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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Structure. Author manuscript; available in PMC Jun 6, 2013.
Published in final edited form as:
PMCID: PMC3373022
NIHMSID: NIHMS372588
Structural Reorganization Triggered by Charging of Lys Residues in the Hydrophobic Interior of a Protein
Michael S. Chimenti,1 Victor S. Khangulov,1 Aaron C. Robinson,1 Annie Heroux,3 Ananya Majumdar,1 Jamie L. Schlessman,2 and E. Bertrand García-Moreno1*
1Department of Biophysics, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218 USA
2Department of Chemistry, U.S. Naval Academy, 572 Holloway Rd., Annapolis, MD 21402, USA,
3Brookhaven National Laboratory, Bldg. 463, P.O. Box 5000, Upton, NY 11973, USA
*Corresponding Author: Tel: (410) 516-4497, FAX: (410) 516-4118, bertrand/at/jhu.edu
Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied the ionization of an internal Lys unfolded the protein globally. In 5 cases the internal charge triggered localized changes in structure and dynamics, and in 3 cases they promoted partial or local unfolding. Remarkably, in 15 proteins the ionization of the internal Lys resulted in no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pKa calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.
Keywords: electrostatics, proteins, pKa values, buried charged NMR spectroscopy, staphylococcal nuclease
Internal ionizable groups in proteins are essential for biological energy transduction processes (Lanyi and Luecke, 2001; Jiang et al., 2003; Rastogi and Girvin, 1999). Internal groups usually titrate with anomalous pKa values, the determinants of which are poorly understood (Isom et al., 2010, 2011). The pKa values can be influenced by the many factors that determine the dielectric response of proteins, including electronic polarizability, reorientation of backbone and side chain dipoles, Coulomb interactions, the reaction field of bulk water, water penetration, and local or subglobal conformational reorganization. Even the global thermodynamic stability (ΔG°H2O) of the protein may influence the properties of internal ionizable groups (García-Moreno et al., 1997; Karp et al., 2010). In this study we examined structural and physical origins of the dielectric response of a protein using X-ray crystallography to describe microenvironments of internal Lys residues in staphylococcal nuclease (SNase) and NMR spectroscopy to survey structural consequences of their ionization.
Charges are incompatible with hydrophobic environments; therefore, the ionization of a group buried in the hydrophobic interior of a protein can alter its structure. When the ionization of an internal residue triggers structural reorganization, the major determinant of its pKa is the reorganization proper. In general, the inability of structure-based calculations to reproduce experimentally measured pKa values of internal groups stems from the failure to account correctly for structural reorganization (Karp et al., 2007). Several groups have made recent progress on this important problem (Arthur et al, 2011; Ghosh and Cui, 2008; Gunner et al, 2011; Kamerlin et al, 2009, Kato and Warshel, 2006; Meyer et al, 2011; Song, 2009; Wallace et al, 2011; Williams et al, 2011; Witham et al, 2011;Yoo and Cui, 2008; Zheng et al., 2008). To improve our understanding of determinants of pKa value of internal groups in proteins it is necessary to describe experimentally the character of the reorganization that is triggered by the ionization of internal groups.
The present studies were performed with 25 variants of staphylococcal nuclease (SNase) with Lys at internal positions. The pKa values of most of these internal Lys ionizable groups are anomalous and depressed, some quite substantially relative to the normal pKa values of Lys in water (Isom et al., 2008; Isom et al., 2010, 2011). To reproduce the experimental pKa values of internal ionizable groups in SNase with structure-based continuum electrostatics calculations using static structures and the finite difference solution of the linearized Poisson-Boltzmann equation, the protein has to be treated with arbitrarily high dielectric constants between 8 and 30 (Dwyer et al., 2000; Fitch et al., 2002; Harms et al., 2009; Harms et al., 2008; Isom, 2010, 2011; Karp et al., 2007). The hypothesis that was examined in this study is that these high dielectric constants reported by the internal Lys residues in SNase reflect structural reorganization coupled to the ionization of the buried groups.
Almost all continuum electrostatics methods require artificially high dielectric constants to reproduce pKa values of internal ionizable groups (Warwicker, 2011; Word and Nicholls, 2011). These high dielectric constants are comparable to the dielectric constants of highly polarizable materials, and much higher than the dielectric constants of 2 to 4 of dried protein powders commonly used in continuum electrostatics calculations (Bashford and Karplus, 1990; Gilson et al., 1988). The need for empirically high protein dielectric constants has been justified by assuming that these values reproduce implicitly the structural reorganization and water penetration that is coupled to the ionization of internal groups (Warshel and Russell, 1984).
The possibility that the ionization of internal residues in SNase triggers conformational reorganization was first suggested by pH titrations that were monitored with optical spectroscopy in 75 variants of SNase with internal Lys, Asp and Glu residues. In most cases the ionization of the internal groups had no detectable structural consequences, but in some cases, local, subglobal, and even global structural changes were apparent (Harms et al., 2011; Isom et al., 2008; Isom et al., 2010; 2011). Detailed NMR spectroscopy studies of variants with Lys, Asp and Glu at positions 38 and 66 confirmed that in some cases the ionization of an internal group did not affect structure while in others it could trigger localized structural reorganization consistent with increased local fluctuations and with local unfolding (Chimenti et al., 2011; Harms et al., 2009; Harms et al., 2008; Karp et al., 2007).
Using NMR spectroscopy we have now surveyed the range of structural reorganization that can be triggered by the ionization of internal Lys residues at 25 internal positions in SNase. The goal was to catalog the character of possible structural changes that are possible and to identify interesting cases for subsequent detailed study. Crystal structures of many variants were determined to describe the conformations of internal Lys side chains and microenvironments of their ionizable moieties. This study contributes systematic and detailed molecular description of structural origins of high apparent dielectric constants inside proteins.
Crystal structures
The crystal structures of variants of SNase with internal Lys-66, Lys-38 and Lys-92 were published previously (Harms et al., 2008; Nguyen et al., 2004; Stites et al., 1991). Structures for 8 other variants with internal Lys have now been solved, in buffers at a pH in which the Lys side chains should have been neutral (Table 1 and Table 2). The substitution of internal positions with Lys did not have an appreciable effect on any of the structures. The backbone of the 12 variants with internal Lys were almost perfectly superimposable and have an average Cα RMSD of less than 0.6 Å relative to the reference protein (Fig. 1A and Table 2). The internal Lys side chains were ordered and visible in the electron density maps of all proteins. Most differences between structures are limited to the Ω loop (residues 44 to 49), which was not present in all the proteins studied. The observation that neutral Lys can be buried in the protein interior without inducing structural reorganization is consistent with previous studies of the resilience of SNase towards substitution of internal positions with bulky, flexible, and non-native amino acids (Wynn et al., 1996; Daopin et al., 1991).
Table 2
Table 2
Microenvironment of internal Lys residues in staphylococcal nuclease
Figure 1
Figure 1
Figure 1
(A) Stereo view of the superposition of crystal structures of twelve variants of SNase with internal Lys residue, overlaid on the structure of the Δ+PHS protein (PDB ID: 3BDC). (B) Composite stereo view of the side chains of twelve internal Lys (more ...)
Internal Lys residues with shifted pKa values decrease the thermodynamic stability of proteins (Isom et al., 2008; Isom, 2010b). This could have been mitigated through small structural changes to enhance penetration of water to hydrate the internal side chain or to allow extrusion of the internal side chain to bulk water, but this was not observed, perhaps because osmotic and packing forces in the crystal prevent this from happening.
The side chain of Lys-92 was poorly defined in the crystal structure (Nguyen et al., 2004), but all other internal Lys side chains had strong and continuous electron density for all atoms. The internal Lys side chains could be modeled without clashes with nearby atoms and without requiring reorganization of the core of the protein. In a few cases (e.g. Lys-72) the conformation of the side chain is likely influenced by extensive contacts between the Nζ atom and polar backbone atoms. In all cases, the B factors of the internal Lys side chains were comparable to those of other nearby atoms. The B factors of the proteins with internal Lys residues were comparable to those of other SNase variants at comparable resolution. The rotameric states of these internal Lys side chains are not common (Dunbrack and Karplus, 1993).
Solvent-accessible surface area (SASA) and depth of burial calculations showed that with the exception of Lys-72, all the Lys side chains and their Nζ atoms were completely inaccessible to water and buried, some quite far from bulk water (Table 2). The side chains of Lys-92 and Lys-104 exist in more than one conformation, suggesting that even when buried the Lys side chains can have considerable mobility. Most Lys side chains point either towards the naturally occurring microcavity at the primary hydrophobic core (Lys-25, Lys-36, Lys-62, Lys-66, Lys-92), or towards the secondary hydrophobic core of SNase (Lys-103, Lys-104, Lys-125) (Fig. 1B). The microenvironments of the internal Lys side chains can be very different in different proteins (Fig. 2 and Table 2). Seven of the internal Lys side chains were packed into predominantly hydrophobic regions with few or no polar atoms near the ionizable moiety (e.g. Lys-23, Lys-25, Lys-34, Lys-36, Lys-66, Lys-92, and Lys-125 have no polar atoms within 3.5 Å of the Nζ atom of lysine). Five lysines (Lys-38, Lys-62, Lys-72, Lys-103 and Lys-104) have Nζ atom within 3.5 Å of 1 to 3 polar groups or crystallographic water molecules (Table 2).
Figure 2
Figure 2
Microenvironment of the ionizable moieties of 11 internal Lys residues. (A) Δ+PHS SNase; (B) Lys-125 (PDB ID: 3c1e); (C) Lys-25 (PDB ID: 3erq); (D) Lys-36 (PDB ID: 3eji); (E) Lys-104 (PDB ID: 3c1f); (F) Lys-62 (PDB ID: 3dmu); (G) Lys-103 (PDB (more ...)
Lys-72 is unique among these internal Lys residues because it is buried shallowly. Its Nζ atom is only 7.9% accessible, 3.5 Å from a crystallographic water molecule, and within hydrogen bonding distance of three backbone carbonyl residues. Surprisingly, despite this highly polar microenvironment, Lys-72 titrates with an anomalous pKa of 8.6. This contrasts with the case of Lys-38, which has a normal pKa despite its Nζ atom being fully buried (Harms et al., 2008). Crystal structures did not reveal a clear connection between measured shifts in pKa values and the depth of burial or polarity of the microenvironments of Nζ atoms, suggesting that other structural properties must be the critical determinants of the pKa values of these internal groups.
Structural consequences of substitution with neutral Lys
1H-15N HSQC spectra of the 25 Lys-containing variants were first measured under conditions of pH where the internal Lys residues were mostly neutral (Table S1 and Fig. S1–S3). Twenty-four out of 25 variants exhibited chemical shift dispersion and well-resolved cross peaks characteristic of folded proteins, consistent with results from CD and Trp fluorescence (Isom et al., 2008). The exception was the N100K variant, whose 1H-15N HSQC spectrum showed a significant loss of peaks consistent with some unfolding.
Assignment of HSQC spectra and other types of NMR spectroscopy experiments will be required to describe on a site-by-site basis the structural and dynamic consequences of substitution of internal positions with Lys residues. However, to identify the proteins worthy of further detailed study it was first necessary to characterize the structural consequences of substitution with and ionization of internal Lys residues in the 25 variants. To examine 1H-15N HSQC spectra qualitatively at pH values where the internal Lys was neutral we focused on the subset of resonances of each variant that are different from resonances in the spectrum of the reference protein at the same pH. The subset of resonances that was affected by the substitution differed from variant to variant. For qualitative purposes, peaks that overlapped those in the reference spectrum unambiguously were assigned by visual inspection. The total number of peaks assigned in this manner was normalized by the total number of assigned peaks in the 1H-15N HSQC spectrum of the reference protein. This provided a measure of how similar the HSQC spectrum of a variant was to that of the reference protein (Table 3). Although this analysis is strictly only qualitative, it contributed significant new insight into the type, magnitude and distribution of structural reorganization coupled to the presence and ionization of internal Lys residues.
Table 3
Table 3
Summary of global thermodynamic stability, pKa values of internal Lys residues and effects of pH on the HSQC spectra of variants of SNase with internal Lys residues
The 24 variants that are folded under conditions of pH where the Lys residues are supposed to be neutral had spectra with 42% to 87% similarity to the reference protein (Table 3). On average 65% of all peaks in the Lys-containing variants were superimposable with peaks in the reference protein. The relatively low level of overlap between the spectra of the Lys-containing variants and the reference protein reflects primarily the sensitivity of the amide chemical shift (Wishart and Case, 2001). On the other hand, the absence of major differences between the 1H-15N HSQC spectra of the reference protein and of the Lys-containing variants is consistent with the similarity between crystal structures. Note that 1H-15N HSQC spectra of some variants of SNase with internal ionizable groups have been assigned and analyzed in terms of distribution of ϕ/ψ angles, and that in those cases the NMR data are fully consistent with the distribution of secondary structure observed in the crystals (Harms et al., 2009; Harms et al., 2008). In fact, the resonances that were affected by the substitutions (e.g.V66K or V66D) were not affected by the ionization of the internal Lys-66 or Asp-66 (Chimenti et al., 2011).
The highest level of similarity between the reference protein and a Lys-containing variant was for the variants with the most solvent exposed Lys (I72K variant with 87% similarity with the spectrum of the reference protein), and for the variant with a Lys in a loop region (N118K variant, with 81% similarity). The variants that introduce Lys into the primary hydrophobic core of SNase at positions 25, 36, 62, and 66 also had HSQC spectra with 70–85% peak overlap with the reference spectrum, suggesting that the region of the protein between helix-1 and β-sheets 2 and 3 tolerate substitutions with Lys better than the region near helix-2 and helix-3, where substitutions with Lys yielded more highly perturbed HSQC spectra. No other general, qualitative trends were observed in the similarities in HSQC before and after substitution of internal positions with Lys.
Structural consequences of the ionization of internal Lys
1H-15N HSQC spectra were measured for each variant at pH values that bracketed the apparent pKa of the internal Lys. The exception was the V23K, for which the highest pH studied corresponded to a form in which Lys-23 was only expected to be 50% neutral. The dispersion and resolution of the 1H-15N HSQC spectra indicate that 23 of the 25 variants were fully folded when the internal Lys residues were charged (Fig. 3D shows representative 1H-15N HSQC spectra before and after the ionization of an internal Lys). In general, the difference in amide proton chemical shifts (Δδ(1HN)) in each variant between conditions where the internal Lys was charged and neutral were quite small (less than ~0.1 ppm). This is illustrated for three specific cases chosen to represent proteins with internal Lys residues with small (N118K), medium (I72K), and large (L125K) depressions in their pKa (Fig. 4). With the exception of the variant with I92K discussed ahead, the amide shifts did not show evidence of any significant structural reorganization upon ionization of the internal Lys, not even in cases where the internal Lys residue has a highly depressed pKa. This is consistent with results with optical spectroscopy showing that all but the I92K variant remained folded when the internal Lys residues were charged (Isom et al., 2008; 2011). The variants appear to retain their native folded structure under conditions of pH where the internal Lys residues are charged.
Figure 3
Figure 3
1H-15N HSQC spectra acquired at pH values above (black) and below (color) the pKa value of internal Lys residues in four proteins. (A) Example of slow exchange with a fully unfolded form in the I92K variant (pKa ~ 5.3) when Lys-92 is ionized at pH 4 (red). (more ...)
Figure 4
Figure 4
ΔΔδ 1HN (ppm) calculated as ΔΔδ(ppm) = [δ(variant)neutral − δ(variant)charged] − [δ(Δ+PHS)neutral − δ(Δ+PHS)charged] (eqs. 2.1) and (more ...)
Local reorganization coupled to the ionization of internal Lys residues
The ionization of five Lys residues (Lys-25, Lys-34, Lys-58, Lys-66 and Lys-125) led to intermediate exchange behavior, consistent with increased dynamics or conformational fluctuations localized to a small part of the protein near the internal Lys side chain (Fig. 3B and 3C). These cases are of special interest because they have information about the roles of conformational reorganization and dynamics as determinants of the pKa values of internal groups.
The ionization of internal Lys residues can increase the frequency and amplitude of their conformational fluctuations (Damjanovic et al., 2005; 2007; 2008). The fluctuation of a charge around nearby amides may contribute to the line-broadening that was observed with some of the variants. However, the observation that the affected resonances do not remain in fast exchange and instead enter intermediate exchange is consistent with underlying slower local fluctuations of the backbone on the order of micro- to milliseconds, which promote conformational states where the internal charged Lys can become dynamic and solvated by water. This has been characterized in detail in the V66K variant (Chimenti et al., 2011; Kitahara, 2010); the Lys-66 side chain is internal when neutral, but still exchanges with water at a rapid rate of ~90 sec−1 at −1 °C, consistent with local unfolding or water penetration (Takayama et al., 2008).
The L25K and L125K variants responded to the ionization of the internal Lys with the same intermediate exchange behavior observed in the V66K variant. Extreme line-broadening and large chemical shift changes were found in the HSQC spectra for a subset of resonances around a region immediately adjacent to the side chains of Lys-25 and Lys-125 (Figure 3B and 3C). Only ~20 resonances broadened upon ionization of the internal lysine of V66K and L125K. As many as 50 resonances broadened in response to the ionization of Lys-25, consistent with a larger dynamic response to ionization of this Lys residue. Many of the affected resonances in the L25K spectrum lost intensity without experiencing chemical shift changes, suggesting a purely dynamic process without structural rearrangement.
The F34K and V39K variants also showed significant intermediate exchange in the HSQC spectra. These variants had stability greater than 3 kcal/mol when the Lys was ionized, indicating that at certain positions in the protein, ionization of Lys can promote increased fluctuations even when the protein is relatively stable. Lys-39 is located in a loop region where fluctuations of the backbone may be normally less restricted. This observation of intermediate exchange dynamics in these more stable variants is counter to the idea that in the more stable variants the ionization of the internal Lys side chain does not increase local fluctuations. On the other hand, the majority of the more stable variants displayed no observable exchange behavior upon titration of the internal lysine (Table 3). Further studies will be necessary to examine further the relationship between ionization of internal Lys residues, promotion of local fluctuations, and global, thermodynamic stability.
Global unfolding coupled to the ionization of internal Lys
The 1H-15N HSQC spectra of the I92K and N100K variants at pH 6.7 and 9.4 respectively, where Lys-92 and Lys-100 are neutral, showed slow exchange behavior between the native state (in the case of I92K) or a partially folded form containing some native peaks (in the case of N100K), and an unfolded form that was characterized by a region of poorly-defined, broadened resonances from 8.0 to 8.4 ppm in 1HN (Figure 3A). The stability of I92K is ~0.2 kcal/mol at pH 4.9 (where Lys-92 is ionized) and the stability of N100K is ~1.3 kcal/mol at pH 7.0 (where Lys-100 is ionized). In both cases, when the Lys residue is >90% ionized, the unfolded form is the only form observable in the 1H-15N HSQC spectrum. In both cases, peaks from the native state in the 1H-15N HSQC did not show substantial chemical shift changes upon titration, indicating that the data are reporting a two-state exchange between folded (or partially folded, in the case of N100K) and unfolded forms without significant intermediates. The NMR data show unequivocally that ionization of Lys-92 and Lys-100 led to the global unfolding of the protein. The high apparent dielectric constants reported by Lys-92 and Lys-100 is simply a reflection of dielectric breakdown.
The HSQC spectra of the A58K, T62K, and V99K variants exhibited slow exchange behavior of a different type. In the case of T62K and V99K, the vast majority of the peaks remained at full intensity throughout the titration. However, at the low pH endpoint of the titration, new resonances appeared in the unfolded region of the 1H-15N HSQC spectrum. These resonances had lower intensity than the native peaks, consistent with the presence of a minor, partially unfolded form of the protein. In the case of the variant with Lys-58 several new resonances appeared at low pH, but they were generally found near native peaks, not in the unfolded region. This could indicate that the protein is switching between two very similar folded conformations on a slow timescale.
Relationship between global thermodynamic stability and the dielectric response
The type of structural response triggered by the ionization of Lys residues in different parts of the protein is summarized in Fig. 5. It is striking that the majority of the variants, including many (L36K, V23K, V74K, V104K, L103K, I72K, A90K, Y91K, A109K, and T41K) in which the Lys titrates with a depressed pKa value, showed no evidence of any major conformational changes coupled to the ionization of the internal Lys.
Figure 5
Figure 5
Summary of consequences of ionization of internal Lys residues in Δ+PHS SNase observed in 1H-15N HSQC data. (●) Ionization of Lys at these locations induced minor or no changes in chemical shifts and no slow or intermediate exchange behavior; (more ...)
All the variants with ΔG°H2O less than 3 kcal/mol at the pH where the internal Lys became fully ionized exhibited some form of exchange behavior in the HSQC spectra, consistent with increased fluctuations between alternative states (Fig. 3A–C, Table 3). These proteins can be divided into two classes depending on the nature of the response. Variants in one class (I92K, N100K, and to a lesser extent, T62K and V99K) exhibited exchange with a fully unfolded form. The A58K variant could be included in this set although the exchange behavior appears to affect only a small subset of peaks. The members of a second class (L25K, V39K, F34K, V66K, L125K) exhibit intermediate exchange only at a subset of residues consistent with exchange with a locally or partially unfolded form.
Whereas the structure of the native state is independent of global stability, the extent of the structural response of the protein to the ionization of an internal group appears to be somewhat dependent on the global thermodynamic stability of the protein. Partially unfolded states in which the internal charged moiety can be solvated through contact with protein or water should be populated more readily when the global stability of a protein is low. That is, in the case of variants with internal Lys residues with depressed pKa the probability of populating excited states, or partially unfolded states, increases with decreasing pH because the energy gap between the native and the unfolded state decreases with decreasing pH. A similar inverse relationship between ΔG°H2O and dynamic backbone fluctuations has been observed previously in SNase by NMR relaxation methods (Alexandrescu et al., 1996) and native-state hydrogen exchange (Maity et al., 2003).
Structural origins of the high apparent dielectric constant in the protein interior
The Lys side chains introduced in SNase are internal and buried deeply when they are neutral (Fig. 1B). The majority of them are embedded in highly hydrophobic microenvironments that are inconsistent with the high apparent dielectric reported by the measured pKa values. For this reason it was natural to invoke increased dynamics or structural reorganization to explain the high dielectric constants consistent with the experimental data. The crystal structures showed no evidence of structural reorganization. Relying on the atomic resolution afforded by NMR spectroscopy we have now shown that 15 out of 25 internal Lys residues in SNase ionize without triggering detectable changes in chemical shifts or in exchange behavior. Neither the substitution of a core position with a Lys nor the ionization of the Lys had any detectable structural or dynamic perturbations in these 15 cases. Six of these 15 Lys residues titrate with a normal pKa (L38K, A132K, A58K, L37K, G20K, N118K) and with the exception of Lys-38, which is known to be buried, these could well end up being trivial cases where the Lys side chains are not actually buried. Crystal structures are needed to establish this. However, the other nine Lys side chains have depressed pKa values. Regardless of what the origins of the apparent dielectric constants reported by these Lys residues might be, they are sufficiently subtle so as to be invisible in HSQC spectra. Three of the four proteins where the amino moiety of the internal Lys residue makes contacts with polar atoms fall under this category; in these cases this high local polarity might be sufficient to solvate an internal charged group thereby preventing conformational changes in response to the ionization event. The lack of evidence for processes in the intermediate exchange regime in the 1H-15N HSQC spectra of a majority of Lys-containing variants of SNase can only be used to exclude very slow and intermediate timescale fluctuations as important elements of the dielectric response probed by the internal Lys residues. Contributions from very fast dynamics or from minor structural forms in solution (<10% populated) can still play a role; this is being examined.
Another possibility is that in the 15 cases where there is no evidence of structural or dynamic perturbation, the Lys side chain is embedded in microenvironments where buried water molecules hydrate the internal ionizable moiety, essentially creating a favorable dielectric environment for the polar group. Internal water molecules near the internal ionizable moieties were absent near most internal Lys residues, but have been observed near other ionizable groups (Schlessman et al., 2008). Alternatively, the minor chemical shift changes upon ionization of the Lys residues in these 15 variants may reflect local electrostatic effects or increased local dynamics on the fast timescale that would be invisible in the 1H-15N HSQC data. Molecular dynamics (MD) simulation on the crystal structure of the L38K variant provided strong evidence of local unfolding and water penetration to solvate the Lys-38 side chain on ns timescales (Harms et al., 2008). A more rigorous experimental characterization of these 15 proteins with relaxation measurements and native-state hydrogen exchange will be needed to obtain more detailed insight into mechanisms of dielectric relaxation.
The substitution of internal positions with Lys decreases the thermodynamic stability of SNase. The 1H-15N HSQC spectra of the most destabilized variants were the ones that exhibited exchange behavior consistent with either increased dynamics at backbone amides or global unfolding in response to the ionization of the group. This suggests that the character of the apparent dielectric response of a protein can be influenced by its global stability (Karp et al., 2010). In the proteins with the lowest thermodynamic stability the partially unfolded states in which the ionized side chains of internal Lys can contact bulk water can be sampled more readily through increased local fluctuations. Because the local fluctuations occur on the intermediate or slow timescales, this increase in local fluctuations was not observed directly but was manifested indirectly in the 1H-15N HSQC spectra. Many peaks in these spectra also exhibited large chemical shift changes upon ionization that were consistent with small structural perturbations or electrostatic effects emanating from the fluctuating electrostatic field from the fluctuating Lys side chain. Efforts are underway to demonstrate that the pKa values of internal ionizable groups are sensitive to the global thermodynamic stability and that for this reason stability can affect the apparent dielectric effect. If this idea is borne out by further experimental data, it has the potential to redirect efforts in structure-based calculation of electrostatic energies.
The increased dynamics and intermediate or slow exchange observed in 10 of 25 variant proteins (variants I92K, V66K, L125K, L25K, V99K, F34K with low stability and T62K, N100K, V39K, A58K with high stability) is consistent with the idea that the high apparent polarizibility reported by the internal Lys residues in some of these proteins is partly a reflection of increased dynamics or structural reorganization concomitant with the ionization event. This type of structural response to the ionization of an internal group is essential for biological function in protein such as bacteriorhodopsin (Lanyi and Luecke, 2001), in ATPase (Rastogi and Girvin, 1999), and in the photoactive yellow protein (Xie et al., 2001). Our smaller and more tractable protein will allow benchmarking of algorithms need to examine mechanism of H+-driven conformational changes in many of the proteins that use internal ionizable groups for purposes of biological energy transduction.
This NMR spectroscopy survey of structural consequences of the ionization of internal Lys residues in SNase contributes novel insight into the range of possible structural reorganization that can be triggered by the ionization of internal groups, and how this is affected by details of the microenvironment of the ionizable moiety or by thermodynamic properties of the protein. It also contributes a set of constraints useful to benchmark the performance and accuracy of computational methods for structure-based calculations of pKa values of ionizable groups in dehydrated environments. The ideas about structural origins of dielectric effects and molecular determinants of pKa values of internal groups that emerge from this study have important, general implications for computational efforts. Computational methods are going to have to be able to identify cases in which the ionization of an internal group can trigger structural rearrangement or increased dynamics, and they should be capable of identifying the specific conformational states stabilized through the ionization of buried groups. The possibility that the structural response to the ionization of an internal group is actually governed by the global stability of the protein raises several challenges for algorithms for structure-based pKa calculations. The most challenging problem is the need for improved sampling capabilities so alternative conformational states that might be populated when an internal ionizable group becomes charged are actually sampled during a calculation. On-going efforts by some groups suggest this is still a daunting task (Kato and Warshel, 2006). Another challenge is the need to be able to discriminate among alternative states and to identify the ones that are relevant. Structure-based calculation of pKa values has in general been focused exclusively on accurate calculation of electrostatic forces. If the suggestion that the global thermodynamic stability of a protein can affect pKa values is confirmed by experimentation, computational methods will have to involve accurate calculation of thermodynamic stability (i.e. ΔG°H2O). Free energy determines the probability of population of a conformational state relative to another; therefore, this thermodynamic parameter will be necessary to elucidate molecular determinants of pKa values and mechanisms of H+-driven conformational transitions in proteins.
Protein expression and purification
Lys-containing variants were made using either the stable PHS form of nuclease made with substitutions P117G, H124L, and S128A, or the Δ+PHS stable variant, which consists of PHS nuclease with additional G50F, V51N substitutions, and a deletion of residues 44–49 (García-Moreno et al., 1997). Mutagenesis was performed with the QuikChange kit from Stratagene (La Jolla, CA). NMR spectroscopy studies were performed with the Δ+PHS protein whereas both Δ+PHS and PHS were used for crystallography. Proteins were expressed and purified as described previously (Shortle and Meeker, 1989). The protein was determined to be > 98% pure by SDS-PAGE analysis. The concentration was determined using an extinction coefficient of 15,600 M−1cm−1 at 280 nm.
Crystallography
The proteins were crystallized using the hanging drop vapor diffusion method. The reservoir solution contained 25 mM potassium phosphate and 2-methyl-2,4-pentanediol (MPD) (Sigma-Aldrich Corp., St. Louis, MO). Proteins were mixed in a 1:1 ratio with reservoir solutions prior to suspension over the reservoir solution and incubation at 277 K, except for the L103K variant, which was incubated at 298K. For the L25K, I72K, L103K, V104K, and L125K variants, the protein was pre-incubated with calcium chloride and pdTp in a 1:3:2 ratio prior to mixing with the reservoir solution. Crystals appeared over a period of several weeks to months.
Each crystal was suspended with mother liquor in a nylon loop mounted on a copper base (CryoLoops™ and CrystalCap Copper Magnetic™ from Hampton Research, Aliso Viejo, CA) and flash-cooled in liquid nitrogen prior to data collection. Diffraction data were collected at cryogenic temperatures from a single crystal of each variant. PHS/T62K and Δ+PHS/L36K data were collected using a Bruker Proteum diffractometer system (Bruker AXS, Madison, WI). All other data were collected using a Bruker ApexII diffractometer system (Bruker AXS, Madison, WI) or at beamline X25 of the NSLS at BNL. Each data set was indexed, integrated, scaled and merged using manufacturer’s software to yield a set of unique reflections.
Initial phasing for all structures was obtained by maximum likelihood-based molecular replacement method with Phaser software (McCoy et al., 2005) within the CCP4 (Collaborative Computational Project Number 4, 1994) suite version 6.0.2, using the structure of the Δ+PHS form of SNase (PDB accession code 3bdc) as a search model. Prior to molecular replacement, the starting model was modified by truncating the substituted amino acid for the appropriate variant to Ala, removing all water molecules, and resetting all B-factors to 20.00 Å. For the Δ+PHS/L25K data set, amino acids 112–116 were also truncated to Ala. Rigid-body and positional refinement produced interpretable electron density maps for each variant. Model building (Emsley and Cowtan, 2004) and refinement (Murshudov et al., 1997) were performed iteratively to yield the final models. Twin refinement of Δ+PHS/L103K was performed with Refmac v5.5.0036. R-work and R-free residuals were monitored throughout the refinements. Water molecules were added during model building to reflect spherical electron density in 2Fo-Fc maps that was within 3.5 Å of a hydrogen bonding partner in the protein model. Final checks of the structure were done using the SFCHECK (Vaguine et al., 1999) and PROCHECK (Laskowski et al., 1993).
NMR spectroscopy
Samples were prepared as described previously (Castañeda et al., 2009; Chimenti et al., 2011). Acetate buffer was used at pH values in the range of pH 4.5–5.9, phosphate at pH values in the range of 6.0–7.9, and borate in the range of 8.0–10.2. Above pH 10.2 the protein behaved as a buffer, but the sample was still prepared with borate buffer for consistency. Following several exchanges into buffer, samples were concentrated to between 0.5 and 1.0 millimolar (mM) protein. pH was checked and adjusted as described previously (Chimenti et al., 2011).
Two-dimensional 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of Δ+PHS and 25 internal lysine variants were acquired at 25°C (actual temperature). The temperature was calibrated as described previously (Castañeda et al., 2009). HSQC experiments were collected as described previously (Chimenti et al., 2011). Data conversion and processing was performed using the nmrPipe software suite (Delaglio et al., 1995). Linear prediction and zero-filling were used to improve digital resolution in the indirect dimensions. Spectroscopic visualization and analysis was done using Sparky (Kneller and Kuntz, 1993).
Supplementary Material
01
Acknowledgements
This work was supported by NIH grant GM-065197 to B.GM. E. Helpful advice form Drs. M.J. Harms, A. Damjanovic and J. T. J. Lecomte is gratefully acknowledged. Crystallographic data were measured at beamline X25 of the National Synchrotron Light Source supported by the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the National Institutes of Health grant number P41RR012408. M.S.C received additional support from the Chapman Charitable Trust.
Footnotes
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