this study, we demonstrate that the highly anomalous, apparent pKa values of buried ionizable residues in staphylococcal nuclease (SNase) can be altered with additional mutations that bias the conformational states accessed by a protein. Alternative conformations of SNase were observed directly via nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. The study is focused on a variant containing the Lys-66 residue, which is buried deep in the hydrophobic core of the protein. Previous studies have shown that in the highly stable form of SNase known as Δ+PHS, the buried Lys-66 titrates with an apparent pKa of 5.727 and its ionization is accompanied by a conformational change in the Cterminus of helix 1.28 Here, we examine the hypothesis that substitutions that disrupt the last turn of helix 1 should increase the population of conformations in which Lys-66 is exposed to water, leading to a shift in the pKa of Lys-66 toward the more normal value of 10.4 for a Lys residue in water. This can be achieved without altering the nature of the conformational states that determine the pKa; only the relative populations of these states are affected. The data from this study further our understanding of the physical basis of pH sensing and other protein functions governed by pH-driven conformational changes in proteins, such as energy transduction29−31 and catalysis.32 The case of Lys-66 constitutes a useful benchmark

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MATERIALS AND EXPERIMENTAL DETAILS Variants were engineered into the highly stable form of the protein, Δ+PHS, and expressed using molecular cloning and protein purification techniques as described previously.15 Crystal structures of Δ+PHS/V66K and Δ+PHS/K64G/ V66K/E67G were determined by molecular replacement using Phaser33 with Δ+PHS (3BDC34) as a search model. Iterative refinement and model building were performed using Refmac535 within the CCP4 suite36 and COOT.37 NMR data were collected on a Bruker Avance II-600 instrument equipped with a cryoprobe, and spectra were processed and analyzed with NMRPipe38 and Sparky,39 respectively. Equilibrium experiments were performed by monitoring the intrinsic fluorescence of Trp-140, and data were collected on an Aviv (Lakewood, NJ) model 107 titrating fluorometer as described previously.15 Far-ultraviolet (far-UV) circular dichroism (CD) spectra were collected on an Aviv model 420 circular dichroism spectrophotometer.

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RESULTS Lys-66 in SNase is buried in the hydrophobic core of the protein.27 The pKa of Lys-66 is 5.7, highly depressed relative to the normal pKa of 10.4 for Lys in water. To determine the effects of local stability and backbone flexibility on the pKa of internal residues, both Gly and Ala substitutions were introduced at positions 64 and 67 in the Δ+PHS/V66K variant studied previously. Ten variants were constructed by engineering the V66K substitution in variants with other single (K64G, K64A, E67G, and E67A) or double (K64G/E67G) substitutions. Thermodynamic Stability. Substitution with Gly or Ala at position 64 in Δ+PHS resulted in only a minor loss of stability (ΔΔG°H2O = ΔG°H2O,var − ΔG°H2O,ref) of 0.9 or 0.3 kcal/mol, respectively, at pH 7 (Table 1 and Table S1). The larger ΔΔG°H2O for the K64G substitution is consistent with the low helix propensity of Gly, especially relative to Ala. The Gly and B

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Figure 1. Thermodynamic stability of Ala or Gly variants of SNase (△) and the Lys-containing variants thereof (□) as a function of pH as measured by fluorescence-monitored GdnHCl denaturation at 25 °C (left axis). The difference between these curves and the pH dependence of stability of the appropriate background protein (●) and fits of this difference to eq 1 (solid line) are also shown (right axis). Data are shown for the following variants of the Δ+PHS form of SNase: (A) K64A and K64A/V66K, (B) E67A and V66K/E67A, (C) Δ+PHS and V66K, (D) K64G and K64G/ V66K, (E) E67G and V66K/E67G, and (F) K64G/E67G and K64G/V66K/E67G. Error bars (standard deviation) are on the order of the symbol size unless otherwise noted.

equilibrium between charged and neutral forms of the ionizable group, including structural reorganization (see Discussion). However, the sign and magnitude of the pH dependence of ΔG° along with the known pKa values measured via NMR spectroscopy of the His, Asp, and Glu residues in SNase suggest that the differential pKa values of Lys-66 when it is buried or exposed to solvent are responsible for the apparent pKa value. The pKapp of Lys-66 is 5.7 in the Δ+PHS/V66K variant of SNase. Furthermore, titration of Lys-66 in Δ+PHS/V66K is coincident with a loss of structure at the C-terminus of helix 1 observed via NMR spectroscopy.28 The introduction of Gly substitution(s) near Lys-66 was therefore expected to shift the pKapp of Lys-66 toward a more normal value because this substitution would promote the disruption of helix 1 and concomitant exposure of Lys-66 to bulk solvent. In each of the five Gly- or Ala-containing variants with Lys-66, the pKapp of Lys-66 was depressed below the normal pKa of Lys, consistent with burial of the Lys-66 side chain in the hydrophobic interior of the protein, even in the presence of substitutions that destabilize the helix near position 66 (Figure 1). The pKapp of Lys-66 in the K64G/V66K variant was 6.3 ± 0.3, depressed relative to the normal value of Lys in water, but 0.6 pKa unit above that of Lys-66 in Δ+PHS/V66K. The pKapp of Lys-66 was also measured in K64A/V66K to determine if its increase was the result of increased flexibility from the Gly-64 substitution or the loss of an unfavorable Coulombic interaction with Lys-64. In the K64A/V66K variant, the pKapp of Lys-66 was 5.7 ± 0.2, in agreement with pKapp for Lys-66 in Δ+PHS. Together, these data suggest that the shift in the pKapp observed in the K64G/V66K protein resulted from the presence of Gly-64 and not from the loss of a repulsive interaction with Lys-64.

Ala substitutions had an equally deleterious effect at position 67 (ΔΔG°H2O = 1.1 kcal/mol). In this context, the identical loss of stability for both Gly and Ala substitutions may reflect the loss of a stabilizing helix salt bridge between Lys-63 and Glu-67.40 The K64G/E67G variant destabilized the protein by 2.0 kcal/ mol, suggesting additive effects for the Gly-64 and Gly-67 substitutions. The introduction of Lys-66 into each of these Gly- or Alacontaining variants further decreased the thermodynamic stability of the protein. The average ΔΔGmut (ΔGvar − ΔGref) near pH 10.4, the pH corresponding to the normal pKa of Lys in water, was 3.6 kcal/mol, ranging from 3.3 to 3.8 kcal/mol. Because the pKa value of Lys-66 in the different proteins is depressed relative to the normal pKa of 10.4 for Lys in water, at this pH the substitution reflects the non-electrostatic cost of inserting a neutral Lys into the hydrophobic environment in the interior. pKa Values of Lys-66. The pKa value of Lys-66 in each variant was measured by analysis of the pH dependence of protein stability using eq 1. ΔΔG°H2O(pH) = ΔΔG°mut − RT ⎡ 1 + e z × 2.303(pH − pKaD) ⎤ ⎥ ln⎢ N ⎢⎣ 1 + e z × 2.303(pH − pKa ) ⎥⎦

(1)

where ΔΔG°mut describes the pH-independent free energy for substituting an ionizable group into the protein when the side chain is neutral. The right-most term describes the pHdependent energy difference between the pKa value of an ionizable group in the native (pKNa ) and denatured (pKDa ) states. The pKa values measured in this manner are apparent, pKapp, in that they reflect any molecular processes that affect the C

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Biochemistry In contrast, the pKapp values of Lys-66 in the V66K/E67G and V66K/E67A variants were 6.4 ± 0.2 and 6.6 ± 0.2, respectively. In these cases, substitution with Gly was not necessary to elevate the pKapp of Lys-66 relative to its value in Δ+PHS/V66K; removal of Glu-67 was sufficient to increase the pKapp of Lys-66 as evidenced by the V66K/E67A variant. As in the E67G and E67A reference proteins, disruption of the i, i + 4 intrahelix salt bridge between Lys-63 and Glu-67 likely destabilizes helix 1 and increases the pKa of Lys-66 in parallel with the loss of stability of their respective background proteins. In the K64G/V66K/E67G variant, the pKapp of Lys-66 was 7.1 ± 0.2, 1.4 pKa units higher than in Δ+PHS/V66K. The effects of the simultaneous substitution of both Gly-64 and Gly-67 appeared to be roughly additive to the pKapp of Lys-66. Crystal Structures. Crystallographic structures of the V66K and K64G/V66K/E67G variants of SNase were determined at pH 9 and 6, respectively (Table S2). The structure of the V66K variant closely resembled that of the Δ+PHS background protein (3BDC),34 with a root-mean-square deviation (RMSD) of 0.2 Å for all Cα atoms (Figure 2A). In Δ+PHS, Val-66 is located on the C-terminal turn of helix 1, facing into the main hydrophobic core of SNase (Figure 2B). In the V66K variant, the ionizable side chain of Lys-66 is buried without disruption of the surrounding secondary structure or the side chain packing of residues that constitute the hydrophobic core (Figure 2C). The only polar contact for Nζ of Lys-66 is a single, internal water molecule, 3.2 Å away. This water molecule is also observed in the Δ+PHS structure, suggesting that its presence is not a result of the Lys-66 substitution. Two molecules were found in the asymmetric unit of the crystal of the K64G/V66K/E67G variant. Both chains adopted the SNase fold, but residues 63−70 in chain A and residues 63−66 in chain B were different, reflecting small yet significant structural reorganization relative to Δ+PHS and Δ+PHS/V66K (Figure 2A). Electron density for residues 63−70 in chain A was clearly interpretable, whereas residues 67−70 in chain B could not be modeled reliably because of a lack of clear electron density. For this reason, they were excluded from the model. In contrast with the Δ+PHS/V66K variant, substitution of Lys-66 in the presence of the two Gly substitutions, K64G and E67G, resulted in a conformational rearrangement in the final turn of helix 1 (Figure 2D,E). Specifically, the ψ angles of residues 63− 65 shift from the mean value for an α-helix (−41°) to values more representative of a 310-helix (−18°).41,42 The change in backbone dihedral angles is accompanied by a shift in the Hbonding pattern of residues 62−67 from an i, i + 4 to an i, i + 3 arrangement. The primary consequence of overwinding the Cterminus of helix 1 is shifting of the register of residues 65 and 66 in the K64G/V66K/E67G variant, removing Lys-66 from the protein interior and placing it in contact with bulk water. Met-65 is also reoriented so that it packs into the hydrophobic core of SNase. Thus, the presence of Gly residues, which presumably enhance backbone flexibility in the region near Lys66, promotes the solvent-exposed configuration of Lys-66, increasing its apparent pKa. Spectroscopic Probes of Structural Reorganization. Structural reorganization coupled to the ionization of Lys-66 in the presence of flanking Gly or Ala residues was further probed by Trp fluorescence (FL), CD, and NMR spectroscopies. Titration with HCl from pH 8 to 2 monitored by Trp-FL exhibited no evidence of a global conformational change coincident with the titration of Lys-66 in any of the singlesubstitution variants studied (Figure S1). A predenaturational

Figure 2. (A) Stereoview of the crystal structures of the V66K (blue, 3HZX) and K64G/V66K/E67G (orange, 5CV5) variants of the Δ+PHS form of SNase, overlaid on the structure of Δ+PHS (white, 3BDC) used as a reference. Microenvironment of (B) Val-66 in Δ+PHS, (C) Lys-66 in Δ+PHS/V66K, and (D and E) Lys-66 in chains A and B of Δ+PHS/K64G/V66K/E67G. Side chains are shown in ball-and-stick representation. Hydrophobic groups are colored yellow. Oxygen atoms are colored red. Nitrogen atoms are colored dark blue. Sulfur atoms are colored green. Residue 66 is colored light blue. Residues 64−69, which undergo structural reorganization, are colored red. Residues not modeled are shown as a red, dashed line. βStrands 1−3 in panels B−E and the final turn of helix α1 in panels B and C are shown in loop representation for the sake of clarity and do not represent a loss of secondary structure.

transition was observed in the K64G/V66K/E67G variant by Trp-FL, beginning near pH 7, coincident with the pKa of Lys66 in this variant. However, even in this variant, the pHmid of the global unfolding transition was unperturbed relative to those of the single-site variants (Table 1). Far-UV CD spectra collected from pH 5 to 8 were identical for all of the Glycontaining variants, further suggesting that the ionization of Lys-66 had no detectable effect on the global structure in any of the variants studied (Figure S2). To determine the extent of local structural reorganization in response to the ionization of Lys-66 in the presence of the K64G and/or E67G substitutions, 1H−15N heteronuclear single-quantum coherence (HSQC) spectra were collected at 0.3−0.4 pH unit intervals over the range of pH two units above and below the pKapp of Lys-66 of each Gly-containing variant. At or above their respective pKapp of Lys-66, all three variants displayed well-dispersed spectra with sharp peaks, indicative of nativelike protein (Figure 3 and Figure S3). Below the pKapp of Lys-66, the spectra displayed behavior consistent with local D

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Figure 3. (A) HSQC spectra of the K64G/V66K/E67G variant at pH 5.98 (red) and pH 8.18 (black) in 100 mM KCl. Backbone amide assignments were made at pH 4.94 and 7.44 and transferred to all other pH values by visual inspection. Approximately 95% of resonances observed in the spectra of the Δ+PHS reference protein at a given pH were present in the spectra for K64G/V66K/E67G. (B) Structure of chain A of Δ+PHS/K64G/ V66K/E67G. The Lys-66 side chain is shown in green ball-and-stick representation with the Nζ atom colored dark blue. The Cα positions of Gly-64 and Gly-67 are shown as cyan spheres. Residues that broaden beyond detection below the pKapp of Lys-66 and do not reappear at lower pH values are colored red. Residues that broaden below the pKapp of Lys-66 but could be followed at pH values above the global acid unfolding of the protein are colored orange.

reorganization of the C-terminus of helix 1. Specifically, residues 14−18 in strand β1 and residues 61−70 in helix 1 showed large changes in chemical shifts (Δδ) and exchange broadening beyond detection over a range of one pH unit below the pKapp (Figure 3B). As the pH was decreased further, most resonances reappeared prior to a global alteration in the HSQC spectrum consistent with acid unfolding of the protein (Figure S4). Several resonances in strands β1−β3 also experienced large Δδ values and exchange broadening below pKapp, albeit to a lesser extent than observed in helix 1 (Figure S3). In the K64G/V66K/E67G variant, there was also evidence of conformational heterogeneity as demonstrated by the presence of a number of broad peaks in the denatured region of the HSQC spectra collected below pH 7.1.

Figure 4. Thermodynamic cycle for the pH-driven conformational change. Legend: NH+op, open state with Lys-66 protonated; Noop, open state with Lys-66 deprotonated; NH+cl, closed state with Lys-66 protonated; Nocl, closed state with Lys-66 deprotonated. Equilibrium constants for acid dissociation, Ka (horizontal), in the open and closed forms and folding reaction, Kcl‑op (downward), in the charged and neutral forms are denoted by the superscripts op, cl, +, and o, respectively.

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DISCUSSION Understanding the molecular determinants of the pKa values of ionizable groups buried in the dehydrated and hydrophobic interior of proteins is of considerable interest, as it would contribute further insight into the molecular mechanisms of energy transduction and motifs that govern pH-switch proteins. To this end, we are engaged in a systematic study of molecular determinants of pKa values of buried residues in SNase and in particular of the role of conformational reorganization coupled to the ionization process. In the V66K variants of SNase, ionization of Lys-66 results in partial unfolding of the C-terminal turn of helix 1, allowing the Lys side chain to contact bulk water without disrupting the overall protein fold.28 This suggests that the propensity of the protein to reorganize in response to a change in pH, no matter how subtle, constitutes an important determinant of the pKa values of buried groups. This can be described exactly with a thermodynamic linkage scheme between pKa and conformational reorganization, originally described by Wyman43 and for the specific case of pH-driven conformational change by Tanford.9 This scheme has been invoked recently in the context of structure-based simulations44−47 (Figure 4).

In this model, the V66K protein is presumed to exist in two conformations in equilibrium: a closed conformation in which Lys-66 is buried in the hydrophobic core of SNase (Ncl) and an open conformation in which the side chain is exposed to bulk solvent (Nop). In the closed conformation, the pKa of Lys-66 is presumably depressed below the apparent pKa measured with eq 1. In the open conformation, the pKa of Lys-66 is presumably close to the normal pKa of 10.4. The Nop → Ncl transition may be described by the conformational equilibrium constants Kcl+‐op = Kclo‐op =

[NH+cl] [NH+op] [N ocl] [N oop]

(2)

in which Lys-66 can be in either the protonated (Lys+) or deprotonated (Lys°) form. The acid/base equilibria for Lys-66 in the closed and open states are described by the microscopic dissociation constants E

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K acl = K aop =

[N ocl][H+] [NH+cl]

ization of SNase when Lys-66 is ionized or neutral is proportional to ΔpKa. If Nop is stabilized by Lys+ to a greater extent than by Lyso (i.e., K+cl‑op < Kocl‑op in Figure 4), then Kcla > cl op Kop a (i.e., pKa must be lower than pKa ). Combining eqs 5 and 7 yields

[N oop][H+] [NH+op]

(3)

From this model, the equilibrium describing the pH-driven conformational change NH+op → Nocl may be described by the phenomenological constant, Kapp a , as K aapp

=

K aapp =

[N oop + N ocl][H+] [NH+op + NH+cl]

=

1 + Kclo‐op 1 + Kcl+‐op

K aop

(5)

Finally, because of the large self-energy associated with burial of Lys+ in a hydrophobic environment,48−51 K+cl‑op ≪ 1, and Kapp a may be approximated by K aapp ≈ (1 + Kclo‐op)K aop

(6)

Kapp a ,

From eq 6, it follows that and therefore the measured pKa, depends on the conformation-specific dissociation constant in the open state, Kop a , and the equilibrium between Noop and Nocl when Lys-66 is neutral, Kocl‑op. These pKapp values are the experimental pKa values reported for Lys-66 in this paper. The introduction of Gly or Ala at position 64 and 67 of SNase resulted in increased flexibility of helix 1, reducing Kocl‑op and increasing the pKapp of Lys-66, in accordance with the proposed model. The specific Gly substitutions that were studied were chosen because they were likely to affect the equilibrium between Nocl and Noop with little or no change in the structural nature of those states.52−54 The conformationcl specific pKa values of Lys-66, pKop a and pKa , depend on the microenvironment of the ionizable moiety in the Nop and Ncl states, respectively. In the Nop state, the favorable solvation of Lys+ in bulk solvent is dominant, while in the Ncl state, desolvation of Lys+ in the hydrophobic environment is dominant. The chemical properties of neither microenvironment are expected to be affected by the substitution of surface residues. In principle, K+cl‑op could also be affected by the substitutions with Gly or Ala; however, as noted above, K+cl‑op already strongly favors NH+op because of the large, unfavorable cost of burying Lys+. Substitutions that further stabilize NH+op were therefore thought to be unlikely to shift the equilibrium appreciably between NH+op and NH+cl. It is clear from the pH dependence of thermodynamic stability of the V66K variants that pKop a for Lys-66 is higher than pKcla . For Lys-66 to drive a conformational transition of SNase in response to a change in pH, the energy required to shift the pKa of the ionizable moiety, 1.36|ΔpKa|, must exceed the net free energy difference between Noop and Nocl. This may be deduced from rearrangement of the expression for the two paths describing the pH-driven unfolding of SNase, Nocl → NH+op ⎛ K op ⎞ cl op Kcl+‐op = Kclo‐op⎜⎜ acl ⎟⎟ = Kclo‐op(10 pKa − pKa ) ⎝ Ka ⎠

cl

op

1 + Kclo‐op(10 pKa − pKa )

K aop (8)

Equation 8 shows that for any value of pKcla < pKop a , there exists a value of Kocl‑op such that a pH-driven conformational change will occur. We do not mean to suggest from this analysis that the value of pKcla is unimportant. For a pH-driven conformational change to occur over any particular pH range, both Kocl‑op and pKcla must be optimized to achieve a specific pKapp.46 If ΔpKa is too small, the corresponding ΔG to shift the pKa of the ionizable group will be insufficient to trigger a conformational change, and pKapp reduces to the conformation-specific value pKacl. Conversely, once ΔpKa is large enough to trigger a conformational change, increasing ΔpKa further will have no effect on pKapp. In the case of a basic group, as long as pKcla is ≤1 pH unit below the desired pKapp, the particular pH at which conformational switching occurs will be governed by Kocl‑op, not by the particular value of pKcla . In its most general formulation, Kaapp describes the equilibrium between Nocl and the conformational state with the lowest free energy that is capable of stabilizing Lys+, here the state in which helix 1 reorganizes. Although this particular “open” state was sufficient to solvate Lys+, in principle, it is not the only state capable of serving this function. Previous studies involving internal ionizable groups in SNase have demonstrated other “open” states that expose the hydrophobic core to bulk solvent through the release of strands β1 and β2.55 Although these are not the lowest-energy states in which the side chain of Lys-66 can be solvated,28 the work presented here suggests that strategic introduction of Gly substitutions into the β-barrel of SNase and/or substitutions that stabilize the C-terminus of helix 1 could alter the conformational landscape of the protein. Such alterations might transform one of these alternative highenergy states into the new lowest-energy conformation capable of solvating the side chain of Lys-66 when it is in the charged form. Conversely, in a variant in which all partially unfolded states are suppressed, Kapp reduces to Keq, which describes a global unfolding of the protein, the only remaining conformation capable of exposing Lys-66 to bulk solvent. Previous studies have demonstrated that the ionization of the deeply buried Lys-92, which titrates with a pKapp of 5.3, triggers global unfolding of the protein, suggesting that there is no other alternative, partially folded conformation in which the charged Lys-92 can be solvated.56,57 NMR spectroscopy studies of the buried Lys-25, which titrates with a pKapp of 6.3, have revealed yet another type of conformational reorganization, intermediate between global unfolding triggered by ionization of Lys-92 and the subtle overwinding of a helix triggered by ionization of Lys66. In the case of the variant with Lys-25, information about the time scale of pH sensitive fluctuations coupled to the conformational reorganization is also emerging.58 The data presented here offer a unique opportunity to evaluate the ability of structure-based electrostatics calculations to reproduce pH-driven conformational changes by correctly describing the free energy landscape of all relevant conformational states of a protein with sufficient structural detail to

(4)

Combining eqs 2−4, we may express Kapp in the form (see a the Supporting Information for derivation) K aapp

1 + Kclo‐op

(7)

From eq 7, it is evident that the difference between the equilibrium constants describing the conformational reorganF

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Biochemistry evaluate whether the simulated conformational changes are realistic. Many features of pH-driven conformational change in several SNase variants with buried ionizable residues were captured recently with microsecond long simulations and a variety of different force fields.59 Constant-pH molecular dynamics (CpHMD) methods22,23,25,60 offer a more comprehensive approach to simulating pH-driven conformational change with atomic detail. Recent studies have demonstrated the usefulness and promise of CpHMD, which in conjunction with experimental data can be useful for elucidating the mechanism of pH-driven conformational changes.46,60−62 CpHMD methods have been less successful, however, in predicting pKapp values for buried ionizable groups a priori by modeling the relevant conformational states that determine the pKapp and the equilibria between them.45,47 In a survey of 10 SNase variants with internal Lys substitutions, pKapp values calculated using CpHMD had an average unsigned error of 1.3 pKa units,47 and it is not obvious that the conformational changes observed in the simulations correspond to the ones observed by NMR spectroscopy or even X-ray crystallography. To calculate the pKa values for buried residues with CpHMD methods, both the relevant alternate conformation(s) and the equilibrium between the relevant conformations, i.e., Kocl‑op, must be determined accurately, even when pKapp is known. If the variants in the study presented here (Table 1) do indeed modulate Kocl‑op without affecting the conformations of Ncl and Nop, it is not obvious that the pKapp for the individual variants in Table 1 could be predicted accurately with CpHMD; even determining the relative rank ordering of pKapp for the variants may prove to be difficult. Furthermore, a useful CpHMD method will have to sample the entire range of pH-driven conformational transitions that have been observed with SNase variants thus far, ranging from a modest fraying or overwinding of a helix to release of β-strands from the β-barrel55 to global unfolding.56 This remains a challenging proposition.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (410) 516-4498. ORCID

Aaron C. Robinson: 0000-0001-6410-7147 Author Contributions

M.T.P., G.O.-Q., and A.C.R. contributed equally to this work. Funding

This work was supported by National Institutes of Health Grant GM 061597 to B.G.-M.E. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS NMR spectroscopy experiments were performed in the BioNMR facility at Johns Hopkins University. REFERENCES

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CONCLUSION pH-driven conformational reorganization is an inherent property of any protein that contains one or more ionizable residues with conformation-specific pKa values. Although a conformational state with an anomalous pKa value is necessary for pH switching to occur, the energy stored in the ΔpKa between the conformation-specific pKa values merely sets a limit on the range of pH over which a pH-driven conformational change may occur. The actual pH at which conformational switching occurs, no matter how subtle the conformational change might be, corresponds to the observed or apparent pKa measured. This pKapp is determined by the free energy difference between the conformations when the ionizable group is in its neutral state, Kocl‑op. Here we have demonstrated that the apparent pKa of Lys-66 in SNase, which reflects modest overwinding of helix 1, may be altered in a predictable way through targeted substitution with Gly to facilitate the specific conformational reorganization that determines the pKa of this buried Lys residue.

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Supplementary crystallography methods, thermodynamic stability versus pH for Lys-66 variants (Table S1), crystallographic collection and refinement statistics (Table S2), acid unfolding of Lys-66 variants monitored by Trp-FL (Figure S1), far-UV CD spectra of Lys-66 variants (Figure S2), HSQC spectra of K64G/V66K and V66K/E67G variants (Figure S3), HSQC spectra of specific residues in the K64G/V66K/E67G variant (Figure S4), and derivation of the thermodynamic cycle (PDF)

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DOI: 10.1021/acs.biochem.7b00678 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

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DOI: 10.1021/acs.biochem.7b00678 Biochemistry XXXX, XXX, XXX−XXX