J Biomol NMR
DOI 10.1007/s10858-014-9862-y

ARTICLE

pH-dependent random coil 1H, 13C, and 15N chemical shiftsof the ionizable amino acids: a guide for protein pKa
measurements
Gerald Platzer • Mark Okon • Lawrence P. McIntosh

Received: 29 July 2014 / Accepted: 9 September 2014
Ó Springer Science+Business Media Dordrecht 2014

Abstract The pKa values and charge states of ionizable
residues in polypeptides and proteins are frequently
determined via NMR-monitored pH titrations. To aid theinterpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the1
H, 13C, and 15N nuclei in the seven common ionizableamino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg)
within the context of a blocked tripeptide, acetyl-Gly-XGly-amide. Alanine amide and N-acetyl alanine were used
as models of the N- and C-termini, respectively. Together,
this study provides an essentially complete set of pHdependent intra-residue and nearest-neighbor reference
chemical shifts to help guide protein pKa measurements.
These data should also facilitate pH-dependent corrections
in algorithms used to predict the chemical shifts of random
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10858-014-9862-y) contains supplementarymaterial, which is available to authorized users.
G. Platzer  M. Okon  L. P. McIntosh (&)
Department of Biochemistry and Molecular Biology, Life
Sciences Centre, 2350 Health Sciences Mall, University of
British Columbia, Vancouver, BC V6T 1Z3, Canadae-mail: mcintosh@chem.ubc.ca
G. Platzer  M. Okon  L. P. McIntosh
Department of Chemistry, University of British Columbia,
Vancouver, BC V6T 1Z1, Canada
G. Platzer  M. Okon  L. P. McIntosh
Michael Smith Laboratories, University of British Columbia,
Vancouver, BC V6T 1Z4, Canada
Present Address:
G. Platzer
Department of Structural and Computational Biology, Max F.
Perutz Laboratories, University of Vienna, Vienna Biocenter
Campus 5, 1030 Vienna, Austria

coil polypeptides. In parallel, deuterium isotope shifts forthe side chain 15N nuclei of His, Lys, and Arg in theirpositively-charged and neutral states were also measured.
Along with previously published results for Asp, Glu, Cys,
and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.
Keywords Protein electrostatics  pH titration  Chemical
shift  Scalar coupling  Deuterium isotope shift  Hydrogenexchange

Introduction
Electrostatic interactions are central to the structures,
dynamics, and functions of proteins (Creighton 2010).
These interactions are established in large part by the pHdependent protonation states of their termini and ionizableside chains. Although substantial progress has been madein theoretical methods to predict the charges and aciddissociation equilibrium constants (pKa values) of thesemoieties within the context of a folded protein (Alexov
et al. 2011; Nielsen et al. 2011), experimental measurements remain critical. This is particularly true for functionally important residues, which often have significantly
perturbed pKa values (Forsyth et al. 2002; Harris andTurner 2002) and may be involved in complex, coupledionization equilibria (Forman-Kay et al. 1992; Lindman
et al. 2006; Ludwiczek et al. 2013). Crystallographicallyand NMR spectroscopically-determined structures areinvaluable for understanding protein electrostatics. However, in the absence of neutron (Blakeley et al. 2008;
Niimura and Bau 2008) or very high-resolution X-ray
diffraction data (Lecomte et al. 2008), the protonation

123

J Biomol NMR

states of residues in these structures are often experimentally undefined and thus inferred from physicochemicalarguments. In favorable cases, the ionization states and pKavalues associated with selected side chains can be determined using approaches spanning potentiometry (Parsons
and Raftery 1972) to pH-dependent stability (Fitch et al.
2002), chemical reactivity (Tolbert et al. 2005), andenzymatic measurements (Knowles 1976). Given suitable
chromophores/fluorophores, various forms of absorption/
emission spectroscopy can also be used. Of all such
experimental methods, NMR spectroscopy is the mostpowerful technique for investigating the residue-specificcharge states and pKa values of proteins and theircomplexes.
The observation of the 1H-NMR signal(s) from the acidic
proton(s) of an ionizable functional group provides unambiguous evidence that it is indeed protonated. However,labile nitrogen-, oxygen- and sulfur-bonded protons typically exchange rapidly with those of water (Englander andKallenbach 1983; Wüthrich and Wagner 1979). Thus, theirdirect detection usually requires conditions of low temperature, pH, and general acid/base buffer concentrations(Liepinsh and Otting 1996; Liepinsh et al. 1992), combinedwith pulse sequences that minimally perturb water magnetization (Gueron et al. 1991; Zheng and Price 2010). Even so,
observable acidic protons also tend to be protected fromexchange (HX) by hydrogen bonding and burial within theinterior of a protein or protein complex. As a result of such
structure-specific environments, the associated residues
often have perturbed pKa values and their acidic protons may
resonate over a wide range of chemical shifts relative to thosefound for model reference compounds. This is advantageous
for simple 1H-NMR measurements if yielding well resolved
signals downfield of *10 ppm (Baturin et al. 2011; Schubert
et al. 2007). However, labile protons with less perturbed
chemical shifts may remain undetected by one-dimensional(1D) 1H-NMR approaches due to spectral overlap within theenvelope of water and protein signals, and by conventional
2D 15N/13C heteronuclear correlation experiments due totheir optimization for amides and aromatic/aliphatic groups,
respectively.
When properly optimized, acidic protons on arginine,
histidine and lysine side chains, as well as the N-terminal
amine, can often be detected in a 15N-HSQC or -HMQC
spectrum due to a strong one-bond 1JNH coupling to thedirectly attached 15N (provided that the HX rate constantkex \ 1JNH to allow coherence transfer (Henry and Sykes
1990; Segawa et al. 2008)). These functional groups areeasily recognized by their diagnostic 15N shifts, which areclearly resolved from those of the protein amides and indoles(Blomberg et al. 1976; Blomberg and Rüterjans 1983; Cohenet al. 1983). In addition, the number of slowly exchanging
protons on the 15N can be determined unambiguously from123

1

JNH multiplet patterns in spectra recorded without 1H
decoupling in the indirect dimension. This is particularly
helpful for distinguishing –NH3? versus –NH2 groups (Poonet al. 2006; Takayama et al. 2008a, b). In contrast, oxygenand sulfur-bonded protons lack such useful one-bond couplings. Thus, direct detection of these protons requires theuse of less sensitive homo- or heteronuclear multiple-bondscalar correlation experiments or potentially ambiguous
NOE approaches (Baturin et al. 2011; Liepinsh et al. 1992;
Nordstrand et al. 1999). Alternatively, oxygen- and sulfurbonded protons may be observed in 1D 1H-NMR spectra
recorded while filtering against protons directly bonded to13
C and 15N nuclei in a protein uniformly labeled with these
two isotopes (Brockerman et al. 2014). In the event that anacidic proton cannot be observed directly because of rapid
HX or because of spectral overlap that cannot be resolved
using scalar or NOE correlations, its presence may still be
revealed through deuterium isotope shift measurements(Hansen 2000, 2007; Ladner et al. 1975; Led and Petersen
1979; Takeda et al. 2009, 2010, 2014; Tugarinov 2014;
Wang et al. 1996).
The pKa values and hence protonation states of ionizable
residues in proteins are most frequently determined via
NMR-monitored pH titrations. Over the course of thetitration, one generally measures the signals from selected
non-exchangeable 1H, 13C or 15N nuclei within a given
residue, whose chemical shifts (or scalar couplings) are
assumed to report the protonation state of that residue.
Fitting the chemical shift versus pH data to a suitable
equation for a chemical equilibrium in the fast exchange
limit yields the apparent pKa values governing the observed
titrations (McIntosh et al. 2011). However, a titration curve
may deviate from that expected for a simple acid/baseequilibrium as reflected by the familiar Hendersen-Hasselbalch
equation. This phenomenon can arise from coupled protonation equilibria, such that a residue exhibits multiple
microscopic pKa values that depend upon the exact charge
states of all other interacting residues (McIntosh et al.
2011; Rabenstein and Sayer 1976b; Shrager et al. 1972;
Søndergaard et al. 2008; Surprenant et al. 1980; Szakacs
et al. 2004; Ullmann 2003). Chemical shift perturbations
may also result from other pH-dependent effects, includingchanges in the electric field around a nucleus due to thedeprotonation of neighboring residues (Buckingham 1960;
Kukic et al. 2013), as well as local or global protein conformational transitions (Tomlinson et al. 2010; Wishart2011). Indeed, it is important to stress that, for most nuclei
in a protein, the range of possible chemical shift changes
induced upon folding or ligand binding encompass thosedue to (de)protonation (Ulrich et al. 2008). Thus, caution
must be exercised when trying to infer the charge state of aresidue solely from the chemical shifts of its 13C, 15N, or
non-exchangeable 1H nuclei.

J Biomol NMR

With these caveats in mind, the pKa value(s) of an
ionizable residue is most confidently determined whenseveral nuclei in that residue (particularly if part of theacidic/basic functional group) report co-incident titrations
and exhibit chemical shift changes comparable in magnitude and sign (i.e., upfield or downfield) to those shown byreference random coil polypeptides. Most of these reference values can be found in papers from the pioneeringdays of biological NMR spectroscopy (Batchelor et al.
1975; Blomberg et al. 1976, 1977; Blomberg and Rüterjans
1983; Bundi and Wüthrich 1979a, b; Cohen et al. 1983;
Freedman et al. 1973; Howarth and Lilley 1978; Kanamori
et al. 1978, Keim et al. 1973, 1974; London 1980; London
et al. 1977, 1978; Quirt et al. 1974; Rabenstein and Sayer
1976a; Richarz and Wüthrich 1978; Surprenant et al.
1980). However, these studies were carried out with avariety of amino acid derivatives, polypeptides, or proteins
under a range of experimental conditions and, to add
potential confusion, were reported using several different
chemical shift referencing protocols. Accordingly, we havemeasured the pH-dependent chemical shifts of essentially
all the 1H, 13C, and 15N nuclei in the seven common ionizable amino acids within the context of a blocked tripeptide. The few missing values correspond to rapidly
exchanging protons not detected under alkaline conditions.
Alanine derivatives were used as models of the N- andC-termini. Deuterium isotope shifts for the side chain 15N
nuclei of His, Lys, and Arg in their cationic and neutral
states were also determined. Collectively, this provides analmost complete set of intra-residue and nearest-neighborreference 1H, 13C, and 15N chemical shift data to guide pKa
measurements and to aid in interpreting the NMR-monitored pH titrations of polypeptides and proteins. These data
should also better enable pH-dependent corrections inalgorithms used to predict the chemical shifts of random
coil polypeptides (Wishart 2011).

Materials and methods
SamplesN-acetyl alanine (CAS 97-69-8) and alanine amide (CAS33208-99-0) were obtained from Chem-Impex. A series ofacetyl-Gly-X-Gly-amide (X = Asp, Glu, His, Cys, Tyr,
Lys, and Arg) tripeptides were purchased from Biometik(HPLC purified to [95 %). Uniformly 13C6/15N4-enriched
L-arginine was bought from Sigma-Aldrich.
NMR spectroscopy
The tripeptides and alanine derivatives were prepared at*10 mM in 50 mM NaCl and 5 % D2O (D = 2H). DSS(4,4-dimethyl-4-silapentane-1-sulfonic acid; 1 mM) was
included as a pH-independent internal reference (Demarco
1977; Wishart 2011). For the cysteine tripeptide, 10 mM
TCEP (tris(2-carboxyethyl)phosphine) was also present as
a reductant. Sample pH values were measured at room
temperature (*20 °C) using a Thermal Scientific Orion*
3-Star pH meter with an Orion ROSS micro pH electrode(8220BNWP), and adjusted by addition of HCl or NaOH
(0.1 or 1 M) in small aliquots. Spectra were recorded at
25 °C using Bruker Avance III 500 and 600 MHz spectrometers with TCI CryoProbes. The 1H and 13C signals
were referenced directly to the internal DSS, and the 15N
referenced indirectly via magnetogyric ratios (Wishart2011). Chemical shifts were measured using 1D 1H-NMR
along with 2D 13C-HSQC spectra for protonated carbons,multiple-bond 13C-HMBC spectra for non-protonated carbons, 15N-HSQC spectra for slowly exchanging protonatednitrogens, and multiple-bond 15N-HMBC spectra for
deprotonated or rapidly exchanging protonated nitrogens.
For the latter, samples were in 99 % D2O to minimize
signal loss due to HX, and the reported chemical shifts
were corrected for deuterium isotope shifts. The isotope
shifts were determined by comparing spectra recorded in
95 % H2O versus 99 % D2O solutions under similar pH (orpH*) conditions such that the functional group in question
was effectively fully charged or neutral. Here, pH* denotes
the uncorrected pH meter reading (Krezel and Bal 2004).
The 13C6/15N4-L-arginine was initially 10 or 100 mM in
50 mM NaCl with 1 mM DSS and 5 % D2O, and thesample pH increased using small aliquots of 0.1 or 1 M
KOH. To obtain pH [ 13, solid KOH was added directly tothe solution. For measurements under very alkaline conditions, KOH is preferable over NaOH to minimize electrode errors (Licht 1985; Popov et al. 2006). Samples werein 5 mm NMR tubes, except under very high pH conditions, where a 3 mm tube was used to allow tuning at theresulting elevated ionic strength. Chemical shifts were
measured from 1D 13C-decoupled 1H-NMR, 1H/15Ndecoupled 13C-NMR (with 1H-NOE), and 1H/13C-decoupled 15N-NMR (with 1H-NOE) spectra. The TCI CryoProbe is designed for direct detection of 1H and 13C, but
not 15N, and thus a 100 mM sample of 13C6/15N4-arginine
was required for the latter 15N-NMR measurements.
Data analysis
Spectra were processed using Topspin 3. The pH-dependent 1H, 13C, or 15N chemical shifts were fit separately with
KaleidaGraph or GraphPad Prism to standard equations for
one, or in the case of arginine, two sequential titrations inorder to obtain pKa and limiting chemical shift values(McIntosh et al. 2011). These values also include possible
effects of increasing ionic strength over the course of the123J Biomol NMR

N-termpKa = 8.23

+3.6 / -0.27+1.0 / -0.59

CH3
CH

+H3N

C

-6.6 /+3.2

O+0.4 / H3C
-0.02

+8.5

C

0.0 / +0.01

O

H
N

N
H

C

C

+0.4

CH2

+1.1

NH2

+0.1
/ -0.01O+0.4 /
0.0 , +0.11-0.20+1.5 / -0.17

His
pKa = 6.45

+2.6 / -0.92 (ε1)

HC+

+56 / HN
+4.2

-0.3 / -0.33 (δ2)

C

N
H

C

C

+1.8 / ~-0.2

+0.1

+10.4

C

+0.1 / -0.08

H 2N

+

+0.4

123

+0.0 /+0.01 , -0.01+0.6 /

CH

O
H
N

N
H

C

+1.9

+2.1 / -0.28/ 0.0 , 0.04~-7.5 /

NH3+

CH2

+5.0 / -0.24+0.4 / -0.08 CH
2

C
CH2

+0.1
/ +0.01NH2+0.1/ -0.01O+3.6 /

+0.50

C
CH2

NH2

+0.1 /
H
N

0.0 /
H C
0.00 3
C
0.0

CH2

C

CH

CH2

N
H

-0.1 / 0.0

O+0.1 /+0.3 / -0.04

O

O
H
N
C
O

0.0

C
CH2

+0.5

NH2

+0.0 / -0.01+0.1+0.7 /+0.4 / -0.04

pTyr
pKa = 5.96

pThr
pKa = 6.30

OO

P

+4.0

OH

NH2

C +4.0OO

P

CH2 +1.0 /CH2-1.2 / -0.11

O-

CH
C
O

+0.2

CH
N
+2.8 / H

+0.64

+4.3

OH

O

O

O

+1.0

0.0 / -0.01

CH -2.4 / -0.25-0.4 / -0.05

OCH+6.1 /
+0.78

N
H

P

C

OH

+0.3 /-0.06 H3C

+1.1 / -0.17
C

O

O-

+3.9

CH2 +0.5 / -0.19+0.2 / -0.07

NH2+1.0 / -0.40 CH2

pSer
pKa = 5.96

CH2 +0.9 /H 2NC+0.0 / -0.01+0.3 / -0.06+5.9 / NH+0.4 /

CH2

CH2

O

H
N
O

+0.6 /

+22 /

H
N

C

C

+0.1

/ 0.00+0.1 / -0.10

O/ 0.0 , -0.11

CH -0.1 / -0.17

CH

+0.6

O

-0.4

HC

C

C
CH2

SH

O

NH2

CH +3.3 / -0.28

0.0

Arg
pKa = 13.9

+0.6

HC

N
H

C

+0.1

O

H
N

N
H

+0.0 / +0.01

CCH2+0.0 / +0.02

O

CH2

Lys
pKa = 10.34

O
CH2

CH

+0.1 / H C
3
+0.01
C
+0.1

OH

H
N

C

+0.1
/ -0.06+1.6 / ~-0.2

-6.7
+0.1/
+0.02 H3C

CH2

O

+0.1/ +0.02+1.7 / -0.09

CH2

+1.5

O

0.0

+0.2 /

O

H
N+0.4 /

O
H
N

O

Tyr
pKa = 9.76

+0.2 / 0.00
0.0 /
H C
+0.01 3C
0.0

+0.5 /+2.4 / -0.17

CHOH+1.5 / -0.06

Cys
pKa = 8.49

+8 /

CH

+4.1

+1.0 / +0.12

CH2+0.3 / -0.01
O
H
0.0 /
H3C
N
C
0.00
CH2
0.0 C
-0.1 / -0.01

OC+1.4 / -0.17

NH+5.2 / -0.41+3.4+3.5 / -0.22 CH2
O

CH

CH2

OH
C

O

+3.0 / -0.23

H
N

CH
N
H

+0.1 / -0.03

CH2O+2.3 / -0.21

CH3

Glu
pKa = 4.34

OHC

+0.3 / +0.02+1.3 / -0.09
O-0.70 C

NH2

O

Asp
pKa = 3.86

+0.1 /+0.01 H3C
C
0.0

C-termpKa = 3.55

HCHC

+1.8 / -0.25
C
O

+0.8

CH
CH
C

-0.03 / -0.02 CH2
CH+0.2 / NH+0.02

0.0 / -0.02
C 0.0
OJ Biomol NMR
b Fig. 1 pH-dependent chemical shift changes (Dd in ppm; negative isupfield) upon deprotonation of the ionizable amino acid functionalgroups (atoms and Dd values are colored as: acidic proton, red;oxygens and non-labile protons, black within the named residue andgrey in flanking blocked glycines; carbon, green; nitrogen, blue;
sulfur, yellow; phosphate, magenta). The data are from Table 1 andSupplemental Table S1, or in the case of the phosphoamino acidpeptides (second ionization step), from (Bienkiewicz and Lumb1999). Data for 13C6/15N4-L-arginine are due to deprotonation of theguanidinium moiety in the context of fully deprontonated amino (pKa9.15) and carboxyl groups (Table 1 and Supplemental Table S2).
Values for the neutral forms of the Gly-His-Gly tripeptide andarginine are tautomer averaged

Table 1 pH-dependent chemical shifts (ppm) of the ionizable aminoacids aNucleus type

H

13

C

N

1

H

13

C

15

Dd (A–HA)

H (amine)

8.04

Ha

4.10

3.51

-0.59

Hb (methyl)

1.54

1.27

-0.27

Ca

51.7

52.7

1.0

Cb (methyl)

19.3

22.9

3.6

CO
N (amine)

176.0
40.4

184.6
33.8

8.5
-6.6

N

HN

8.35

7.94

-0.41

Ha

4.33

4.12

-0.21

Hb (methyl)

1.41

1.32

-0.09

Ca

51.4

53.7

2.3

Cb (methyl)

18.8

20.1

1.3

CO

179.6

183.0

3.4

N

110.5

115.7

5.2

Aspartic acid: Ac-Gly-Asp-Gly-NH2 (pKa 3.86)
1

H

HN

8.55

8.38

-0.17

Ha

4.78

4.61

-0.17

2.93

2.70

-0.23

54.3
41.1

1.4
3.0

Hb (avg.)
Hd2 (carboxyl)
13

C

N

Ca
Cb

[10
52.9
38.0

Cc (carboxyl)

177.1

180.3

3.2

CO

175.8

176.9

1.1

N

118.7

120.2

1.5

Glutamic acid: Ac-Gly-Glu-Gly-NH2 (pKa 4.34)
1

H

The pH-dependent chemical shifts of the ionizable aminoacids were measured in the context of a blocked tripeptide,acetyl-Gly-X-Gly-amide. Along with parallel studies of Nacetyl alanine and alanine amide, this provides a near complete database of the conformationally-averaged intra-residue and nearest-neighbor 1H, 13C, and 15N chemical shiftchanges resulting from the deprontonation of terminal andside chain groups. These results are summarized in Fig. 1
and Table 1 (with complete data sets in Supplemental Tables
S1 and S2), and discussed below. A review of key literature
and/or representative studies on each amino acid type is alsod (A)C-terminal carboxylic acid: N-acetyl alanine (pKa 3.55)

15

Results

d (HA)

N-terminal amine: alanine-amide (pKa 8.23)
1

15

titrations due to the addition of acid or base. As expectedwith only a single ionizable group in the alanine derivatives
and blocked tripeptides, all constituent nuclei exhibited coincident titrations. Thus, the cited pKa values, with estimatederrors of ±0.05 units due to pH measurements, are theaverage of the very similar fit values for each nucleus
showing a substantial pH-dependent chemical shift change.
These averaged pKa values agree closely with those reported
previously for the termini and Asp, Glu, His, Cys, Tyr, andLys side chains in alanine pentapeptides (Grimsley et al.
2009). Analysis of the titration curves also yielded plateau
chemical shift values for the tripeptides and arginine in theirfully protonated and deprontonated forms, with fitting errorsof ±0.02 ppm for 1H nuclei, ±0.08 ppm for 13C, and±0.06 ppm for 15N. These chemical shift data have been
deposited in the BioMagResBank (Ulrich et al. 2008).

Nucleus

HN

8.45

8.57

0.12

Ha

4.39

4.29

-0.10

Hb (avg.)

2.08

2.02

-0.06

Hc

2.49

2.27

-0.22

He2 (carboxyl)
13

C

15

N

[10

Ca

56.0

56.9

1.0

Cb

28.5

30.0

1.5

Cc

32.7

36.1

3.5

Cd (carboxyl)

179.7

183.8

4.1

CO

176.5

177.0

0.6

N

119.9

120.9

1.0

123

J Biomol NMR
Table 1 continued
Nucleus type

Nucleus

Table 1 continued
d (HA)d (A)

Dd (A–HA)

Nucleus typeHistidine: Ac-Gly-His-Gly-NH2 (pKa 6.45)c
1

H

13

C

15

N

HN

8.55

*8.35d

*-0.2

Ha*4.75d

4.59

*-0.2

13

C

H

13

C

15

N

H

C

15

N

H

123

He

3.00

Hf (amine)

7.52

2.60

-0.40

Ca

56.4

56.9

0.4

*1–2f

3.25

3.08

-0.17

Cb

32.8

33.2

0.3

6.97

-0.33

Cc

24.7

25.0

0.4

He1

8.60

7.68

-0.92

Cd

28.9

33.9

5.0

Hd1

[10

Ce

42.1

43.1

1.0

He2

[10

CO

177.0

177.5

0.5

N

121.0

121.7b

0.7

32.7

g

*-7.5g

Ca

55.1

56.7

1.6

Cb

28.9

31.3

2.4

Cc
Cd2

131.0
120.3

135.3
120.0

4.2
-0.3

Ce1

136.6

139.2

2.6

CO

174.8

176.2

1.5

N

117.9

119.7b

1.8

Nd1

175.8

231.3b

56

Ne2

173.1

181.1b815NNf (amine)*25.2

Arginine: 13C6/15N4-Arg (pKa 13.9)h
1

H

13

7.81

Ha

3.26

Hb (avg.)

1.60

Hc

1.60

Hd

3.19

He (guan.)

7.22

Hg (guan.)

6.67

3.19

-0.07
\ |-0.1|\ |-0.1|

3.00

-0.19

8.48

Ca

58.4

58.6

0.2

Ha

4.56

4.28

-0.28

Cb

34.4

35.2

0.9

Hb (avg.)

2.97

2.88

-0.09

Cc

27.2

28.1

1.0

Hc (thiol)

*2.0

e

Ca
Cb

58.5
28.0

60.6
29.7

2.1
1.7

CO

175.0

176.9

1.9

118.7

b

3.6

N

C

HN (amine)

HN

122.2

HN

8.16

15

N

Ha

4.55

4.49

-0.06

Hb (avg.)

3.02

2.94

-0.08

Hd

7.14

6.97

-0.17

6.85

6.57

-0.28

Hg (phenol)

*9.3e

Ca

58.0

58.2

0.3

Cb
Cc

38.6
130.5

38.7
123.8

0.1
-6.7

Cd

133.3

133.2

-0.1

Ce

118.4

121.7

3.3

Cf

157.0

167.4

10.4

CO

176.3

176.7

0.4

N

120.1

120.7b

0.6

HN

8.40

Ha

4.34

Cd

43.8

44.3

0.5

Cf (guan.)
CO

159.6
185.8

163.5
186.1

4.0
0.2

N

33.2

33.6

0.4

Ne (guan.)

85.6

91.5

5.9

Ng (guan.)

71.2

93.2

22

a

Lysine: Ac-Gly-Lys-Gly-NH2 (pKa 10.34)
1

Dd (A–HA)

7.30

He
13

d (A)

Hb (avg.)

Tyrosine: Ac-Gly-Tyr-Gly-NH2 (pKa 9.76)
1

d (HA)

Hd2

Cysteine: Ac-Gly-Cys-Gly-NH2 (pKa 8.49)
1

Nucleus

Recorded at 25 °C with 50 mM NaCl and 5 % D2O, unless indicated. Reported are the fit pKa values and end point chemical shifts d(ppm) of the of the acid (HA) and conjugate base (A) forms, alongwith the chemical shift change upon deprotonation (Dd; negative isupfield). The estimated errors are ±0.05 for pKa values (±0.1 forarginine), ±0.02 ppm for 1H nuclei, ±0.08 ppm for 13C, and±0.06 ppm for 15N. Blank values indicate not determined. Prochiral
1 b
H shifts are averaged. Supplemental Table S1 provides data for theflanking blocked glycinesb

Recorded in 99 % D2O and corrected for the deuterium isotope
shift
Data for neutral histidine are an average of *80 % Ne2H and*20 % Nd1H tautomerscd

Estimated from (Kjaergaard et al. 2011)e

From the BioMagResBank (Ulrich et al. 2008)f

From (Takayama et al. 2008a)g

From (Andre et al. 2007)

h

4.30

-0.04

Hb (avg.)

1.82

1.78

-0.04

Hc

1.44

1.36

-0.08

Hd

1.68

1.44

-0.24

Tabulated are the fit chemical shifts for the titration of the guanidinium moiety in 13C6/15N4-L-arginine with its a-carboxyl and aamine deprotonated (with the exception of the nitrogen-bonded 1H at
pH *7). Supplemental Table S2 provides full data including the aamine titration. Due to bond rotations, the two 15Ng and four 1Hg
each yield one broad signal. The shifts for neutral arginine are tautomer averaged

J Biomol NMR

given. For consistency, titrations are described in terms ofincreasing pH and thus deprotonation of the acid species toform its conjugate Brønsted-Lowry base.
N-terminal amine
Alanine amide was used as a simple model of an N-terminal residue, yielding a pKa value of 8.23. The a-amino
15
N shifts by -6.6 ppm (i.e., upfield) upon deprotonation,and thus serves as an obvious reporter nucleus for measuring the pKa value of the N-terminus of a protein. Furthermore, the protonated nitrogen has a relatively uniquechemical shift (*40 ppm), distinct from that of the lysineside chain 15Nf (*33 ppm), thus potentially allowing its
observation as a well-resolved signal in a 1D 15N-NMR
spectrum (Mcintosh et al. 1990; Smith et al. 1987; Zhu
et al. 1995). However, detecting the 15N nucleus indirectly
via a 2D Ha(Ca)N-type experiment recorded with a selectively or uniformly 13C/15N-labeled protein should provide
improved sensitivity (Andre et al. 2007). Alternatively, the1 a 1 b 13 a 13 b
H , H , C , C , and even more so, carbonyl 13CO ofalanine amide all show substantial chemical shift changes
upon deprotonation of the a-aminium group (Brown et al.
1978; Led and Petersen 1979; Quirt et al. 1974; Rabensteinand Sayer 1976a; Surprenant et al. 1980). Thus, whenmonitored by 13C-HSQC or Ha(Ca)CO-type experiments(Kay 1993; Oda et al. 1994), respectively, these nuclei
should also serve as reliable reporters for measuring the
pKa value of the N-terminus of a protein. (Complex heteronuclear NMR experiments are denoted following aloose convention of specifying the measured nuclei, andindicating additional nuclei used for key coupling steps
within parentheses). Although not studied herein, upon aaminium deprotonation, additional side chain 13C nuclei indifferent amino acids are known to show pH-dependentchemical shift changes that vary in sign and magnitude(Quirt et al. 1974; Surprenant et al. 1980). These can also
serve as reporter nuclei, as demonstrated by the recent
measurement of the pKa value of Thr1 in the proteosome
core particle, labeled selectively with 13Cc2H3-threonine(13Cc2 Dd * -1 ppm) for methyl-TROSY spectroscopy(Velyvis and Kay 2013).
C-terminal carboxylic acidN-acetyl alanine was used as a model of a C-terminal
residue, for which a pKa value of 3.55 was measured. The
13
CO of the carboxylic acid functional group shifts
downfield by 3.4 ppm upon deprotonation, and thus should
be a reliable reporter nucleus for pKa measurements via 1D
13
C-NMR or 2D Ha(Ca)CO-type approaches (Kay 1993;
Oda et al. 1994). In addition to significant pH-dependent
1 a 13 a
H , C and 13Cb chemical shift perturbations (Brown

et al. 1978; Led and Petersen 1979; Quirt et al. 1974;
Rabenstein and Sayer 1976a; Surprenant et al. 1980), it isalso notable that the amide 15N and 1HN signals shiftdownfield by 5.2 ppm and upfield by -0.41 ppm, respectively, when the carboxyl ionizes (Bundi and Wüthrich
1979b). Indeed, the 15N-HSQC spectrum of a protein often
has a sharp, downfield-shifted 15N signal arising from theamide of its flexible, charged C-terminal residue. Thus,
15
N-HSQC spectra can also be used for pKa measurementsof a protein’s C-terminus, particularly since amide HX will
be slow under the acidic conditions likely associated withsuch a pH titration. This approach was used to determine
the pKa * 2.7 for the C-terminal Trp185 of Bacillus circulans xylanase (Joshi et al. 1997).
Aspartic and glutamic acid
The carboxylic acid functional groups of Asp and Glu inthe blocked tripeptides have pKa values of 3.86 and 4.34,
respectively. With downfield chemical shift changes of 3.2
and 4.1 ppm, respectively, upon deprotonation, the side
chain carboxyl 13C nuclei are most frequently used asreporter nuclei for pH titrations monitored via 1D 13Cb/c
NMR or 2D Hb/c
)COc/d spectra (McIntosh et al. 1996,
2 (C2011; Yamazaki et al. 1993, 1994). However, the adjacentaliphatic 13Cb of Asp (3 ppm) and 13Cc of Glu (3.5 ppm),
as well as their directly bonded protons, show comparable
chemical shift changes and can also be used for pKa
measurements (London et al. 1978; Pujato et al. 2006;
Quirt et al. 1974; Rabenstein and Sayer 1976a; Richarz andWüthrich 1978; Yamazaki et al. 1994). This could beb/c
accomplished using 2D or 3D versions of the Hb/c
COc/
2 C
d
experiment (Yamazaki et al. 1994). Alternatively, 2D
13
C-HSQC spectra could be used to observe the 13Cb/cH2
signals. However this latter approach would likely require
some form of selective 13C-Asp or -Glu labeling to avoid
spectral overlap from a multitude of additional signals
expected with a uniformly labeled protein. Given that boththe carboxyl and adjacent aliphatic 13C nuclei show substantial pH-dependent chemical shift changes, ‘‘protonless’’ 13C-detected Cb/cCOc/d correlation spectra also
provide an effective route for measuring Asp and Glu pKavalues (Castaneda et al. 2009). Although the most sensitive
approaches for NMR data acquisition generally rely on 1Hdetection, cryogenic probes open the door for the improved
observation of such 13C nuclei (Felli and Brutscher 2009).
Signals from the rapidly exchanging oxygen-bonded Hd2of Asp or He2 of Glu (or the C-terminal a-carboxylic acidH00 proton) are very rarely seen in the NMR spectra ofproteins. Furthermore, with the exception of2
JCOH * 7 Hz and 4JHCCOH * 1 Hz measured for acetic
acid in liquid Freon at 110 K (Tolstoy et al. 2004), no
useful scalar couplings have been reported for these acidic

123

J Biomol NMR
Table 2 Useful deuterium isotope shifts for characterizing ionizable
functional groups
Amino acid

Exchangeable sitea

Asp/Glu

13

Isotope shift (ppm)a

Cc/dO2D / 13Cc/dO2H

13

c/d

-

c/d

13

C O2 /

0.23b

-

C O2

0

Nd1/e2D? / 15Nd1/e2H?

0.95c,dCbScD / 13CbScH

0.12e

CbSc- / 13CbSc-

0

CfOgD / 13CfOgH

0.13f

CfOg- / 13CfOg-

0

NfD3? / 15NfH3?

1.05g,iNfD2 / 15NfH2*1.9h,i

NeD? / 15NeH?

1.0d,j

His

15

Cys

13
13

Tyr

13
13

Lys

15

15

Arg

15

NeDx / 15NeHx

15

15

g

15

g

13

f

e/g

13

f

e/g

?

N D2 /N Dy /
CNCN

1.8d,jg

15

1.4d,j,k

?

N H2

15

g

1.7d,j

N Hy

?

D5 /
D4 /

f

13

13

e/g

CN
f

CN

e/g

H5

0.19j

H4

0.08j

?

a

Detected nucleus underlined and shift changes are upon transferfrom D2O to H2O. Paired rows correspond to the acid (upper) andconjugate base (lower) formsb
From (Ladner et al. 1975; Led and Petersen 1979; Wang et al.
1996). A similar value is expected for the C-terminal carboxylic acidc

Average value measured herein at pH/pH* * 3.6 for the imidazolium nitrogens in the blocked histidine tripeptide. Not determined
for the neutral side chain due to tautomer averagingdValues due to one-bond and possible three-bond contributionse

From (Takeda et al. 2010). The 13C also experiences three-bondisotope shift upon exchange of the amide DN to HNf

From (Takeda et al. 2009)binding (Frey 2001; Hanoian et al. 2010; Langkilde et al.
2008; Saito and Ishikita 2012). However, most of thesecarboxylic acid-associated protons are also involved in
short strong or low barrier hydrogen bonds, for which thepredominant role of the Asp/Glu as a proton donor or
acceptor is difficult to define.
Although the acidic proton of an Asp, Glu, or C-terminus may be exchanging too rapidly to be detected directly,
the observation of a two-bond isotope shift of *0.23 ppm
for the carboxyl carbon (13CO2H vs. 13CO2D; Table 2)
upon transfer from D2O to H2O provides very good evidence that the functional group is predominantly neutral
under the experimental conditions (Ladner et al. 1975; Led
and Petersen 1979; Wang et al. 1996). In contrast, theunambiguous lack of an isotope shift indicates that thegroup is deprotonated (although the a-carboxylate of the
C-terminal residue could exhibit a three-bond isotope shiftof *0.03 ppm upon protonation of its deuterated amide(Ladner et al. 1975). Such deuterium isotope shifts are
particularly useful when other reporter nuclei do not show
a titration, making it unclear if the carboxylic acid has a
pKa less than the lowest or greater than the highest pH
tested (Joshi et al. 1997). Under conditions when the carboxylic acid is neutral (pH  pKa), intermediate deuterium isotope shifts might result from strong hydrogenbonding (Dziembowska and Rozwadowski 2001; Guo et al.
2012; Hansen 2007; Joshi et al. 2000).
It is also noteworthy that the 1JCC *50 Hz to the side
chain carboxyl of Asp/Glu and to the C-terminal carboxyl
decrease by *3 to 4 and *6 Hz, respectively, upon deprotonation (London et al. 1978). This offers anotherpossible route for pKa measurements and ionization state
determination.

g

Measured herein at pH/pH* *4, and in agreement with (Tomlinson
et al. 2009; Ullah et al. 2011)
Estimated from Dd of -8.3 and *-7.5 ppm upon 15Nf deprotonation in D2O (herein) and H2O (Andre et al. 2007), respectively,
combined with the total 1.05 ppm deuterium isotope shift for the
protonated f-aminium of the blocked lysine tripeptidehiIsotope shifts of 1.07 ppm for the a-aminium (pH 7 versus pH* 5.8)
and 1.86 ppm for the neutral a-amine (pH 15.25 versus pH* 15.1)
were measured with 13C6/15N4-L-argininejValues measured for the side chain of 13C6/15N4-L-arginine in its
guanidinium (pH 7 versus pH* 5.8) and guanidine (pH 15.25 versus
pH* 15.1) forms. The latter are tautomer averaged and the degree ofsite-specific protonation (x B 1, y B 2) is undefinedk

A value of 1.2 ppm was reported by (London et al. 1977)

protons in a polypeptide or protein. Thus, the very limited
number of carboxylic acid protons identified to date, whichhave chemical shifts in the range of *10 to 22 ppm,
appear to have been assigned via homonuclear correlations(e.g., for samples in aprotic solvents (Volpon et al. 2007))or via perturbations resulting from mutations or ligand

123

Histidine
Histidine is undoubtedly the most studied ionizable aminoacid by NMR spectroscopists for many reasons. These
include its frequent roles in ligand binding and enzymatic
catalysis, its ionization typically occurring over a physiological pH range (blocked tripeptide pKa 6.45), and its
wealth of reporter nuclei. Early studies of histidines inproteins often focused on the aromatic carbon-bonded 1Hd2
and 1He1, which shift upfield by *-0.3 and -0.9 ppm,
respectively, upon deprotonation (Markley 1975). However, essentially all of the 15N and 13C nuclei in this aminoacid show substantial pH-dependent chemical shift andJ-coupling changes. In particular, the ring 15Nd1 and 15Ne2
have dramatically different chemical shifts whether protonated or deprotonated (or post-translationally modified,including phosphorylation (Rajagopal et al. 1994; van Dijk
et al. 1990)), thus providing very strong evidence for theionization state of a histidine side chain (Blomberg et al.

J Biomol NMR
137 ppm(222 Hz)173 ppm

HC
HN
176 ppm

NH+C

CH
120 ppm(202 Hz)+2.6 ppm(-13 Hz)HC-9 ppmε1

N δ1+73 ppm+73 ppm

HC

NH

ε2

N

ε1

δ2 CH

C

+2.6 ppm(-13 Hz)-2.1 ppm(-10 Hz)ε2

HN δ1-9 ppmδ2 CH

C+7.1 ppm(-10 Hz)131 ppm~ +6.8 ppm~ -2.3 ppm

imidazolium

Nε2H tautomer

Nδ1H tautomer

Fig. 2 Determining the charge and tautomeric state of a histidine.
(left) Chemical shifts and 1JCH couplings of the ring 13C (green) and15
N (blue) nuclei in the protonated side chain (protons, red and black).
Also shown are the chemical shift and J-coupling changes upon
deprotonation to the Ne2H (middle) or Nd1H (right) neutral tautomer.
The data are from Table 1 or averaged for model compounds reported
in the following references for 15N shifts (Bachovchin 1986;
Bachovchin and Roberts 1978; Blomberg et al. 1977; Munowitz

et al. 1982; Pelton et al. 1993; Roberts et al. 1982; Witanowski et al.
1972), 13C shifts (Goux and Allerhand 1979; Quirt et al. 1974;
Reynolds et al. 1973; Reynolds and Tzeng 1977), and 1JCH
(Bachovchin et al. 1981; Day et al. 2003; Hansen and Kay 2014;
Hunkapiler et al. 1973; Wasylishen and Tomlinson 1975). The 1JCH
coupling for 13Ce1 has been confirmed to be essentially independentof tautomer form (Hansen and Kay 2014), and a similar behavior for
13 d2
C is assumed

1977; Kawano and Kyogoku 1975). Accordingly, a wide
variety of 1D 1H-, 13C-, and 15N-NMR or 2D single- andmultiple-bond heteronuclear correlation experiments canbe used to measure histidine pKa values (Betz et al. 2004;Löhr et al. 2002, 2005; Ludwiczek et al. 2013; Pelton et al.
1993; Plesniak et al. 1996; Schubert et al. 2007; Spitzner
et al. 2001; Sudmeier et al. 1996; Yu and Fesik 1994).
Although the labile nitrogen-bonded 1Hd1 and 1He2 of arandom coil histidine exchange rapidly with water, whensufficiently protected from HX within a folded protein,
they also yield signals typically downfield of 10 ppm.
These rather distinct signals are readily detectable in 1D
1
H-NMR or 2D 15N-HSQC spectra (Bachovchin 1985;
Connelly and McIntosh 1998; Plesniak et al. 1996; Robillard and Shulman 1972; Schubert et al. 2007; Wu et al.
1984).
The neutral imidazole of histidine exists in a pH-independent equilibrium between two tautomers, with the Ne2H
form favored by *4 to 7-fold over the Nd1H form in thefree amino acid (Blomberg et al. 1977). However, thisequilibrium can be readily perturbed by interactions suchas hydrogen bonding, and thus it is often important todetermine the tautomeric state of a neutral histidine ingiven protein. Based on NMR spectroscopic studies ofseveral histidine derivatives (Fig. 2), upon neutralization of
the imidazolium ring, the 15N that retains a proton shifts by*-9 ppm, whereas the deprotonated 15N shifts dramatically by *73 ppm (Bachovchin 1986; Bachovchin andRoberts 1978; Munowitz et al. 1982; Roberts et al. 1982;
Witanowski et al. 1972). Therefore, the tautomeric state (orequilibrium distribution of tautomers) of a neutral histidine
can be deduced immediately upon assignment of its ring15
N signals. Although obtainable via 1H/13C/15N triple
resonance experiments (Löhr et al. 2005; Sudmeier et al.
1996), these assignments are most frequently determinedusing qualitative patterns of small pH-dependent two- andthree-bond 1H–15N scalar couplings detected in long-range
15
N-HSQC or 15N-HMBC spectra (Blomberg et al. 1977;
Pelton et al. 1993). A one-bond deuterium isotope shift of*0.95 ppm on the ring nitrogens (Table 2) could also be
used to identify the charge and tautomeric state of a histidine. However, given that 15N shift alone is so diagnosticof protonation, deuterium isotope shift measurements may
be more useful for probing other histidine properties, suchas hydrogen bonding and HX kinetics (Chevelkov et al.
2010; Tugarinov 2014). In the case of a detectable nitrogen-bonded proton, the magnitude of 1JNd1H may also
correlate to the neutral Nd1H tautomer ([97 Hz) or charged
imidazolium form (\94 Hz), whereas 1JNe2H appears to beuniformly*99 Hz regardless of charge (Schubert et al.
2007). However, this correlation is derived from a very
limited data set and is unlikely to be a robust criterion for
determining the tautomerization state of a histidine due toadditional structure-dependent factors that could affectthese couplings (Bachovchin 2001).
Histidine ionization and tautomerization can also be
determined by measuring the chemical shifts of the ring 13C
nuclei using direct 13C or indirect 1H–13C heteronuclear correlation approaches (Ludwiczek et al. 2013). These are generally more sensitive that the 15N-directed experiments
discussed above. Again, based on model compounds (Fig. 2),
deprotonation of the histidine side chain to its Ne2H tautomer
leads to chemical shift changes of approximately 6.5 to
6.8 ppm for the 13Cc nucleus and -2.1 ppm for the 13Cd2,
whereas the opposite changes of -2.3 to -2.5 and 7.1 ppm,
respectively, result upon formation of its Nd1H tautomer(Goux and Allerhand 1979; Reynolds et al. 1973). Thus, thedifference between the chemical shift changes exhibited bythese two nuclei with increasing pH provides a robust indication of the resulting tautomeric distribution of a given

123

J Biomol NMR

histidine. Consistent with these data, it has been noted that a
13 d2
C chemical shift[122 ppm is diagnostic of a predominant
Nd1H tautomer (Sudmeier et al. 2003). Parenthetically, due toits sensitivity to tautomerization, 13Cd2 shift is a reliable signature of the zinc coordination mode of a histidine (Barraud
et al. 2012). Two- and three-bond 1H–13C scalar couplings(Wasylishen and Tomlinson 1977), as well as one-bond
13
C–15N couplings (Alei et al. 1980; Blomberg et al. 1977;
Shimba et al. 1998; Sudmeier et al. 1996) within the imidazole
ring can also be used to distinguish histidine tautomers. In
contrast, the chemical shifts of carbon-bonded 1Hd2 and 1He1
alone are not useful reporters of tautomerization (Tanokura
1983). The readily measurable strong 1JCH couplings(*200 Hz) of the ring 13Ce1 and 13Cd2 also decrease by*10 Hz upon deprotonation with little apparent dependence
upon tautomeric state (Fig. 2). This provides a complementaryroute for pKa measurements and ionization state determination(Bachovchin et al. 1981; Day et al. 2003; Hunkapiler et al.
1973; Wasylishen and Tomlinson 1975). Indeed, this couplinghas been exploited to measure the pKa values of histidines insparsely populated unfolded states of proteins (Hansen and
Kay 2014). Solid-state NMR methods have also been developed to characterize the charged form and the neutral tautomers of histidine (Li and Hong 2011; Miao et al. 2014).
As a closing point, the values listed in Fig. 1 andTable 1 for the blocked Gly-His-Gly tripeptide are tautomer averaged. Assuming fast exchange between thelimiting 15N and 13C chemical shift changes of Fig. 2, theneutral histidine in the tripeptide is in equilibrium between
major Ne2H (*80 %) and minor Nd1H (*20 %) forms.
Accordingly, the observed macroscopic pKa of 6.45 fordeprotonation of either nitrogen is a weighted sum of the
microscopic pKa values of 6.55 and 7.15 for forming these
two species, respectively (Tanokura 1983).
Cysteine
The side chain thiol of cysteine in the blocked tripeptide has a
pKa value of 8.49. In addition to having highly diagnostic
chemical shifts for a disulfide-bridged side chain(*41 ppm) versus a reduced thiol (*28 ppm) (Wishart2011), the 13Cb signal moves downfield by 1.67 ppm upon
deprotonation to the thiolate anion. Accordingly, the pKavalues of cysteines in several proteins selectively labeledwith 13Cb-Cys have been measured via 13C-HSQC approaches (Chivers et al. 1997; Jeng et al. 1995; Mavridou et al.
2007; Mossner et al. 2000; Roos et al. 2013; Wilson et al.
1995). It is notable that the 1Ha, 13Ca and amide 15N showeven larger pH-dependent chemical shift changes and thus
can also be used as reporter nuclei (Forman-Kay et al. 1992).
However, under the alkaline conditions likely associatedwith cysteine deprotonation, rapid amide HX may well
preclude a 15N-HSQC approach (Lim et al. 2012).

123

Although the labile sulfur-bonded 1Hc of cysteine is
generally not observable for a random coil polypeptide, it
can be detected when protected from rapid HX within thecontext of a structured protein. Of course, the observationof such a signal provides unambiguous evidence that acysteine is predominantly in its neutral state. The BioMagResBank (Ulrich et al. 2008) currently lists an averagechemical shift of 2.0 ± 1.3 ppm for a small number ofthiol 1Hc signals. However, significant shift deviations are
possible depending upon the environment of the proton(Nordstrand et al. 1999). The signals from these slowlyexchanging 1Hc protons were likely assigned via 3JHbHc
scalar (Nordstrand et al. 1999) or interproton NOE interactions (Takeda et al. 2010). Alternatively, by exploiting
an expected 3JCaHc * 3 to 7 Hz (Hansen 1981), the thiol
proton should be detectable and assignable using a longrange 13C-HSQC experiment as demonstrated for serine/
threonine hydroxyl protons (Brockerman et al. 2014). This
strategy would likely require selective 13C-Cys labeling(ideally 13Ca only) because a uniformly enriched protein
will yield a myriad of complicated long-range couplings.
The presence of the 1Hc can be confirmed via a twobond deuterium isotope shift of *0.12 ppm (13CbScH vs.
13 b cC S D; Table 2) measured via 13C-NMR in proteins
selectively labeled with 13CbD2-Cys (Takeda et al. 2010).
This isotope shift has also been exploited to quantitate HX
rates and protium-deuterium fractionation factors for thethiol moiety (Takeda et al. 2010).
Tyrosine
Within the context of a blocked tripeptide, tyrosine has a
pKa value of 9.76. Thus, this residue is neutral in mostproteins under physiological conditions. Upon ionization of
the phenolic oxygen, the ring 13Cc and 13Cf shift substantially by -6.7 and 10.4 ppm, respectively (Norton andBradbury 1974; Richarz and Wüthrich 1978). In contrast to
the smaller changes exhibited by the other ring 13C and 1H
nuclei, these ionization-dependent shift changes are larger
than those typically accompanying protein folding or
ligand binding (Baturin et al. 2011). Therefore, the 13Cc
and 13Cf both serve as very reliable reporter nuclei for pKa
measurements. However, neither is directly protonated,thus requiring lower sensitivity approaches such as 2D
He(Ce)Cf or Hb2 (Cb)Cc correlation spectroscopy (Baturin
et al. 2011; Oktaviani et al. 2012; Prompers et al. 1998;
Yamazaki et al. 1993).
As with other oxygen-bonded protons, the labile 1Hg oftyrosine is usually observable only when protected fromrapid HX, such as via hydrogen bonding within a folded
protein (Liepinsh and Otting 1996; Liepinsh et al. 1992).
Although the BioMagResBank (Ulrich et al. 2008) lists anaverage chemical shift of 9.3 ± 1.3 ppm for a small

J Biomol NMR

number of assigned 1Hg signals, a substantially larger
range is possible depending on the structural environmentof the tyrosine (Baturin et al. 2011; Werner et al. 1997). In
addition to homonuclear NOE approaches, one unambiguous method to assign these signals relies on a weakdihedral angle-dependent 3JCeHg coupling of *4 to 8 Hz
(Borisov et al. 1998; Bystrov 1976; Hansen 1981) that is
detectable in long range 13C-HSQC spectra (Baturin et al.
2011; Werner et al. 1997). The observation of the 1Hg
either directly or indirectly via a two-bond deuterium isotope shift of *0.13 ppm (13CfOgH vs. 13CfOgD; Table 2)(Takeda et al. 2009) also provides clear evidence that atyrosine is neutral. Once detected, NOE and J-couplingmeasurements can be used to obtain distance and dihedral
angle restraints, respectively, to define the structural features of tyrosine 1Hg protons in proteins. Complementary
HX studies can be carried out to help characterize theirdynamic properties (Baturin et al. 2011; Liepinsh et al.
1992; Takeda et al. 2009).
LysineLysine in the blocked tripeptide has a pKa value of 10.34.
Hence, unless in a highly unusual environment, lysine Nfamino groups in proteins are most likely positively-charged
under neutral pH conditions (Daopin et al. 1991; Isom et al.
2011). Similar to an N-terminal amine, the lysine side
chain 15Nf shifts by *-7.5 ppm upon deprotonation(Andre et al. 2007), thus allowing reliable pKa measurements by 1D 15N-NMR (Zhu et al. 1995) or 2D He2(Ce)Nf
approaches (Andre et al. 2007; Tomlinson et al. 2009;
Yamazaki et al. 1993). In contrast to changes of -0.4 and-0.24 ppm for the non-labile 1He and 1Hd, respectively,
and a change of only 1 ppm for the nitrogen-bonded 13Ce,the ‘‘once removed’’ 13Cd shifts substantially by 5 ppm
upon lysine neutralization (Batchelor et al. 1975; Keimet al. 1974; Kesvatera et al. 1996; Richarz and Wüthrich1978; Surprenant et al. 1980). Thus, as exemplified bystudies of the lyase domain of DNA Pol b (Gao et al.
2006), the 13Cd aliphatic carbon can be used as a very
reliable reporter nucleus for pKa measurements via sensitive 2D 13C-HSQC experiments. However, selective
labeling with 13C-lysine (ideally 13Cd only) is likely
required to avoid spectral overlap with other side chain
signals, as would arise with a uniformly 13C-enriched
protein.
The labile amino protons of lysines (or the N-terminal
amine) can also be observed directly, particularly when
protected from rapid HX due to their environment within afolded protein and/or under conditions of low temperature,
pH, and general acid/base concentration (Esadze et al.
2014; Iwahara et al. 2007; Liepinsh and Otting 1996;
Liepinsh et al. 1992; Poon et al. 2006; Zandarashvili et al.

2013). Strikingly, a chemical shift of 0.8 ppm has beenreported for the nitrogen-bonded 1Hf of a neutral buriedlysine in a staphylococcal nuclease mutant (Takayama
et al. 2008a). This stands in contrast to a shift of *7.5 ppm
typically found for the protons in the charged f-aminium
group. Thus, if detectable, the 1Hf chemical shift of a lysineside chain will be highly diagnostic of its charge state.
Alternatively, the 15N nuclei in –15NH3? versus –15NH2
groups show distinctly different multiplet patterns due to1
JNH couplings of *74 Hz (quartet) and *64 Hz (triplet),respectively (Poon et al. 2006; Takayama et al. 2008a, b;
Tomlinson et al. 2009). This provides unambiguous evidence for the protonation of a lysine residue. The detectionof the lysine –15NH3? also opens the doors to dynamic
studies of this side chain in proteins and protein complexes(Anderson et al. 2013; Esadze et al. 2011; Zandarashvili
et al. 2011, 2013).
The lysine 15Nf aminium group has a combined onebond deuterium isotope shift of 3 9 0.35 = 1.05 ppm(15NH3? vs. 15ND3?; Table 2) (Tomlinson et al. 2009). In
contrast, a value of *2 9 0.95 = 1.9 ppm can be estimated for the combined isotope shift of –15NH2 versus–15ND2 in the neutral amine (Table 2). Similar results were
found for the a-amine of 13C/15N-arginine (Table 2).
Therefore the deuterium isotope shifts, which reflect electronic structure and bonding (Hansen 2000), are substantially different for the cationic and neutral states of thelysine 15Nf nucleus and the N-terminal amine. This parallels data reported for the ammonium ion versus ammonia(Wasylishen and Friedrich 1987). Smaller pH-dependentchanges in the multiple-bond deuterium isotope shifts on
the side chain 13C nuclei of lysine have also been measured(Led and Petersen 1979). However, it may be difficult touse deuterium isotope shifts as a criterion for characterizing the charge of a lysine or N-terminus because they also
vary with hydrogen bonding and counterions (Hansen and
Lycka 1989; Tomlinson et al. 2009; Ullah et al. 2011). This
stands in contrast to the simple presence or absence of a
deuterium isotope shift for the carboxyl, thiol, and hydroxyl groups in their neutral versus anionic states,
respectively.
Arginine
The pKa value of the arginine side chain has recently beenmeasured as 13.9 (in preparation). This is considerably
higher than the commonly cited values of *12 to 12.5
(Creighton 2010), and thus arginines are invariably positively-charged in proteins under physiological conditions.
To the best of our knowledge, an arginine in a measurably
populated neutral state has never been unambiguously
observed within the context of a protein or protein complex, and hence a corresponding pKa value has never been123

J Biomol NMR

determined (Baillargeon et al. 1980; Grissom et al. 1987;
Harms et al. 2011; Xiao and Braiman 2005). Nevertheless,such studies are very interesting to pursue, particularly forproteins in which the guanidinium moiety becomes posttranslationally modified (Smith and Denu 2009) or ispotentially involved in a proton transfer cascade (Hutson
et al. 2000; McMahon et al. 2004; Petkova et al. 1999;
Xiao et al. 2004), or for enzymes that appear to use thisside chain as a general acid/base catalyst (Schlippe andHedstrom 2005). As with all pKa measurements, control
experiments would be absolutely necessary to ensure thatthe protein remains in its native state and not hydrolyzed orotherwise chemically modified over the entire pH titration
range studied (which, for most systems, seems unlikely
given the extreme alkaline conditions required for arginine
deprotonation).
Arginine side chains have been characterized extensively with NMR spectroscopy, although almost always in
the cationic state (Blomberg et al. 1976; Blomberg andRüterjans 1983; Kanamori et al. 1978; Kanamori andRoberts 1983; Keim et al. 1974; Legerton et al. 1981;
London et al. 1977; Oldfield et al. 1975a, b; Pregosin et al.
1971; Richards and Thomas 1974; Richarz and Wüthrich1978; Surprenant et al. 1980; Yavari and Roberts 1978). To
enable this characterization, many pulse sequences havebeen developed to detect and assign the 1H, 13C, and 15N
signals of the guanidinium moiety (Andre et al. 2007;
Iwahara and Clore 2006; Pellecchia et al. 1997; Rao et al.
1996; Vis et al. 1994; Wittekind et al. 1993; Yamazaki
et al. 1993, 1995). In particular, due to strong 1JNeH and
1
JNgH couplings of *93 Hz, the nitrogen-bonded protons
of arginines are often readily observable in 15N-HSQC
spectra recorded under conditions to minimize HX. However, unless restrained by interactions such as hydrogenbonding, rotation about the Ne–Cf and Cf–Ng partial
double bonds generally leads to broad, degenerate signals
from the two 15Ng and four 1Hg nuclei (Henry and Sykes
1995; Kanamori and Roberts 1983). Note that, due to thisconjugated bonding, the guanidinium group is planar(Raczynska et al. 2003).
We initially attempted to measure the pKa value and pHdependent chemical shift changes of the guanidinium
moiety in a blocked tripeptide. However, due to hydrolysis
at high pH, reliable data could only be obtained for the side
chain 13Cf and 1Hd nuclei (Supplemental Table S1).
Therefore, we used a sample of 13C6/15N4-arginine toobtain the desired results, which after correction for shift
perturbations due to the titration of the a-amine (Supplemental Table S2), agreed well with those for the tripeptide.
As summarized in Fig. 1 and Table 1, the 13Cf, 15Ne, and15 g
N all show substantial chemical shift changes upon
deprotonation, and thus serve as potential reporter nuclei
for arginine pKa measurements. The titration Dd values

123

measured herein for these nuclei are consistent with, but
generally larger in magnitude, than those published previously (Baillargeon et al. 1980; Kanamori et al. 1978;
Kanamori and Roberts 1983; London et al. 1977; Suzuki
et al. 1974; Xiao and Braiman 2005). This is most likely
due to difficulties in extrapolation to high pH plateau
chemical shifts. Alternatively, since arginine can dimerize(Kubickova et al. 2011; Vondrasek et al. 2009), this mightreflect differences in experimental conditions. However,similar shift changes were determined with 10 and
100 mM samples. Under highly alkaline conditions, theguanidinium protons will undergo rapid HX, and thus thesereporter nuclei would have to be observed by directly via
15
N- or 13C-NMR or indirectly via scalar correlations with
the non-exchangeable side chain protons. Observing the
15 g
N nuclei would be most challenging due to their terminal positions and potentially broad signals resulting fromrotation about the Ne–Cf bond or from tautomerization(Kanamori and Roberts 1983). This is unfortunate as the
15 g
N nuclei, with dramatically different chemical shifts of*71 and 93 ppm in charged versus neutral arginine,
should best serve as reporters of its ionization state.
However, any arginine with an unusually low pKa value
will likely be in a very unusual environment, which may
also lead to strongly perturbed chemical shifts. Indeed, thechemical shift differences between the charged and neutral
arginine side chain nitrogens are substantially smaller in
nonpolar solvents than in water (Xiao and Braiman 2005).
It is also worth noting that the neutral side chain guanidine moiety is non-planar and can exist in five possible
tautomeric forms, each lacking one of the five different
nitrogen-bonded protons (Raczynska et al. 2003). Experimental (Kanamori and Roberts 1983) and theoretical calculations (Norberg et al. 2005) suggest that thesetautomers, which can interconvert rapidly via bond rotations or proton transfer, are roughly iso-energetic and exist
in an equilibrium distribution with *1/3 deprotonated at
Ne. This is also supported by the observation that the 15Ne
and two 15Ng nuclei still have similar chemical shifts in the
deprotonated state of 13C6/15N4-arginine (with the 15Ng
being degenerate). Significantly different shifts would be
expected for non-interconverting sp2 (C=N bonded) andsp3 (C–N bonded) hybridized nitrogens (Witanowski et al.
1976).
To test this prediction, we also measured deuterium
isotope shifts for the 15Ne and 15Ng nuclei. Based oncomparative titrations of 13C6/15N4-arginine in H2O and
D2O, at low (high) pH, deuterium isotope shifts of 1.0 ppm(1.8 ppm) were measured for the 15Ne and 1.4 ppm(1.7 ppm) were measured for the 15Ng (Table 2). Thus, asseen with the amino group, deuterium isotope shifts are
generally larger for the neutral guanidine than the charged
guanidinium species despite the reduced number of

J Biomol NMR

hydrogens. However, these measurements are complicatedby possible incomplete (and differing levels of) deprotonation under extremely high pH conditions, particularly
since pH meter readings and the pKa value of arginine
likely differ in H2O versus D2O solutions (Krezel and Bal
2004). In addition, multiple three-bond isotope shifts could
add to the one-bond shifts. Although many such caveats
exist, these measurements do indicate that the Ne and Ng
nuclei all remain at least partially protonated in the neutral
tautormers of free arginine. Of course, if indeed present ina folded protein, the relative populations of these tautomers
could change in response to structure-dependent intermolecular interactions, such as hydrogen bonding. Conversely, without better characterization, deuterium isotope
shift measurements are not likely useful for determining
the charge and tautomeric form of an arginine side chain,even if restrained within a protein.
Phosphoamino acids
For completeness, we have also included the pH-dependentchemical shift changes of phosophoserine (second pKa5.96), phosphothreonine (pKa 6.30) and phosphotyrosine(pKa 5.96) in Fig. 1. These data were measured by Bienkiewicz and Lumb for these commonly phosphorylatedamino acids (X) in the context of the blocked pentapeptide,acetyl-Gly-Gly-X-Gly-Gly-amide at 25 °C (Bienkiewicz
and Lumb 1999). Similar results were reported earlier for
unblocked Gly-Gly-X-Ala peptides containing these threephosophoamino acids (Hoffmann et al. 1994). Data havealso been published for phosphohistidine (Kalbitzer andRosch 1981) and phosphoaspartate (Schlemmer et al.
1988).
Certainly, 31P-NMR monitored pH-titrations of thephosphate moieties provide an obvious route for pKa
measurements. However, several 1H, 13C and 15N nuclei inthese amino acids show diagnostic changes upon phosphorylation, as well as upon deprotonation to the dianion.
The former can be used to identify modification sites
within a protein (Bienkiewicz and Lumb 1999; Hoffmannet al. 1994; Lau et al. 2012; McIntosh et al. 2009; SmetNocca et al. 2013), whereas the latter can also be monitored
for pKa measurements. Akin to phosphorylation, many
additional post-translational modifications involve ionizable side chains or introduce ionizable groups into protein.
These can also be characterized by NMR spectroscopy,
including biologically relevant in vivo approaches (Theillet
et al. 2012).
AmidesThe main chain amide 1HN, 15N and 13CO nuclei of theblocked tripeptides exhibit chemical shift changes due toside chain deprotonation. As expected for both throughbond inductive and through-space electric field interactions, the magnitudes of these changes generally increase
with decreasing separation to the acid/base functionalgroup. Thus, the intra-residue 1HN and 15N of the peptide
preceding and the 13CO of the peptide following the ionizable side chain show the largest pH-dependent shift
perturbations, whereas the flanking glycines report smallerchanges. Nevertheless, the latter nearest-neighbor amide
titration shifts can still be substantial, particularly for Asp,
His, and Cys (Fig. 1 and Supplemental Table S1). It is alsointeresting that the intra-residue 1HN signal of N-acetyl
alanine, Asp, His, and Cys shift upfield upon deprotonation, whereas those of Glu, phosphoserine, and phosphothreonine shift downfield. In the case of Glu, the downfield
shift has been attributed to a transient intra-residuehydrogen bond between the amide and the d-carboxyl thatstrengthens when the latter is deprotonated (Bundi andWüthrich 1979b; Mayer et al. 1979). Similar seven-membered rings could be formed by the two phosphorylatedamino acids.
The amide 15N-HSQC spectrum of a protein serves as its‘‘fingerprint’’ due to the presence of one 1HN-15N crosspeakfrom each non-proline or non-N-terminal residue that does
not undergo rapid HX. Robust methods have also beendeveloped to rapidly assign the signals from the mainchain
1
H, 13C and 15N nuclei of a protein, which in turn are
stepping stones to its side chain nuclei (Sattler et al. 1999).
Accordingly, it is often simple and convenient to monitor
the pH-titration of a protein using 15N-HSQC or HNCOtype spectra. Indeed, in numerous cases, pKa values ofionizable groups have been extracted from pH-dependent
intra-residue and nearest-neighbor amide 1HN, 15N and, toa lesser extent, 13CO shifts in both folded (Anderson et al.
1993; Betz et al. 2004; Forman-Kay et al. 1992; Lindman
et al. 2007; Tomlinson et al. 2010) and unfolded proteins(Pujato et al. 2006). However, caution must be exercised ininterpreting such data because the amide of an acid/baseresidue may also report the ionization of other residues in afolded protein. Indeed, amides of non-titrating residues
often show pronounced apparent titrations that can arise
from many reasons. In addition to pH-dependent structuralchanges (Kukic et al. 2010; Tomlinson et al. 2010), interresidue hydrogen bonding between an amide and an ionizable side chain can lead to diagnostic downfield 1HN shiftchanges upon deprotonation (Betz et al. 2004; Clark et al.
2007; Ebina and Wüthrich 1984; Haruyama et al. 1989;
Khare et al. 1997; Schaller and Robertson 1995). Furthermore, the chemical shifts of the polarizable peptide areparticularly sensitive to their local electric fields (Buckingham 1960), and these fields can change upon the titrations of even relatively distal groups in a protein. Thissensitivity has been exploited to probe protein

123

J Biomol NMR

electrostatics, including extracting dielectric constants (Anet al. 2014; Boyd et al. 2003; Hass et al. 2008; Kukic et al.
2013).

Concluding remarks
In this paper, we report the pH-dependent chemical shiftsof essentially all the 1H, 13C, and 15N nuclei associatedwith the acid/base functional groups in a protein. The few
missing values correspond to the rapidly exchanging protons of the conjugate base forms of the ionizable residues
that could not be detected under alkaline conditions. These
data, which are consistent with and extend upon those ofnumerous pioneering studies, were measured in the context
of a blocked tripeptide under a common set of experimental
conditions. This unified set of reference chemical shifts
should help guide protein pKa studies.
Measuring pH-dependent chemical shifts by NMR
spectroscopy is often straightforward for a protein, provided that it is well behaved across the necessary experimental conditions. However, interpreting the resulting
titration curves to yield residue-specific pKa values andionization states can be very difficult (Lindman et al. 2006;
McIntosh et al. 2011; Søndergaard et al. 2008; Szakacs
et al. 2004; Ullmann 2003; Webb et al. 2011). This difficulty results from the complex nature of electrostaticinteractions in proteins, combined with the sensitivity ofchemical shifts to many pH-dependent factors and the often
unappreciated fact that most titration curves are experimentally underdetermined for model fitting (Shrager et al.
1972). The confidence by which one can assign observed
titrations to the ionization of a specific residue in a protein
certainly increases if several 13C, 15N or non-exchangeable1
H nuclei closely associated with that residue all reportcoincident pH-dependent chemical shift changes. Importantly, these titration shifts should be comparable in signand magnitude to those exhibited by reference modelcompounds. Practically speaking, the number of suchreporter nuclei that can be monitored will depend upon the
pH-dependent stability of the protein combined with feasibility of the required isotopic labeling strategies and thedispersion, resolution and sensitivity (time requirements)of the corresponding NMR experiments. With this said,
there are numerous published cases where even directly
bonded nuclei within a given residue track very different
titrations. These include amide 15NH (Tomlinson et al.
2010; Webb et al. 2011), Asp, Glu, and Cys 13CH2 (Jenget al. 1995; Oda et al. 1994; Song et al. 2003; Wilson et al.
1995), and even His imidazole 13CH pairs (Ludwiczek
et al. 2013). Based on these and other examples, the 15N
and 13C nuclei are generally the more reliable reporters of
intra-residue ionization, whereas the 1H are frequently

123

sensitive to additional pH-dependent changes in theprotein.
Unfortunately, the chemical shifts of most reporter
nuclei for the protonated and deprotonated forms of theionizable functional groups fall within the range of chemical shifts induced upon protein folding or ligand binding.
For example, the 13Cb and carboxyl 13Cc of an aspartic acidchange from approximately 38 to 41 and 177 to 180 ppm,
respectively, upon deprotonation, whereas structuredependent shifts of 38–44 and 176–183 ppm are listed forthese nuclei in the BioMagResBank (Ulrich et al. 2008).
The latter values represent the mean chemical shifts ±2
standard deviations for all diamagnetic proteins, regardlessof sample conditions. The few exceptions to this point
include the histidine 15Nd1 and 15Ne2, tyrosine 13Cc and
13 f
C , and arginine 15Ng reporter nuclei, which exhibit
rather large, and hence diagnostic, pH-dependent changes
in their resonance frequencies. Accordingly, great caution
should be exercised in inferring ionization states fromchemical shift (or J-coupling) information alone. Strictly
speaking, in the absence of a detectable pH titration, one
can only conclude that the pKa value of a residue is either
less than the lowest sample pH value examined (and thus
deprotonated throughout the titration) or greater than thehighest pH value examined (protonated). Fortunately,
several NMR approaches can be used to resolve thisproblem by providing complementary insights into theionization states of protein. In particular, the direct observation of a slowly exchanging acidic proton via chemical
shift or J-coupling measurements is clearest evidence that afunctional group is in its conjugate acid form. Alternatively, for groups with well-populated but rapidly
exchanging acidic protons, deuterium isotope shift measurements can help define their charged states. Of course,
parallel studies with biophysical techniques other than
NMR spectroscopy can be highly informative.
The reference data provided herein should also help
enable pH-dependent corrections in algorithms used to
predict the ‘‘random coil’’ chemical shifts of a polypeptide sequence. These reference shifts are of great importance to NMR spectroscopists for numerous reasons,
including determining the secondary/tertiary structure anddynamics of a protein or protein complex from chemical
shift information, as well as characterizing transient
conformations within otherwise intrinsically disorderedregions of these biomolecules (Wishart 2011). Broadly
speaking, these algorithms have been developed from thestatistical analyses of protein chemical shift databases (DeSimone et al. 2009; Tamiola et al. 2010; Wang and Jardetzky 2002) or from studies of various reference polypeptides (Braun et al. 1994; Kjaergaard et al. 2011;
Prestegard et al. 2013; Schwarzinger et al. 2001; Thanabal
et al. 1994; Wishart et al. 1995). The latter were generally

J Biomol NMR

carried out under acidic conditions, and have been adapted to account for the ionization of Asp, Glu, and His.
The current tripeptide data provide corrections for theremaining termini and Cys, Tyr, Lys, and Arg residues, aswell as an essentially complete summary of the pHdependent 1H, 13C, and 15N chemical shifts of all themain chain and side chain nuclei of these ionizable aminoacids in proteins.
Acknowledgments G. P. was supported by the Austrian Science
Fund (FWF). This research was funded by a Natural Sciences andEngineering Research Council of Canada (NSERC) Discovery Grant
to L.P.M. Instrument support was provided by the Canadian Institutesfor Health Research (CIHR), the Canadian Foundation for Innovation(CFI), the British Columbia Knowledge Development Fund
(BCKDF), the UBC Blusson Fund, and the Michael Smith Foundationfor Health Research (MSFHR).

References
Alei M, Morgan LO, Wageman WE, Whaley TW (1980) pHdependence of 15N NMR shifts and coupling-constants inaqueous imidazole and 1-methylimidazole—comments on estimation of tautomeric equilibrium-constants for aqueous histidine. J Am Chem Soc 102:2881–2887
Alexov E et al (2011) Progress in the prediction of pKa values inproteins. Proteins 79:3260–3275
An L, Wang Y, Zhang N, Yan S, Bax A, Yai L (2014) Protein
apparent dielectric constant and its termperature dependence
from remote chemical shift effects. J Am Chem Soc 136, 9 Sept2014 [Epub ahead of print]
Anderson DE, Lu J, McIntosh LP, Dahlquist FW (1993) The folding,stability and dynamics of T4 lysozyme: a perspective using
nuclear magnetic resonance. In: Clore GM, Gronenborn AM(eds) NMR of Proteins, MacMillan Press, London, pp 258–304
Anderson KM, Esadze A, Manoharan M, Bruschweiler R, Gorenstein
DG, Iwahara J (2013) Direct observation of the ion-pairdynamics at a protein-DNA interface by NMR spectroscopy.
J Am Chem Soc 135:3613–3619
Andre I, Linse S, Mulder FAA (2007) Residue-specific pKa
determination of lysine and arginine side chains by indirect
15
N and 13C NMR spectroscopy: application to apo calmodulin.
J Am Chem Soc 129:15805–15813
Bachovchin WW (1985) Confirmation of the assignment of the lowfield proton-resonance of serine proteases by using specifically15
N labeled enzyme. Proc Natl Acad Sci USA 82:7948–7951
Bachovchin WW (1986) 15N NMR spectroscopy of hydrogen-honding
interactions in the active-site of serine protease—evidence for amoving histidine mechanism. Biochemistry 25:7751–7759
Bachovchin WW (2001) Contributions of NMR spectroscopy to thestudy of hydrogen bonds in serine protease active sites. Magn
Reson Chem 39:S199–S213
Bachovchin WW, Roberts JD (1978) 15N nuclear magnetic-resonancespectroscopy—state of histidine in catalytic triad of alpha-lytic
protease—implications for charge-relay mechanism of peptidebond cleavage by serine proteases. J Am Chem Soc 100:8041–8047
Bachovchin WW, Kaiser R, Richards JH, Roberts JD (1981) Catalytic
mechanism of serine proteases—re-examination of the pHdependence of the histidyl 1J13C2H coupling-constant in the
catalytic triad of alpha-lytic protease. Proc Natl Acad Sci
78:7323–7326

Baillargeon MW, Laskowski M, Neves DE, Porubcan MA, Santini
RE, Markley JL (1980) Soybean trypsin-inhibitor (kunitz) and its
complex with trypsin—13C nuclear magnetic-resonance studiesof the reactive site arginine. Biochemistry 19:5703–5710
Barraud P, Scubert M, Allain FHT (2012) A strong 13C chemical shift
signature provides the coordination mode of histidines in zincbinding proteins. J Biomol NMR 53:93–101
Batchelor JG, Feeney J, Roberts GCK (1975) 13C NMR protonation
shifts of amines, carboxylic-acids and amino acids. J Magn
Reson 20:19–38
Baturin SJ, Okon M, McIntosh LP (2011) Structure, dynamics, andionization equilibria of the tyrosine residues in Bacillus circulans xylanase. J Biomol NMR 51:379–394
Betz M, Löhr F, Wienk H, Rüterjans H (2004) Long-range nature of
the interactions between titratable groups in Bacillus agaradhaerens family 11 xylanase: pH titration of B-agaradhaerens
xylanase. Biochemistry 43:5820–5831
Bienkiewicz EA, Lumb KJ (1999) Random-coil chemical shifts ofphosphorylated amino acids. J Biomol NMR 15:203–206
Blakeley MP, Langan P, Niimura N, Podjarny A (2008) Neutron
crystallography: opportunities, challenges, and limitations. Curr
Opin Struct Biol 18:593–600
Blomberg F, Rüterjans H (1983) Nitrogen-15 NMR in biologicalsystems. Biol Magn Reson 21–73
Blomberg F, Maurer W, Rüterjans H (1976) 15N nuclear magnetic
resonance investigations on amino-acids. Proc Natl Acad Sci
USA 73:1409–1413
Blomberg F, Maurer W, Rüterjans H (1977) Nuclear magnetic
resonance investigation of 15N-labeled histidine in aqueoussolution. J Am Chem Soc 99:8149–8159
Borisov EV, Zhang W, Bolvig S, Hansen PE (1998) nJ(13C, O1H)
coupling constants of intramolecularly hydrogen-bonded compounds. Magn Reson Chem 36:S104–S110
Boyd J, Domene C, Redfield C, Ferraro MB, Lazzeretti P (2003)
Calculation of dipole-shielding polarizabilities (r1abc): the influence of uniform electric field effects on the shielding of
backbone nuclei in proteins. J Am Chem Soc 125:9556–9557
Braun D, Wider G, Wüthrich K (1994) Sequence-corrected 15N
random coil chemical shifts. J Am Chem Soc 116:8466–8469
Brockerman JA, Okon M, McIntosh LP (2014) Detection andcharacterization of serine and threonine hydroxyl protons in
Bacillus circulans xylanase by NMR spectroscopy. J Biomol
NMR 58:17–25
Brown LR, Demarco A, Richarz R, Wagner G, Wüthrich K (1978)
Influence of a single salt bridge on static and dynamic features ofglobular solution conformation of basic pancreatic trypsininhibitor—1H and 13C NMR studies of native and transaminatedinhibitor. Eur J Biochem 88:87–95
Buckingham AD (1960) Chemical shifts in the nuclear magnetic
resonance spectra of molecules containing polar groups. Can J
Chem 38:300–307
Bundi A, Wüthrich K (1979a) 1H-NMR parameters of the common
amino-acid residues measured in aqueous-solutions of the linear
tetrapeptides H-Gly-Gly-X-L-Ala-OH. Biopolymers 18:285–297
Bundi A, Wüthrich K (1979b) Use of amide 1H-NMR titration shifts
for studies of polypeptide conformation. Biopolymers 18:299–311
Bystrov VF (1976) Spin-spin coupling and the conformational statesof peptide systems. Prog Nuc Mag Reson Spect 10:41–81
Castaneda CA, Fitch CA, Majumda A, Khangulov V, Schlessman JL,
Garcia-Moreno B (2009) Molecular determinants of the pKavalues of Asp and Glu residues in staphylococcal nuclease.
Proteins Struct Funct Design 15:570–588
Chevelkov V, Xue Y, Rao DK, Forman-Kay JD, Skrynnikov NR(2010) 15N H/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured
drkN SH3. J Biomol NMR 46:227–244

123

J Biomol NMR
Chivers PT, Prehoda KE, Volkman BF, Kim B-M, Markley JL,
Raines RT (1997) Microscopic pKa values of Escherichia
thioredoxin. Biochemistry 36:14985–14991
Clark AT, Smith K, Muhandiram R, Edmondson SP, Shriver JW(2007) Carboxyl pKa values, ion pairs, hydrogen bonding, andthe pH-dependence of folding the hyperthermophile proteins
Sac7d and Sso7d. J Mol Biol 372:992–1008
Cohen JS, Hughes L, Wooten JB (1983) Observations of amino acidside chains in proteins by NMR methods. In: Cohen JS (ed)Magnetic resonance in biology, vol 2. Wiley Interscience, New
York, pp 130–147
Connelly GP, McIntosh LP (1998) Characterization of a buried
neutral histidine in Bacillus circulans xylanase: internal dynamics and interaction with a bound water molecule. Biochemistry
37:1810–1818
Creighton TE (2010) The biophysical chemistry of nucleic acids andproteins. Helvetian Press, Oxford
Daopin S, Anderson DE, Baase WA, Dahlquist FW, Matthews BW(1991) Structural and thermodynamic consequences of burying acharged residue within the hydrophobic core of T4 lysozyme.
Biochemistry 30:11521–11529
Day RM et al (2003) Tautomerism, acid-base equilibria, andH-bonding of the six histidines in subtilisin BPN’ by NMR.
Protein Sci 12:794–810
De Simone A, Cavalli A, Hsu STD, Vranken W, Vendruscolo M(2009) Accurate random coil chemical shifts from an analysis ofloop regions in native states of proteins. J Am Chem Soc
131:16332–16333
Demarco A (1977) pH-dependence of internal references. J Magn
Reson 26:527–528
Dziembowska T, Rozwadowski Z (2001) Application of the deuterium isotope effect on NMR chemical shift to study protontransfer equilibrium. Curr Org Chem 5:289–313
Ebina S, Wüthrich K (1984) Amide proton titration shifts in bullseminal inhibitor IIa by two-dimensional correlated 1H nuclear
magnetic-resonance (COSY)—manifestation of conformational
equilibria involving carboxylate groups. J Mol Biol 179:283–288
Englander SW, Kallenbach NR (1983) Hydrogen exchange andstructural dynamics of proteins and nucleic acids. Q Rev
Biophys 16:521–655
Esadze A, Li DW, Wang TZ, Bruschweiler R, Iwahara J (2011)
Dynamics of lysine side-chain amino groups in a protein studied
by heteronuclear 1H-15N NMR spectroscopy. J Am Chem Soc
133:909–919
Esadze A, Zandarashvili L, Iwahara J (2014) Effective strategy toassign 1H-15N heteronuclear correlation NMR signals fromlysine side-chain NH3? groups of proteins at low temperatures.
J Biomol NMR 60:23–27
Felli IC, Brutscher B (2009) Recent advances in solution NMR: fast
methods and heteronuclear direct detection. ChemPhysChem
10:1356–1368
Fitch CA, Karp DA, Lee KK, Stites WE, Lattman EE, Garcia-Moreno
EB (2002) Experimental pKa values of buried residues: analysis
with continuum methods and role of water penetration. Biophys
J 82:3289–3304
Forman-Kay JD, Clore GM, Gronenborn AM (1992) Relationshipbetween electrostatics and redox function in human thioredoxin—characterization of pH titration shifts using 2-dimensional homonuclear and heteronuclear NMR. Biochemistry 31:3442–3452
Forsyth WR, Antosiewicz JM, Robertson AD (2002) Empirical
relationships between protein structure and carboxyl pKa values
in proteins. Proteins Struct Func Bioinform 48:388–4034
Freedman MH, Lyerla JR, Chaiken IM, Cohen JS (1973) 13C nuclear
magnetic-resonance studies on selected amino acids, andproteins. Eur J Biochem 32:215–226

123

Frey PA (2001) Strong hydrogen bonding in molecules and enzymatic
complexes. Magn Reson Chem 39:S190–S198
Gao GH, Prasad R, Lodwig SN, Unkefer CJ, Beard WA, Wilson SH,
London RE (2006) Determination of lysine pK values using[5-13C]lysine: application to the lyase domain of DNA Pol b.
J Am Chem Soc 128:8104–8105
Goux WJ, Allerhand A (1979) Studies of chemically modified
histidine residues of proteins by 13C nuclear magnetic resonancespectroscopy. Reaction of hen egg white lysozyme with iodoacetate. J Biol Chem 254:2210–2213
Grimsley GR, Scholtz JM, Pace CN (2009) A summary of themeasured pK values of the ionizable groups in folded proteins.
Protein Sci 18:247–251
Grissom CB, Chinami MA, Benway DA, Ulrich EL, Markley JL(1987) Staphylococcal nuclease active-site amino-acids—pHdependence of tyrosine and arginine as determined by NMR and
kinetic-studies. Biochemistry 28:2116–2124
Gueron M, Plateau P, Decorps M (1991) Solvent signal suppression in
NMR. Prog Nuc Mag Reson Spect 23:135–209
Guo J, Tolstoy PM, Koeppe B, Golubev NS, Denisov GS, Smirnov
SN, Limbach HH (2012) Hydrogen bond geometries and proton
tautomerism of homoconjugated anions of carboxylic acids
studied via H/D isotope effects on 13C NMR chemical shifts.
J Phys Chem A 116:11180–11188
Hanoian P, Sigala PA, Herschlag D, Hammes-Schiffer S (2010)
Hydrogen bonding in the active site of ketosteroid isomerase:electronic inductive effects and hydrogen bond coupling.
Biochemistry 49:10339–10348
Hansen PE (1981) Carbon-hydrogen spin-spin coupling-constants.
Prog Nucl Magn Reson Spect 14:175–296
Hansen PE (2000) Isotope effects on chemical shifts of proteins andpeptides. Magn Reson Chem 38:1–10
Hansen PE (2007) Isotope effect on chemical shifts in hydrogenbonded systems. J Labelled Compd Rad 50:967–981
Hansen AL, Kay LE (2014) Measurement of histidine pKa values andtautomer populations in invisible protein states. Proc Natl Acad
Sci U S A 111:E1705–E1712
Hansen PE, Lycka A (1989) A reinvestigation of one-bond deuterium-isotope effects on nitrogen and on proton nuclear shielding
for the ammonium ion. Acta Chem Scand 43:222–232
Harms MJ, Schlessman JL, Sue GR, Garcia-Moreno B (2011)
Arginine residues at internal positions in a protein are always
charged. Proc Natl Acad Sci USA 108:18954–18959
Harris TK, Turner GJ (2002) Structural basis of perturbed pKa valuesof catalytic groups in enzyme active sites. IUBMB life 53:85–98
Haruyama H, Qian YQ, Wüthrich K (1989) Static and transienthydrogen-bonding interactions in recombinant desulfatohirudin
studied by 1H nuclear magnetic resonance measurements of
amide proton exchange rates and pH-dependent chemical shifts.
Biochemistry 28:4312–4317
Hass MA, Jensen MR, Led JJ (2008) Probing electric fields inproteins in solution by NMR spectroscopy. Proteins 72:333–343
Henry GD, Sykes BD (1990) Hydrogen-exchange kinetics in amembrane-protein determined by 15N NMR spectroscopy—useof the INEPT experiment to follow individual amides in detergentsolubilized M13 coat protein. Biochemistry 29:6303–6313
Henry GD, Sykes BD (1995) Determination of the rotationaldynamics and pH-dependence of the hydrogen-exchange ratesof the arginine guanidino group using NMR spectroscopy.
J Biomol NMR 6:59–66
Hoffmann R, Reichert I, Wachs WO, Zeppezauer M, Kalbitzer HR(1994) 1H and 31P NMR spectroscopy of phosphorylated model
peptides. Int J Pept Protein Res 44:193–198
Howarth OW, Lilley DMJ (1978) Carbon-13 NMR of peptides andproteins. Prog Nuc Magn Reson Spect 12:1–40

J Biomol NMR
Hunkapiler MW, Smallcombe SH, Whitaker DR, Richards JH (1973)
Carbon nuclear magnetic-resonance studies of histidine residue
in alpha-lytic protease—implications for catalytic mechanism ofserine proteases. Biochemistry 12:4732–4743
Hutson MS, Alexiev U, Shilov SV, Wise KJ, Braiman MS (2000)
Evidence for a perturbation of arginine-82 in the bacteriorhodopsin photocycle from time-resolved infrared spectra. Biochemistry 39:13189–13200
Isom DG, Castaneda CA, Cannon BR, Garcia-Moreno BE (2011)
Large shifts in pKa values of lysine residues buried inside aprotein. Proc Natl Acad Sci USA 108:5260–5265
Iwahara J, Clore GM (2006) Sensitivity improvement for correlations
involving arginine side-chain Ne/He resonances in multi-dimensional NMR experiments using broadband 15N 180o pulses.
J Biomol NMR 36:251–257
Iwahara J, Jung YS, Clore GM (2007) Heteronuclear NMR spectroscopy for lysine NH3 groups in proteins: unique effect of waterexchange on 15N transverse relaxation. J Am Chem Soc
129:2971–2980
Jeng MF, Holmgren A, Dyson HJ (1995) Proton sharing between
cysteine thiols in Escherichia coli thioredoxin—implications forthe mechanism of protein disulfide reduction. Biochemistry34:10101–10105
Joshi MD, Hedberg A, McIntosh LP (1997) Complete measurementof the pKa values of the carboxyl and imidazole groups in
Bacillus circulans xylanase. Protein Sci 6:2667–2670
Joshi MD, Sidhu G, Pot I, Brayer GD, Withers SG, McIntosh LP(2000) Hydrogen bonding and catalysis: a novel explanation for
how a single amino acid substitution can change the pH optimumof a glycosidase. J Mol Biol 299:255–279
Kalbitzer HR, Rosch P (1981) The effect of phosphorylation of thehistidyl residue in the tetrapeptide Gly-Gly-His-Ala—changes ofchemical shift and pK values in 1H-NMR and 31P-NMR spectra.
Org Magn Reson 17:88–91
Kanamori K, Roberts JD (1983) A 15N NMR study of the barriers toisomerization about guanidinium and guanidino carbon nitrogenbonds in L-arginine. J Am Chem Soc 105:4698–4701
Kanamori K, Cain AH, Roberts JD (1978) Studies of pH and anion
complexation effects on L-arginine by natural abundance 15N
nuclear magnetic resonance spectroscopy. J Am Chem Soc
100:4979–4981
Kawano K, Kyogoku Y (1975) Nitrogen-15 nuclear magneticresonance of histidine—effect of pH. Chem Lett 12:1305–1308
Kay LE (1993) Pulsed-field gradient-enhanced 3-dimensional NMR
experiment for correlating 13Ca/b, 13C’, and 1Ha chemical-shifts
in uniformly 13C-labeled proteins dissolved in H2O. J Am Chem
Soc 115:2055–2057
Keim P, Vigna RA, Morrow JS, Marshall RC, Gurd FRN (1973) 13C
nuclear magnetic-resonance of pentapeptides of glycine containing central residues of serine, threonine, aspartic and glutamic
acids, asparagine, and glutamin. J Biol Chem 248:7811–7818
Keim P, Vigna RA, Nigen AM, Morrow JS, Gurd FRN (1974) 13C
nuclear magnetic-resonance of pentapeptides of glycine containing central residues of methionine, proline, arginine, andlysine. J Biol Chem 249:4149–4156
Kesvatera T, Jonsson B, Thulin E, Linse S (1996) Measurement andmodelling of sequence-specific pKa values of lysine residues incalbindin D9k. J Mol Biol 259:828–839
Khare D, Alexander P, Antosiewicz J, Bryan P, Gilson M, Orban J(1997) pKa measurements from nuclear magnetic resonance for
the B1 and B2 immunoglobulin G-binding domains of protein G:
comparison with calculated values for nuclear magnetic resonance and X-ray structures. Biochemistry 36:3580–3589
Kjaergaard M, Brander S, Poulsen FM (2011) Random coil chemical
shift for intrinsically disordered proteins: effects of temperatureand pH. J Biomol NMR 49:139–149

Knowles JR (1976) The intrinsic pKa values of functional groups inenzymes: improper deductions from the pH-dependence ofsteady-state parameters. CRC Crit Rev Biochem 4:165–173
Krezel A, Bal W (2004) A formula for correlating pKa values
determined in D2O and H2O. J Inorg Biochem 98:161–166
Kubickova A, Krizek T, Coufal P, Wernersson E, Heyda J, Jungwirth
P (2011) Guanidinium cations pair with positively charged
arginine side chains in water. J Phys Chem Lett 2:1387–1389
Kukic P, Farrell D, Søndergaard CR, Bjarnadottir U, Bradley J,
Pollastri G, Nielsen JE (2010) Improving the analysis of NMR
spectra tracking pH-induced conformational changes: removing
artefacts of the electric field on the NMR chemical shift. Proteins
Struct Func Bioinform 78:971–984
Kukic P et al (2013) Protein dielectric constants determined from NMR
chemical shift perturbations. J Am Chem Soc 135:16968–16976
Ladner HK, Led JJ, Grant DM (1975) Deuterium-isotope effects on
13
C chemical shifts in amino acids and dipeptides. J Magn Reson
20:530–534
Langkilde A et al (2008) Short strong hydrogen bonds in proteins: acase study of rhamnogalacturonan acetylesterase. Acta Crystallogr D 64:851–863
Lau DKW, Okon M, McIntosh LP (2012) The PNT domain from
Drosophila Pointed-P2 contains a dynamic N-terminal helix
preceded by a disordered phosphoacceptor sequence. Protein Sci
21:1716–1725
Lecomte C, Jelsch C, Guillot B, Fournier B, Lagoutte A (2008)
Ultrahigh-resolution crystallography and related electron densityand electrostatic properties in proteins. J Synchrotron Rad
15:202–203
Led JJ, Petersen SB (1979) Deuterium-isotope effects on 13C
chemical-shifts in selected amino-acids as function of pH.
J Magn Reson 33:603–6170
Legerton TL, Kanamori K, Weiss RL, Roberts JD (1981) 15N NMR
studies of nitrogen metabolism in intact mycelia of Neurospora
crassa. Proc Natl Acad Sci 78:1495–1498
Li SH, Hong M (2011) Protonation, tautomerization, and rotameric
structure of histidine: a comprehensive study by magic-anglespinning solid-state NMR. J Am Chem Soc 133:1534–1544
Licht S (1985) pH measurement in concentrated alkaline-solutions.
Anal Chem 57:514–519
Liepinsh E, Otting G (1996) Proton exchange rates from amino acidside chains—implications for image contrast. Magn Reson Med
35:30–42
Liepinsh E, Otting G, Wüthrich K (1992) NMR spectroscopy ofhydroxyl protons in aqueous solutions of peptides and proteins.
J Biomol NMR 2:447–465
Lim JC, Gruschus JM, Kim G, Berlett BS, Tjandra N, Levine RL (2012)
A low pKa cysteine at the active site of mouse methionine
sulfoxide reductase A. J Biol Chem 287:25596–25601
Lindman S, Linse S, Mulder FA, Andre I (2006) Electrostatic
contributions to residue-specific protonation equilibria andproton binding capacitance for a small protein. Biochemistry45:13993–14002
Lindman S, Linse S, Mulder FA, Andre I (2007) pKa values for sidechain carboxyl groups of a PGB1 variant explain salt and pHdependent stability. Biophys J 92:257–266
Löhr F, Katsemi V, Betz M, Hartleib J, Rüterjans H (2002) Sequencespecific assignment of histidine and tryptophan ring 1H, 13C and
15
N resonances in 13C/15N- and 2H/13C/15N-labelled proteins.
J Biomol NMR 22:153–164
Löhr F, Rogov VV, Shi MC, Bernhard F, Dötsch V (2005) Tripleresonance methods for complete resonance assignment ofaromatic protons and directly bound heteronuclei in histidine
and tryptophan residues. J Biomol NMR 32:309–328
London RE (1980) Correlation of carboxyl carbon titration shifts andpK values. J Magn Reson 38:173–177

123

J Biomol NMR
London RE, Walker TE, Whaley TW, Matwiyoff NA (1977) 15N
NMR studies of 13C, 15N labeled arginine. Org Magn Reson
9:598–600
London RE, Walker TE, Kollman VH, Matwiyoff NA (1978) Studies
of pH-dependence of 13C shifts and carbon-carbon couplingconstants of [U-13C]aspartic and glutamic acids. J Am Chem Soc
100:3723–3729
Ludwiczek ML et al (2013) Strategies for modulating the pHdependent activity of a family 11 glycoside hydrolase. Biochemistry 52:3138–3156
Markley JL (1975) Observation of histidine residues in proteins bymeans of nuclear magnetic-resonance spectroscopy. Acc Chem
Res 8:70–80
Mavridou DAI, Stevens JM, Ferguson SJ, Redfield C (2007) Activesite properties of the oxidized and reduced C-terminal domain of
DsbD obtained by NMR spectroscopy. J Mol Biol 370:643–658
Mayer R, Lancelot G, Spach G (1979) Side chain-backbonehydrogen-bonds in peptides containing glutamic-acid residues.
Biopolymers 18:1293–1296
McIntosh LP, Wand AJ, Lowry DF, Redfield AG, Dahlquist FW (1990)
Assignment of the backbone 1H and 15N NMR resonances ofbacteriophage T4 lysozyme. Biochemistry 29:6341–6362
McIntosh LP et al (1996) The pKa of the general acid/base carboxylgroup of a glycosidase cycles during catalysis: a 13C-NMR studyof Bacillus circulans xylanase. Biochemistry 35:9958–9966
McIntosh LP, Kang HS, Okon M, Nelson ML, Graves BJ, Brutscher
B (2009) Detection and assignment of phosphoserine andphosphothreonine residues by 13C-31P spin-echo difference
NMR spectroscopy. J Biomol NMR 43:31–37
McIntosh LP, Naito D, Baturin SJ, Okon M, Joshi MD, Nielsen JE(2011) Dissecting electrostatic interactions in Bacillus circulans
xylanase through NMR-monitored pH titrations. J Biomol NMR
51:5–19
McMahon BH et al (2004) FTIR studies of internal proton transfer
reactions linked to inter-heme electron transfer in bovine
cytochrome c oxidase. Biochim Biophys Acta 1655:321–331
Miao Y, Cross TA, Fu R (2014) Differentiation of histidine
tautomeric states using 15N selectively filtered 13C solid-state
NMR spectroscopy. J Magn Reson 245C:105–109
Mossner E, Iwai H, Glockshuber R (2000) Influence of the pKa valueof the buried, active-site cysteine on the redox properties ofthioredoxin-like oxidoreductases. FEBS Lett 477:21–26
Munowitz M, Bachovchin WW, Herzfeld J, Dobson CM, Griffin RG(1982) Acid-base and tautomeric equilibria in the solidstate—15N NMR spectroscopy of histidine and imidazole.
J Am Chem Soc 104:1192–1196
Nielsen JE, Gunner MR, Garcia-Moreno BE (2011) The pKa
Cooperative: a collaborative effort to advance structure-based
calculations of pKa values and electrostatic effects in proteins.
Proteins 79:3249–3259
Niimura N, Bau R (2008) Neutron protein crystallography: beyond
the folding structure of biological macromolecules. Acta Cryst
Section A 64:12–22
Norberg J, Foloppe N, Nilsson L (2005) Intrinsic relative stabilities of
the neutral tautomers of arginine side-chain models. J Chem
Theory Comput 1:986–993
Nordstrand K, Aslund F, Meunier S, Holmgren A, Otting G, Berndt KD(1999) Direct NMR observation of the Cys-14 thiol proton of
reduced Escherichia coli glutaredoxin-3 supports the presence of anactive site thiol-thiolate hydrogen bond. FEBS Lett 449:196–200
Norton RS, Bradbury JH (1974) 13C nuclear magnetic-resonance studyof tyrosine titrations. J Chem Soc Chem Comm 21:870–871
Oda Y, Yamazaki T, Nagayama K, Kanaya S, Kuroda Y, Nakamura
H (1994) Individual ionization-constants of all the carboxylgroups in ribonuclease HI from Escherichia coli determined by
NMR. Biochemistry 33:5275–5284

123

Oktaviani NA, Pool TJ, Kamikubo H, Slager J, Scheek RM, Kataoka
M, Mulder FAA (2012) Comprehensive determination of protein
tyrosine pKa values for photoactive yellow protein using indirect
13
C NMR spectroscopy. Biophys J 102:579–586
Oldfield E, Norton RS, Allerhand A (1975a) Studies of individualcarbon sites of proteins in solution by natural abundance carbon
13 nuclear magnetic-resonance spectroscopy—relaxation behavior. J Biol Chem 250:6368–6380
Oldfield E, Norton RS, Allerhand A (1975b) Studies of individualcarbon sites of proteins in solution by natural abundance carbon
13 nuclear magnetic-resonance spectroscopy—strategies for
assignments. J Biol Chem 250:6381–6402
Parsons SM, Raftery MA (1972) Ionization behavior of the catalytic
carboxyls of lysozyme. Effects of ionic strength. Biochemistry
11:1623–1629
Pellecchia M, Wider G, Iwai H, Wüthrich K (1997) Arginine side
chain assignments in uniformly 15N-labeled proteins using thenovel 2D He(Ne)HcHH experiment. J Biomol NMR 10:193–197
Pelton JG, Torchia DA, Meadow ND, Roseman S (1993) Tautomericstates of the active-site histidines of phosphorylated andunphosphorylated III(Glc), a signal-transducing protein from
Escherichia coli, using 2-dimensional heteronuclear NMR
techniques. Protein Sci 2:543–558
Petkova AT, Hu JG, Bizounok M, Simpson M, Griffin RG, Herzfeld J(1999) Arginine activity in the proton-motive photocycle ofbacteriorhodopsin: solid-state NMR studies of the wild-type andD85N proteins. Biochemistry 38:1562–1572
Plesniak LA, Connelly GP, Wakarchuk WW, McIntosh LP (1996)
Characterization of a buried neutral histidine residue in Bacillus
circulans xylanase: NMR assignments, pH titration, and hydrogen exchange. Protein Sci 5:2319–2328
Poon DKY, Schubert M, Au J, Okon M, Withers SG, McIntosh LP(2006) Unambiguous determination of the ionization state of a
glycoside hydrolase active site lysine by 1H-15N heteronuclear
correlation spectroscopy. J Am Chem Soc 128:15388–15389
Popov K, Ronkkomaki H, Lajunen LHJ (2006) Guidelines for NMR
measurements for determination of high and low pKa values.
Pure Appl Chem 78:663–675
Pregosin PS, Randall EW, White AI (1971) Natural abundance 15N
nuclear magnetic resonance spectroscopy—amino acid derivatives. J Chem Soc Chem Comm 24:1602–1603
Prestegard JH, Sahu SC, Nkari WK, Morris LC, Live D, Gruta C(2013) Chemical shift prediction for denatured proteins. J Biomol
NMR 55:201–209
Prompers JJ, Groenewegen A, Hilbers CW, Pepermans HAM (1998)
Two-dimensional NMR experiments for the assignment of aromaticside chains in 13C-labeled proteins. J Magn Reson 130:68–75
Pujato M, Navarro A, Versace R, Mancusso R, Ghose R, Tasayco ML(2006) The pH-dependence of amide chemical shift of Asp/Glu
reflects its pKa in intrinsically disordered proteins with only
local interactions. BBA-Proteins Proteom 1764:1227–1233
Quirt AR, Lyerla JR, Peat IR, Cohen JS, Reynolds WF, Freedman
MH (1974) 13C nuclear magnetic resonance titration shifts inamino-acids. J Am Chem Soc 96:570–571
Rabenstein DL, Sayer TL (1976a) 13C chemical shift parameters foramines, carboxylic acids, and amino acids. J Magn Reson
24:27–39
Rabenstein DL, Sayer TL (1976b) Determination of microscopic aciddissociation constants by nuclear magnetic-resonance spectrometry. Anal Chem 48:1141–1145
Raczynska ED et al (2003) Consequences of proton transfer in
guanidine. J Phys Org Chem 16:91–106
Rajagopal P, Waygood EB, Klevit RE (1994) Structural consequencesof histidine phosphorylation—NMR characterization of the phosphohistidine form of histidine-containing protein from Bacillus
subtilis and Escherichia coli. Biochemistry 33:15271–15282

J Biomol NMR
Rao NS, Legault P, Muhandiram DR, Greenblatt J, Battiste JL,
Williamson JR, Kay LE (1996) NMR pulse schemes for thesequential assignment of arginine side-chain He protons. J Magn
Reson Ser B 113:272–276
Reynolds WF, Tzeng CW (1977) Determination of preferred tautomeric form of histamine by 13C NMR spectroscopy. Can J
Biochem Cell B 55:576–578
Reynolds WF, Peat IR, Freedman MH, Lyerla JR Jr (1973)
Determination of the tautomeric form of the imidazole ring of
L-histidine in basic solution by 13C magnetic resonance spectroscopy. J Am Chem Soc 95:328–331
Richards RE, Thomas NA (1974) Nitrogen-14 nuclear magneticresonance study of amino acids, peptides, and other biologicallyinteresting molecules. J Chem Soc Perk T 2:368–374
Richarz R, Wüthrich K (1978) 13C NMR chemical-shifts of common
amino acid residues measured in aqueous-solutions of linear
tetrapeptides H-Gly-Gly-X-L-Ala-OH. Biopolymers 17:2133–2141
Roberts JD, Yu C, Flanagan C, Birdseye TR (1982) A 15N nuclear
magnetic-resonance study of the acid-base and tautomericequilibria of 4-substituted imidazoles and its relevance to thecatalytic mechanism of a-lytic protease. J Am Chem Soc
104:3945–3949
Robillard G, Shulman RG (1972) High-resolution nuclear magneticresonance study of histidine—aspartate hydrogen-bond in chymotrypsin and chymotrypsinogen. J Mol Biol 71:507–511
Roos G, Foloppe N, Messens J (2013) Understanding the pKa ofredox cysteines: the key role of hydrogen bonding. Antioxid
Redox Sign 18:94–127
Saito K, Ishikita H (2012) H atom positions and nuclear magnetic
resonance chemical shifts of short H bonds in photoactive yellow
protein. Biochemistry 51:1171–1177
Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination ofproteins in solution employing pulsed field gradients. Prog Nucl
Mag Reson Specty 34:93–158
Schaller W, Robertson AD (1995) pH, ionic strength, and temperaturedependences of ionization equilibria for the carboxyl groups inturkey ovomucoid third domain. Biochemistry 34:4714–4723
Schlemmer H, Sontheimer GM, Kalbitzer HR (1988) 31P nuclear
magnetic-resonance spectroscopy of the phosphorylated tetrapeptide Gly-Gly-Asp-Ala. Magn Reson Chem 26:260–263
Schlippe YVG, Hedstrom L (2005) A twisted base? The role ofarginine in enzyme-catalyzed proton abstractions. Arch Biochem
Biophys 433:266–278
Schubert M et al (2007) Probing electrostatic interactions along thereaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy. Biochemistry 46:7383–7395
Schwarzinger S, Kroon GJA, Foss TR, Chung J, Wright PE, Dyson
HJ (2001) Sequence-dependent correction of random coil NMR
chemical shifts. J Am Chem Soc 123:2970–2978
Segawa T, Kateb F, Duma L, Bodenhausen G, Pelupessy P (2008)
Exchange rate constants of invisible protons in proteins determined by NMR spectroscopy. ChemBioChem 9:537–542
Shimba N, Takahashi H, Sakakura M, Fujii I, Shimada I (1998)
Determination of protonation and deprotonation forms andtautomeric states of histidine residues in large proteins using
nitrogen-carbon J couplings in imidazole ring. J Am Chem Soc
120:10988–10989
Shrager RI, Sachs DH, Schechte A, Cohen JS, Heller SR (1972)
Nuclear magnetic-resonance titration curves of histidine ring
protons.2. Mathematical models for interacting groups in nuclear
magnetic-resonance titration curves. Biochemistry 11:541–547
Smet-Nocca C, Launay H, Wieruszeski JM, Lippens G, Landrieu I(2013) Unraveling a phosphorylation event in a folded protein by
NMR spectroscopy: phosphorylation of the Pin1 WW domain by
PKA. J Biomol NMR 55:323–337

Smith BC, Denu JM (2009) Chemical mechanisms of histone lysine
and arginine modifications. BBA-Gene Regul Mech 1789:45–57
Smith GM, Yu LP, Domingues DJ (1987) Directly observed 15N
NMR spectra of uniformly enriched proteins. Biochemistry
26:2202–2207
Søndergaard CR, McIntosh LP, Pollastri G, Nielsen JE (2008) Determination of electrostatic interaction energies and protonation state
populations in enzyme active sites. J Mol Biol 376:269–287
Song JK, Laskowski M, Qasim MA, Markley JL (2003) NMR
determination of pKa values for Asp, Glu, His, and Lys mutants
at each variable contiguous enzyme-inhibitor contact position of
the turkey ovomucoid third domain. Biochemistry 42:2847–2856
Spitzner N, Löhr F, Pfeiffer S, Koumanov A, Karshikoff A, Rüterjans
H (2001) Ionization properties of titratable groups in ribonuclease T1—I. pKa values in the native state determined by twodimensional heteronuclear NMR spectroscopy. Eur Biophys Jwith s 30:186–197
Sudmeier JL, Ash EL, Gunther UL, Luo XL, Bullock PA, Bachovchin
WW (1996) HCN, a triple-resonance NMR technique forselective observation of histidine and tryptophan side chains in
13 15
C/ N-labeled proteins. J Magn Reson Ser B 113:236–247
Sudmeier JL, Bradshaw EM, Haddad KE, Day RM, Thalhauser CJ,
Bullock PA, Bachovchin WW (2003) Identification of histidine
tautomers in proteins by 2D 1H/13Cd2 one-bond correlated NMR.
J Am Chem Soc 125:8430–8431
Surprenant HL, Sarneski JE, Key RR, Byrd JT, Reilley CN (1980) 13C
NMR studies of amino acids—chemical-shifts, protonation shifts,
microscopic protonation behavior. J Magn Reson 40:231–243
Suzuki T, Yamaguch.T, Imanari M (1974) 15N FT NMR spectra ofamino acids in natural abundance—pH-dependence of 15N
chemical-shifts for L-arginine. Tetrahedron Lett 20:1809–1812
Szakacs Z, Kraszni M, Noszal B (2004) Determination of microscopic
acid-base parameters from NMR pH titrations. Anal Bioanal
Chem 378:1428–1448
Takayama Y, Castaneda CA, Chimenti M, Garcia-Moreno B, Iwahara
J (2008a) Direct evidence for deprotonation of a lysine side
chain buried in the hydrophobic core of a protein. J Am Chem
Soc 130:6714–6715
Takayama Y, Sahu D, Iwahara J (2008b) Observing in-phase singlequantum 15N multiplets for NH2/NH3? groups with twodimensional heteronuclear correlation spectroscopy. J Magn
Reson 194:313–316
Takeda M, Jee J, Ono AM, Terauchi T, Kainosho M (2009) Hydrogenexchange rate of tyrosine hydroxyl groups in proteins as studied
by the deuterium isotope effect on Cf chemical shifts. J Am
Chem Soc 131:18556–18562
Takeda M, Jee J, Terauchi T, Kainosho M (2010) Detection of thesulfhydryl groups in proteins with slow hydrogen exchange rates
and determination of their proton/deuteron fractionation factors
using the deuterium-induced effects on the 13Cb NMR signals.
J Am Chem Soc 132:6254–6260
Takeda M, Miyanoiri Y, Terauchi T, Yang C-J, Kainosho M (2014) Use
of H/D isotope effects to gather information about hydrogenbonding and hydrogen exchange rates. J Magn Reson 241:148–154
Tamiola K, Acar B, Mulder FAA (2010) Sequence-specific random
coil chemical shifts of intrinsically disordered proteins. J Am
Chem Soc 132:18000–18003
Tanokura M (1983) 1H-NMR study on the tautomerism of theimidazole ring of histidine-residues 1. Microscopic pK values
and molar ratios of tautomers in histidine-containing peptides.
Biochim Biophys Acta 742:576–585
Thanabal V, Omecinsky DO, Reily MD, Cody WL (1994) The 13C
chemical shifts of amino acids in aqueous-solution containing
organic-solvents—application to the secondary structure characterization of peptides in aqueous trifluoroethanol solution.
J Biomol NMR 4:47–59

123

J Biomol NMR
Theillet FX et al (2012) Cell signaling, post-translational protein
modifications and NMR spectroscopy. J Biomol NMR 54:217–236
Tolbert BS, Tajc SG, Webb H, Snyder J, Nielsen JE, Miller BL,
Basavappa R (2005) The active site cysteine of ubiquitinconjugating enzymes has a significantly elevated pKa: functional
implications. Biochemistry 44:16385–16391
Tolstoy PM, Schah-Mohammedi P, Smirnov SN, Golubev NS,
Denisov GS, Limbach HH (2004) Characterization of fluxionalhydrogen-bonded complexes of acetic acid and acetate by NMR:
geometries and isotope and solvent effects. J Am Chem Soc
126:5621–5634
Tomlinson JH, Ullah S, Hansen PE, Williamson MP (2009)
Characterization of salt bridges to lysines in the protein G B1
domain. J Am Chem Soc 131:4674–4684
Tomlinson JH, Green VL, Baker PJ, Williamson MP (2010) Structural
origins of pH-dependent chemical shifts in the B1 domain ofprotein G. Proteins Struct Funct Bioinform 78:3000–3016
Tugarinov V (2014) Indirect use of deuterium in solution NMR
studies of protein structure and hydrogen bonding. Prog Nucl
Magn Reson Spect 77:49–68
Ullah S, Ishimoto T, Williamson MP, Hansen PE (2011) Ab initio
calculations of deuterium isotope effects on chemical shifts ofsalt-bridged lysines. J Phys Chem B 115:3208–3215
Ullmann GM (2003) Relations between protonation constants andtitration curves in polyprotic acids: a critical view. J Phys Chem
B 107:1263–1271
Ulrich EL et al (2008) BioMagResBank. Nucleic Acids Res
36:D402–D408
van Dijk AA, Delange LCM, Bachovchin WW, Robillard GT (1990)
Effect of phosphorylation on hydrogen-bonding interactions of theactive-site histidine of the phosphocarrier protein hpr of thephosphoenolpyruvate-dependent phosphotransferase system determined by 15N NMR spectroscopy. Biochemistry 29:8164–8171
Velyvis A, Kay LE (2013) Measurement of active site ionization
equilibria in the 670 kDa proteasome core particle using methylTROSY NMR. J Am Chem Soc 135:9259–9262
Vis H, Boelens R, Mariani M, Stroop R, Vorgias CE, Wilson KS,
Kaptein R (1994) 1H, 13C, and 15N resonance assignments andsecondary structure-analysis of the hu protein from bacillusstearothermophilus using 2-dimensional and 3-dimensional
double-resonance and triple-resonance heteronuclear magneticresonance spectroscopy. Biochemistry 33:14858–14870
Volpon L et al (2007) NMR structure determination of a synthetic
analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in
the natural iturinic antibiotics. Spectrochim Acta Part A
67:1374–1381
Vondrasek J, Mason PE, Heyda J, Collins KD, Jungwirth P (2009)
The molecular origin of like-charge arginine-arginine pairing inwater. J Phys Chem B 113:9041–9045
Wang YJ, Jardetzky O (2002) Investigation of the neighboring
residue effects on protein chemical shifts. J Am Chem Soc
124:14075–14084
Wang YX et al (1996) Solution NMR evidence that the HIV-1
protease catalytic aspartyl groups have different ionization statesin the complex formed with the asymmetric drug KNI-272.
Biochemistry 35:9945–9950
Wasylishen RE, Friedrich JO (1987) Deuterium-isotope effects on
nuclear shielding constants and spin spin coupling-constants in theammonium ion, ammonia, and water. Can J Chem 65:2238–2243
Wasylishen RE, Tomlinson G (1975) pH-dependence of 13C chemical-shifts and 13C, H coupling-constants in imidazole and Lhistidine. Biochem J 147:605–607
Wasylishen RE, Tomlinson G (1977) Applications of long-range 13C,
H nuclear spin-spin coupling-constants in study of imidazole
tautomerism in L-histidine, histamine, and related compounds.
Can J Biochem Cell B 55:579–582

123

Webb H, Tynan-Connolly BM, Lee GM, Farrell D, O’Meara F,
Søndergaard CR, Teilum K, Hewage C, McIntosh LP, Nielsen
JE (2011) Remeasuring HEWL pKa values by NMR spectroscopy: methods, analysis, accuracy, and implications for theoretical pKa, calculations. Proteins Struct Func Bioinform
79:685–702
Werner MH, Clore GM, Fisher CL, Fisher RJ, Trinh L, Shiloach J,
Gronenborn AM (1997) Correction of the NMR structure of theETS1/DNA complex. J Biomol NMR 10:317–328
Wilson NA, Barbar E, Fuchs JA, Woodward C (1995) Aspartic-acid
26 in reduced escherichia-coli thioredoxin has a pK(a) [9.
Biochemistry 34:8931–8939
Wishart DS (2011) Interpreting protein chemical shift data. Prog Nucl
Magn Reson Spectr 58:62–87
Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) 1H,
13
C and 15N random coil NMR chemical-shifts of the common
amino-acids 1. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81
Witanowski M, Webb GA, Stefania L, Januszew H, Grabowsk Z(1972) Nitrogen-14 nuclear magnetic resonance of azoles andtheir benzo derivatives. Tetrahedron 28:637–653
Witanowski M, Stefaniak L, Szymanski S, Webb GA (1976) 14N
NMR study of isomeric structures of urea and its analogs.
Tetrahedron 32:2127–2129
Wittekind M, Metzler WJ, Mueller L (1993) Selective correlations ofamide groups to glycine alpha-protons in proteins. J Magn Reson
Ser B 101:214–217
Wu XJ, Westler WM, Markley JL (1984) The assignment ofimidazolium NH-H1 peaks in the 1H-NMR spectrum of a protein
by one-dimensional and two-dimensional NOE experiments.
J Magn Reson 59:524–529
Wüthrich K, Wagner G (1979) Nuclear magnetic-resonance of labile
protons in the basic pancreatic trypsin-inhibitor. J Mol Biol
130:1–18
Xiao YW, Braiman M (2005) Modeling amino acid side chains inproteins: 15N NMR spectra of guanidino groups in nonpolar
environments. J Phys Chem B 109:16953–16958
Xiao Y, Hutson MS, Belenky M, Herzfeld J, Braiman MS (2004)
Role of arginine-82 in fast proton release during the bacteriorhodopsin photocycle: a time-resolved FT-IR study of purple
membranes containing 15N-labeled arginine. Biochemistry
43:12809–12818
Yamazaki T, Yoshida M, Nagayama K (1993) Complete assignmentsof magnetic resonances of ribonuclease-H from Escherichia coliby double-resonance and triple-resonance 2D and 3D NMR
spectroscopies. Biochemistry 32:5656–5669
Yamazaki T et al (1994) NMR and X-ray evidence that the HIVprotease catalytic aspartyl groups are protonated in the complex
formed by the protease and a nonpeptide cyclic urea-based
inhibitor. J Am Chem Soc 116:10791–10792
Yamazaki T, Pascal SM, Singer AU, Forman-Kay JD, Kay LE (1995)
NMR pulse schemes for the sequence-specific assignment ofarginine guanidino 15N and 1H chemical-shifts in proteins. J Am
Chem Soc 117:3556–3564
Yavari I, Roberts JD (1978) Differential rates of proton-exchange for
guanidinium nitrogens of L-arginine determined by naturalabundance 15N nuclear magnetic-resonance spectroscopy. Biochem Biophys Res Commun 83:635–640
Yu LP, Fesik SW (1994) pH titration of the histidine-residues ofcyclophilin and FK506 binding-protein in the absence andpresence of immunosuppressant ligands. Bioc Biophys Acta Prot
Struct Molr Enzym 1209:24–32
Zandarashvili L, Li DW, Wang T, Bruschweiler R, Iwahara J (2011)
Signature of mobile hydrogen bonding of lysine side chains fromlong-range 15N-13C scalar J-couplings and computation. J Am
Chem Soc 133:9192–9195

J Biomol NMR
Zandarashvili L, Esadze A, Iwahara J (2013) NMR Studies on the
dynamics of hydrogen bonds and ion pairs involving lysine side
chains of proteins. Adv Protein Chem Str 93:37–80
Zheng G, Price WS (2010) Solvent signal suppression in NMR. Prog
Nuclear Magn Res Spect 56:267–288

Zhu L, Kemple MD, Yuan P, Prendergast FG (1995) N-terminus andlysine side chain pKa values of melittin in aqueous solutions andmicellar dispersions measured by 15N NMR. Biochemistry34:13196–13202

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