9824 Chem. Commun., 2013, 49, 9824--9826 This journal is c The Royal Society of Chemistry 2013

Cite this: Chem. Commun.,2013,49, 9824

Detection of salt bridges to lysines in solution inbarnase†

Mike P. Williamson,*a Andrea M. Hounslow,a Joe Ford,a Kyle Fowler,a

Max Hebditcha and Poul Erik Hansenb

We show that salt bridges involving lysines can be detected by

deuterium isotope effects on NMR chemical shifts of the sidechain

amine. Lys27 in the ribonuclease barnase is salt bridged, and

mutation of Arg69 to Lys retains a partially buried salt bridge.

The salt bridges are functionally important.

Salt bridges in proteins are formed by the interaction ofpositively and negatively charged sidechains.2 Isolated chargeswithin the hydrophobic interior of proteins are of high energy,and therefore buried salt bridges are rare, confer significantstabilisation energy to the protein, and are usually of functionalimportance.3 By contrast, surface-exposed salt bridges are generallyweaker, more variable in their contribution to stability, and moredifficult to predict:4 we showed recently that three salt bridgesexposed on the surface of the protein GB1, which are well-definedin crystal structures, are not present in solution.1 Since all analysesof salt bridges are based on crystal structures (because NMR isusually unable to define the positions of surface-exposed side-chains), it is difficult to assess the real importance of exposedsalt bridges. However when present, exposed salt bridges areusually important for function, not least because of their stronggeometrical dependence,5 for example in directing coiled-coilformation;6 directing the folding pathway;7,8 or stabilisingproteins from thermophiles.9 Intermolecular salt bridges arealso important determinants of specificity,10 where for examplea salt bridge between two kinase monomers was shown to beessential for kinase activity.11

It is therefore important to be able to identify salt bridges insolution. Currently two experimental approaches are used forthe detection of salt bridges in solution. One is to measure thepKa of one or both of the residues involved: the acidic partner

should have a lower pKa than normal, whereas the basic partnershould have a higher pKa than normal.12,13 Site-specific measure-ments of pKa are normally done by NMR, by observation of Asp orGlu, since measurement of Lys or Arg sidechains is difficult,especially at high pH (typically >10).14 The magnitude of thechange in pKa provides a good estimate of the free energy of thesalt bridge.12 The second method is to mutate the residuesinvolved. This is typically done as a double mutant cycle, in whichboth residues are mutated, individually and together, and thestability to unfolding of each mutant is measured.12 The differ-ence in free energy between the double mutant and the sum of thetwo individual mutations is the coupling free energy, which isassumed to equal the free energy of the salt bridge. Both of thesemethods have drawbacks. Measurement of pKa requires thefolded protein to be stable at an extreme of pH, while the doublemutant cycle assumes no sidechain interactions in the unfoldedstate. Therefore we present here an alternative, more direct, andless laborious method.

As a test system, we have used the ribonuclease, barnase(Fig. 1). This is a small and well characterized enzyme thathydrolyses RNA. Salt bridges in barnase have been studied byboth the methods described above, which revealed two saltbridges involving arginines (R83-D75 and R69-D93) plus a third

Fig. 1 The structure of barnase (PDB 1a2p), showing the three salt bridgesdescribed here.

a Dept of Molecular Biology and Biotechnology, University of Sheffield, Firth Court,

Western Bank, Sheffield S10 2TN, UK. E-mail: m.williamson@sheffield.ac.uk;

Fax: +44 (0)114 2224243; Tel: +44 (0)114 2224224b Dept of Science, Systems and Models, 18.1, Roskilde University, Universitetsvej 1,

PO Box 260, DK-4000 Roskilde, Denmark. E-mail: poulerik@ruc.dk;

Fax: +45 46743011; Tel: +45 46742432

† Electronic supplementary information (ESI) available: Lysine assignments,NMR spectra, experimental methods. See DOI: 10.1039/c3cc45602a

Received 23rd July 2013,Accepted 5th September 2013

DOI: 10.1039/c3cc45602a

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This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 9824--9826 9825

involving a lysine (K27-D54). Wild-type barnase is stable tobelow pH 2, allowing the measurement of the pKas of theaspartates involved (D75, D93 and D54) as 3.1, 1.5, and 2.2respectively, compared to approximately 3.9 in the denaturedstate.15 From the relationship DDG = DpKa � 2.303RT, thiscorresponds to free energies of approximately 4.6, 13.8 and9.6 kJ mol�1, respectively. The stabilities of mutants of saltbridge residues are reduced: by 20.9 kJ mol�1 for D75N,10.9 kJ mol�1 for D54N, 13.8 kJ mol�1 for D54A, and 1.7 kJ mol�1

for K27A,15–17 giving an estimated coupling free energy of12.6–14.6 kJ mol�1 for the R83-D75 and R69-D93 salt bridges.7

The salt bridges are usually present in crystal structures. Allthree salt bridges are thus well defined.

The method that we present here to detect salt bridges relieson observation of deuterium isotope effects on NMR signalsfrom the amine group at the end of the lysine sidechain. Lysineamino protons are broadened by rapid exchange with water,and are consequently difficult to observe. We therefore usedlow pH and temperature to slow down the exchange (pH 4.8,3 1C) and used a modification of the HSQC experiment namedHISQC that was developed for application to lysine sidechains,in which 15N transverse magnetization is kept in-phase withrespect to 1H during t1 evolution.18 Fig. 2 shows the HISQCspectrum of barnase, in 10 mM sodium acetate. All eight lysinesidechain signals are observed. In 20% D2O–80% H2O (Fig. 2b)one expects the relative concentrations of NH3

+, NH2D+ andNHD2

+ to be 0.51 : 0.38 : 0.10. In agreement with these ratios,the NH3

+ and NH2D+ peaks are of comparable intensity, whilethe NHD2

+ peak is much weaker.Lysine sidechain signals were assigned by using a CCH-TOCSY

experiment to assign sidechain 1H and 13C signals from the back-bone out to Ce, combined with 2D H3NCECD18 and HSQC-TOCSYexperiments to go from the amino group towards the backbone(ESI†). Chemical shift assignments are given in Table 1, and showthat the highest field 15N signal is from K27.

The deuterium isotope effect is the difference in chemicalshift between NH3

+ and NH2D+ peaks, or equivalently NH2D+

and NHD2+ peaks; the difference in 15N shift is called 1D15N,

while the difference in 1H shift is called 2D1H. The isotopeeffects are defined as 1D15N = dN(H) � dN(D). The deuterium

isotope effect is expected to be different in salt-bridged andnon-salt-bridged amines, because the formation of an N–H� � �Osalt bridge makes the N–H bond longer and weaker, and there-fore changes the perturbation of nuclear shielding caused by theintroduction of the heavier 2H nucleus. Both calculation19,20 andexperiment21 suggest that there should be significant differencesin isotope effects as a result of salt bridging.

The isotope effects are readily measured by studying samples inH2O–D2O mixtures (Fig. 2) and are listed in Table 1. Because theisotope effects are differences between pairs of peaks in the samespectrum, they can be measured with high accuracy. The isotopeeffect for K27 is different from those of all other lysines: 1D15N issmaller while 2D1H is larger (more negative). Measurements of pKasof all 12 acidic residues in barnase15 showed that four (D54, E73,D93 and D101) have pKas lowered significantly compared to typical‘random coil’ values. Crystal structures suggest that D54 has a saltbridge to K27 and D93 to R69, while E73 forms a hydrogen bond toY103 OH, and D101 to T105. Thus, previous data suggest that theonly lysine to be involved in a salt bridge is K27. The unusualisotope effect for K27 is therefore most obviously explained asarising from the salt bridge.

In order to check this hypothesis, we generated another salt-bridged lysine by mutation of R69 to lysine. R69 forms a strong andpartially buried salt bridge to D93, as evidenced by a pKa for D93 ofapproximately 1.5, a coupling free energy of 13.8 kJ mol�1, andconsistent formation of salt bridges in crystal structures.7,15 Themutation was made using PCR and verified by sequencing, andthe mutated protein was purified using the standard protocol forwild-type barnase.22 The HISQC spectrum of lysine sidechains inthe mutant shows the presence of an extra signal (ESI†).

In order to verify that K69 in the mutant is still forming a saltbridge to D93, we measured the pKa of D93 by following thechemical shift of the sidechain carboxyl resonance as a functionof pH, as detected in 2D H(CA)CO spectra modified for aspartatesidechains.23 The results are shown in Fig. 3, and show that D93 hasa pKa of approximately 2.3, with the other aspartates measuredhaving pKas in the normal non-hydrogen-bonded range of 3.2–3.8.Thus, D93 remains strongly salt bridged in the mutant. The R69Kmutant is however significantly less stable at low pH than is the wild-type: the wild-type protein unfolds at pH 2.15,15 whereas R69Kunfolds at pH 2.85, making it difficult to obtain data at low pH.

Fig. 2 HISQC spectrum of lysine sidechains in barnase: (a) in 100% H2O (b) in20% D2O–80% H2O. In (b), for each lysine, the lower signal (larger 15N chemicalshift) is from the NH3

+ form, while the upper one is NH2D+. For three of theresidues the NHD2

+ peak is also visible, with the NH3+–NH2D+ spacing being

equal to the NH2D+–NHD2+ spacing. The residue indicated in green is the salt-

bridged K27.

Table 1 Chemical shifts and isotope effects for lysine residues in barnase(in ppm)

15N shift 1H shift 1D15N 2D1H

K19 32.72 7.68 0.357 �0.0233K27 31.82 7.88 0.348 �0.0335K39 32.46 7.73 0.359 �0.0239K49 32.30 7.60 0.363 �0.0274K62 32.82 7.92 0.362 �0.0260K66 32.48 7.67 0.363 �0.0217K98 32.60 7.52 0.358 �0.0284K108 32.38 7.60 0.354 �0.0260K69a 32.75 7.91 0.356 �0.0318

Chemical shifts are given in ppm relative to DSS, and were measured in10 mM acetate, pH 4.8, 3 1C. For exchange of two deuteriums the totaleffect is twice as large (see Fig. 2b). a In the R69K mutant. All others aremeans from wild-type and R69K.

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9826 Chem. Commun., 2013, 49, 9824--9826 This journal is c The Royal Society of Chemistry 2013

This provides a good illustration of the difficulties of detectingsalt bridges using pKa measurements: if the protein is unstableat low pH, then chemical shifts cannot be followed to the end ofthe pH titration, and pKa measurements will be less precise.

In the R69K mutant, the isotope effects for K69 are similar tothose observed for K27, though slightly less extreme (Table 1).There is thus a small but clear difference in isotope effectsbetween salt bridged and non-salt bridged lysines, providing asimple indicator of the presence of a salt bridge: the 1D15N and2D1H are respectively 0.363 � 0.004 and �0.026 � 0.003 in theabsence of a salt bridge, and 0.352� 0.004 and�0.033� 0.002 inthe presence of a salt bridge, a highly significant difference forboth (even using non-parametric tests), though more obvious for2D1H. The limited data available suggests that isotope effects fromstronger salt bridges are more different. We note that the changein both 1D15N and 2D1H on salt bridge formation is smaller thanthat predicted from calculation,19,20 although it is in the directionpredicted. The magnitudes of 1D15N are also generally less thanpredicted, but similar to those observed previously for proteinG1 and HoxD9 homeodomain.18 We have previously suggestedthat this difference in 1D15N may be due to solvation of the amine;it is likely that solvation is also responsible for reducing thechanges in isotope effects in salt bridges.

In summary, we have shown that salt bridges in solution canbe detected using deuterium isotope effects. The salt bridgeformed by K27 in barnase is functionally important. Mutationof lysine 27 to alanine produces an enzyme with kcat only 0.03%of wild-type activity.24 It is suggested that in the transition statefor RNA hydrolysis, K27 forms a salt bridge with the cleaved

phosphate oxygen.25 In most crystal structures of free barnase,K27 has an unusual folded-back conformation and forms a saltbridge to D54, but in almost all the structures of barnase boundto substrate analogues or inhibitors, K27 is extended and inter-acts with the ligand. The loss of the intramolecular salt bridgeand formation of an alternative salt bridge to the cleavedphosphate oxygen therefore seems to be a crucial part of thestructural rearrangements needed to form and stabilize thetransition state. The salt bridge to Arg69 is also essential fordirecting the folding pathway and stabilising the folded protein.7

In general there is good evidence that salt bridges in proteins,where present, are functionally important. The ability to detectsuch salt bridges is thus crucial.

We thank SURE (Sheffield Undergraduate Research Experi-ence) for funding (KF).

Notes and references1 J. H. Tomlinson, S. Ullah, P. E. Hansen and M. P. Williamson, J. Am.

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Fig. 3 Aspartate sidechain carboxylate chemical shifts as a function of pH in theR69K mutant. Black, D93; brown, D8; blue, D54; orange, D44; green, D12. Thecurves were fitted to single pH dependences.1 For D93, the fits were less reliablebecause less of the titration curve could be sampled. The curve shown was fittedby fixing the chemical shift change on protonation to be similar to those seen forthe other aspartates.

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