Hydrogen Exchange Rate of Tyr -OH Groups in Proteins
A R T I C L E S
environments, the hydroxyl groups of the amino acid side chains
of the buried residues might have much slower hydrogen
exchange rates and could be studied without interference from
trace amounts of contaminants or inorganic buffers, as compared
to those of the solvent-exposed residues in peptides. Therefore,
the microenvironments for the hydroxyl groups of the amino
acid side chains in solution may be characterized by the
detection of the slowly exchanging hydrogen atoms attached
to them.
A number of NMR studies have analyzed the hydrogen
exchange phenomena of side chain hydroxyl groups.15,16 Many
of the hydroxyl protons in proteins, however, were observed at
the chemical shift of water, due to the fact that the chemical
exchange rates between the hydroxyl and water protons are, in
general, fast enough to merge their NMR signals.17,18 Further-
more, the hydroxyl proton can hardly be detected by sensitive
Figure 1. Stable isotope labeling pattern of SAIL (2S,3R)-[ꢀ2,ε1,2
-
2H3;0,R,ꢀ,ꢁ-13C4;15N]-Tyr. 12C atoms are not shown in the figure.
method to detect the correlated hydrogen exchange phenomena
of a protein, which are otherwise difficult to investigate.25
The deuterium isotope shift should, in principle, also be
applicable for studying the hydrogen exchange rates of other
polar groups of the amino acid side chains in a protein. In the
case of Tyr, a sizable two-bond deuterium isotope shift is
expected to be observed for the 13C chemical shifts of the Cꢁ
atoms. Recent progress in cryogenic probe technology28 has
enabled highly sensitive, direct observations of the 13C NMR
spectra, even for relatively small amounts of labeled protein.
However, the uniformly 13C-labeled Tyr is not suitable for
detecting the deuterium isotope effects of the Cꢁ NMR signals,
since the 13C-13C spin couplings between 1J(13Cꢁ-13Cε1/2), ∼70
Hz, and 3J(13Cꢁ-13Cγ), ∼10 Hz, result in complex, broad
signals.29 The lack of robust assignment methods for the 13Cꢁ
signals has further limited the use of the uniformly 13C-labeled
Tyr. In the present study, we describe a new method to observe
and assign the 13Cꢁ signals of the Tyr rings in a protein and to
investigate the hydrogen exchange rates of individual hydroxyl
groups of Tyr residues, by observing the two-bond deuterium
1
indirect-detection NMR measurements, such as H-13C cor-
relation spectroscopy, and thus many of the hydroxyl proton
signals tend to be overlooked, even though they actually appear
as discrete 1H NMR signals. Among the hydroxyl amino acids,
i.e., Thr, Ser, and Tyr, the hydroxyl groups of Tyr residues are
often involved in intermolecular hydrogen bonds within the core
region of a protein.19 Therefore, the identification and charac-
terization of slowly exchanging Tyr hydroxyl protons is expected
to provide valuable information on these auxiliary hydrogen
bonds.
In addition to the direct observation of exchangeable proton
signals, various alternative methods, using deuterium isotope
effects on the chemical shifts of the directly or indirectly bonded
carbons to study the hydrogen exchange rates for backbone
amide groups, have been developed.20-26 The deuterium
substitution usually causes substantial upfield shifts for the nuclei
separated by as many as three covalent bonds.27 Thus, in a 1:1
mixture of H2O and D2O, for slowly exchanging amide protons,
the carbonyl carbon or R-carbon signals of amide groups may
appear as multiple peaks if the hydrogen-deuterium exchange
rates are slower than the inverse of the respective isotope shift
differences. This method was originally applied for small
peptides20,21 and then for proteins.22-24 Simultaneous monitoring
of the proton exchange rates for the amide groups of the ith
and (i + 1)th residues through the two and three bond deuterium
isotope effects, which can be observed for a protein dissolved
in a 1:1 mixture of H2O and D2O, led to the development of a
2
isotope shift, ∆δ13Cꢁ, for the 13Cꢁ signals. In doing so, we
synthesized a new type of stereoarray isotope labeled (SAIL)
amino acid,30 (2S,3R)-[ꢀ2,ε1,2-2H3;0,R,ꢀ,ꢁ-13C4;15N]-Tyr, ꢁ-SAIL
Tyr, which has the optimal isotope-labeling pattern for the
present purpose (Figure 1). We used an E. coli cell-free protein
expression system to incorporate ꢁ-SAIL Tyr into an 18.2 kDa
E. coli protein, peptidyl-prolyl cis-trans isomerase b (EPPIb),
for which we had already established the sequential assignment31
and determined the solution structure.32 By virtue of the optimal
labeling pattern of the ꢁ-SAIL Tyr residues, the three 13Cꢁ atoms
of the Tyr residues in EPPIb were readily assigned and then
used for studying the hydrogen exchange rates of the Tyr
hydroxyl groups.
(15) Liepinsh, E.; Otting, G.; Wu¨thrich, K. J. Biomol. NMR 1992, 2, 447–
465.
(16) Pfeiffer, S.; Spitzner, N.; Lo¨hr, F.; Ru¨terjans, H. J. Biomol. NMR 1998,
11, 1–15.
Materials and Methods
(17) Otting, G.; Wu¨thrich, K. J. Am. Chem. Soc. 1989, 111, 1871–1875.
(18) Otting, G.; Liepinsh, E.; Wu¨thrich, K. J. Am. Chem. Soc. 1991, 113,
4363–4364.
(2S,3R)-[ꢁ2,ε1,2-2H3;0,r,ꢁ,ꢀ-13C4;15N]-Tyr, ꢀ-SAIL Tyr (6). ꢁ-SAIL
Tyr 6 was synthesized according to the scheme described in Figure
2. The [4-13C]4H-pyran-4-one 2 was derived from [2-13C]-acetone
(CIL)1bythemethoddescribedpreviously,withslightmodifications.33,34
Treatment of 2 with diethyl [1,3-13C2]-malonate (CIL) in the
(19) Pace, C. N.; Horn, G.; Hebert, E. J.; Bechert, J.; Shaw, K.; Urbanikova,
L.; Scholtz, J. M.; Sevcik, J. J. Mol. Biol. 2001, 312, 393–404.
(20) Feeney, J.; Partington, P.; Roberts, G. C. K. J. Magn. Reson. 1974,
13, 268–274.
(21) Hawkes, G. E.; Randall, E. W.; Hull, W. E.; Gattegno, D.; Conti, F.
Biochemistry 1978, 17, 3986–3993.
(28) Serber, Z.; Richter, C.; Do¨tsch, V. ChemBioChem 2001, 2, 247–251.
(29) Torizawa, T.; Ono, A. M.; Terauchi, T.; Kainosho, M. J. Am. Chem.
Soc. 2005, 127, 12620–12626.
(22) Kainosho, M.; Tsuji, T. Biochemistry 1982, 21, 6273–6279.
(23) Kainosho, M.; Nagao, H.; Tsuji, T. Biochemistry 1987, 26, 1068–
1075.
(30) Kainosho, M.; Torizawa, T.; Iwashita, Y.; Terauchi, T.; Ono, A. M.;
Gu¨ntert, P. Nature 2006, 440, 52–57.
(24) Markley, J. L.; Kainosho, M. In Stable Isotope Labeling and Resonance
Assignments in Larger Proteins: NMR of Macromolecules; Roberts,
G. C. K., Ed.; Oxford University Press: New York, 1993; pp 101-
152.
(31) Kariya, E.; Ohki, S.; Hayano, T.; Kainosho, M. J. Biomol. NMR. 2000,
18, 75–76.
(32) Takeda, M.; Terauchi, T.; Ono, A. M.; Kainosho, M. J. Biomol. NMR
2009, in press; DOI: 10.1007/s10858-009-9360-9.
(33) Riegel, E. R.; Zwilgmeyer, F. Organic Synthesis; Wiley: New York,
1943 Collect. Vol. 2, p 126.
(25) Uchida, K.; Markley, J. L.; Kainosho, M. Biochemistry 2005, 44,
11811–11820.
(26) Liu, A.; Lu, Z.; Wang, J.; Yao, L.; Li, Y.; Yan, H. J. Am. Chem. Soc.
2008, 130, 2428–2429.
(34) Owen, G. E., Jr.; Pearson, J. M.; Szwarc, M. Trans. Faraday. Soc.
1964, 60, 564–571.
(27) Hansen, P. E. Annu. Rep. NMR Spectrosc. 1983, 15, 105–234.
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