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Anomalous Properties of Lys Residues Buried in the Hydrophobic Interior of a Protein Revealed With N-Detect NMR Spectroscopy 15

Christos M. Kougentakis, Emily M. Grasso, Aaron C Robinson, José A. Caro, Jamie L Schlessman, Ananya Majumdar, and Bertrand Garcia-Moreno Estava J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b02668 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 27, 2017

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The Journal of Physical Chemistry Letters

Anomalous Properties of Lys Residues Buried in the Hydrophobic Interior of a Protein Revealed with 15

N-detect NMR Spectroscopy

Christos M. Kougentakis1,2, Emily M. Grasso2, Aaron C. Robinson1, José A. Caro1, Jamie L. Schlessman3, Ananya Majumdar4, and Bertrand García-Moreno E.1,*. Department of Biophysics1, Program in Molecular Biophysics2, and Biomolecular NMR Facility4, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States. Chemistry Department3, United States Naval Academy, 572M Holloway Rd MS 9B, Annapolis, Maryland 21402, United States.

*Email: [email protected].

ACS Paragon Plus Environment

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ABSTRACT: Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pKa values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues in that titrate with pKa values ranging from 6.2 to 8. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how conformational transitions coupled to their ionization affect their pKa values.

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Ionizable residues buried in the hydrophobic core of proteins are rare but they are essential for energy transduction1, catalysis2, macromolecular assembly3 and other important biochemical processes4-5. These buried residues are necessary for H+ transport and e- transfer, and for promoting conformational reorganization in response to binding of ligands or changes in pH. Their functional importance correlates with their unusual properties, including anomalous pKa values, conformational dynamics, and attenuated hydrogen exchange rates. The pKa values of buried residues can be shifted relative to their normal pKa values in water in the direction that favors the neutral state (i.e. elevated for Asp/Glu and depressed for Lys/His)4-5. Owing to shifts in pKa the ionizable moieties are usually neutral when they are buried, reflecting the incompatibility of charges with hydrophobic environments4-11. Despite their importance, the determinants of the anomalous properties of buried groups, including their pKa values, are not well understood. The pKa values of buried residues cannot be predicted accurately with structurebased calculations. Constant pH MD simulations in which structural relaxation coupled to H+ binding or release is treated explicitly show promise6-11; however, benchmarking of calculations against a set of experimental pKa values measured for Lys, Glu or Asp at 25 internal positions in staphylococcal nuclease (SNase) has revealed how challenging it is to reproduce conformational reorganization coupled to the ionization of buried groups4-11. NMR spectroscopy is uniquely suited to help improve structure-based calculations because it can be used to probe the microscopic determinants of the anomalous pKa values of buried ionizable residues; it is sensitive to protonation, conformational dynamics, and solvent exchange effects at atomic resolution, thus it can contribute the physical insight needed to improve our understanding of these important functional groups.


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Previous NMR spectroscopy studies have shown that the chemical shifts of deprotonated Lys residues are distinct from those of protonated Lys residues12. Lys residues are generally difficult to detect by 1H detect experiments owing to the rapid exchange of the Hζ protons with those from water12-15. Water penetration and backbone relaxation can cause significant “selfdecoupling” with internal Lys residues, requiring the use of low temperatures and specialized pulse sequences to detect them12. Owing to the unfavorable relaxation properties of the relatively rigid, buried Lys residues, the experiments normally used to study surface Lys residues14-15 are not sufficiently sensitive to detect pH dependent changes of the internal Lys Nζ chemical shifts12,16.

Figure 1.

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N 1D spectra of SNase variants

with internal Lys residues. (A) Nomenclature of Lys side chain. (B)

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N spectra at 1 to 2

pH units above the pKa value of the internal Lys. No 1H decoupling was used during data acquisition. (C) Crystal structure of SNase (PDB ID:3BDC) with the five internal positions where Lys substitutions were performed shown with spheres.

Color of

spectra in (B) match color of positions in the structure in (C). To overcome the limitations inherent to 1H detection17-20, 15N 1D experiments were used to identify the Nζ resonance of internal Lys residues and to monitor their titration with pH (Figure 1, S1). The goal was not the measurement of the pKa values of buried Lys residues as it


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was to gain insight into other physical attributes of these internal Lys side chains, including their state of hydration and the conformational dynamics of the Nζ atom. This initial study was performed with five variants of staphylococcal nuclease (SNase) with internal Lys residues. The apparent pKa values of the internal Lys residues in the T62K, V74K, V99K, L25K, and L125K variants, measured by linkage thermodynamics, are 8.1, 7.4, 6.5, 6.3, and 6.2, respectively5. In crystal structures of the five variants the internal Lys

Figure

2.

Crystal

structures

showing

microenvironments of internal Lys residues. (A) Overlay of background protein and the 5 variants of SNase with internal Lys residues. (B-F) Microenvironments of Lys-25 (3ERQ),

side chains are observed sampling different microenvironments (Figure 2 and S2-S6, Table 1). The overall fold of the protein was unaffected by the mutations used to introduce the internal Lys residues (Figure 2A).

Lys-62 (3DMU), Lys-74 (3RUZ), Lys-99 Lys-62, Lys-74, and Lys-99 were all (4HMI), and Lys-125 (3C1E). The Lys side within 3.5 Å of polar atoms, with the latter two chain

is

represented

in

light

blue, being within hydrogen bonding distance of

hydrophobic residues in yellow, and water interfacial or internal water molecules. Lys-25 molecules in cyan. and Lys-125 are both in deeply buried, hydrophobic environments21. Backbone 1H-15N


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HSQC experiments on these variants measured previously showed that changes in pH affect the five proteins very differently (Table 1). The effects range from minor chemical shift perturbations all the way to broadening and increased presence of random coil resonances indicative of partial unfolding, presumably to allow the internal Lys access to bulk water21. The Nζ resonance of the internal Lys residues in the T62K, V74K, and V99K variants were detectable by

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N 1D pulse acquire experiments and assigned using the H2(C)N Nζ-Hε

correlation experiment (Figure S7). The resonance belonging to the internal Lys residue was very weak in the

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N 1D experiment of the L25K variant, and completely absent in the case of the

L125K variant.

Lys-125 was also not

detectable using the H2(C)N experiment. In these two cases, 1H-15N-heteronuclear cross polarization22-27 (HCP) was used to increase the sensitivity of the 1

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N 1D experiment. In

H coupled spectra at pH values where the

internal Lys residue was expected to be neutral, the Lys-25 Nζ resonance displayed a 1:2:1 triplet splitting with a JNH~65 Hz, consistent with a deprotonated Lys and significantly attenuated solvent exchange12 (Figure 3A). In the case of the variant with Lys-125, the resulting resonance in the HCP experiment was broad even at pH values significantly above the apparent pKa value of

Figure 3.

Solvent exchange behavior of

variants with Lys-99, Lys-25 and Lys-125. (A)

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N HCP 1D experiments for the variant

with V99K at pH 7.0, for L25K at pH 7.3, and for L125K at pH 7.6. Stars identify resonances and splitting patterns. (B) 1H-15N Nζ selective HSQC of variants with Lys-25 and Lys-125.


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6.2, although splittings caused by Nζ-Hζ couplings were observed at low temperatures. In cases where splittings were observed in 1H coupled spectra, the Nζ:Hζ resonance could be clearly observed in Nζ selective 1H-15N HSQC experiments13 (Figure 3B). The 1H chemical shifts of the neutral NH2 species are highly upfield shifted relative to the chemical shift of protonated Lys Nζ resonances (6-8 ppm) and consistent with those observed in sp3-type alkyl-NH2 compounds (1-2 ppm)12. The 1H chemical shift of Lys-25 displayed an even greater upfield shift than those of Lys-125 or the previously studied Lys-66 in the V66K variant of SNase12. No splittings were observed in the other variants, consistent with water penetration or backbone relaxation causing significant self-decoupling. The conformational response of the protein to ionization of the internal Lys residues was probed with pH titrations monitored with Nζ chemical shifts (Figure 4, S8-S12). The Nζ resonance of Lys-74 remained sharp throughout the pH titration in both

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N 1D and H2(C)N

experiments. In contrast, the Nζ resonance of Lys-62 was not detectable by the H2(C)N experiment below pH 9.3. In this case, the

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N 1D experiment was necessary to detect the pH

dependence of the Nζ chemical shift, the resonance of which became progressively broader as the apparent pKa value was approached. Lys-99, Lys-25 and Lys-125 exhibited no shift of the Nζ resonance in 15N 1D, 1H-15N HSQC, and 15N HCP 1D experiments, respectively; a pH dependent loss in the intensity of the resonance was observed instead. The pH dependence of the lineshape behavior observed in these experiments provided insight into the protonation dynamics of the internal Lys residues. Chemical exchange, originating from either solvent exchange or conformational exchange between two distinct species, can influence the lineshape of NMR resonances in several ways, depending on the timescales of inter-conversion, populations of states, and differences in chemical shift (referred


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to as ∆ω, where ω is the Larmor frequency)28,29. The titration of Lys-74 offers an example of fast exchange (kex>>∆ω) in which the chemical shift is the population-weighted average of both the protonated and deprotonated forms of the residue. Backbone amide chemical shift perturbations (CSPs) above and below the pKa value of Lys-74 were minor and on the scale observed in the background protein (Figure S13), suggesting that ionization of Lys-74 does not result in significant structural reorganization of the protein. It is noteworthy that the pKa value of Lys-74 determined by analysis of the pH dependence30 of the Nζ chemical shift was shown to be in excellent agreement with that obtained through linkage thermodynamics5 (Figure 4B). In contrast to what was observed with Ly-74, the Nζ resonance of Lys-62 exhibited pH dependent line broadening in both the 15N 1D and H2(C)N experiments. Broadening is indicative

Figure 4.

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N 1D pH titrations of SNase

variants with internal Lys residues. (A) In V74K, Lys-74 shows pH dependent fast exchange behavior. (B) Top: H2(C)N titration of V74K confirms assignment of Lys-74 resonances. Bottom: Fit of pH dependence of the

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N chemical shift of

Lys-74 from 1D titration to a modified Hill equation29. (C) In T62K, Lys-62 shows pH dependent

intermediate

exchange

behavior. (D) In V99K, Lys-99 shows pH dependent slow exchange behavior.


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of intermediate exchange (kex∼∆ω). Consistent with this observation, large backbone amide CSPs were observed in regions adjacent to the amine of Lys-62 (Figure S14)16. The broadening of the Lys-62 Nζ resonance is consistent with an internal Lys that is highly dynamic, while the large backbone CSPs is consistent with structural reorganization in response to the ionization of this Lys residue, or with changes in the electronic environment due to the presence of a partially buried NH3+ group. This is consistent with the observation of peak doubling and broadening of the

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C and 1H resonances belonging to the Lys-62 side chain in triple resonance experiments

(Figure S15). The pH dependent decrease in resonance intensity observed in variants with Lys-25, Lys99 and Lys-125 is caused by slow exchange between two populations (kex

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