3S-Fluoroproline as a probe to monitor proline isomerization during protein folding by 19F-NMR

Article abstract of DOI:10.1039/b821952d

Variable-temperature inversion transfer NMR is used to determine the kinetic and thermodynamic parameters of cis-trans isomerization of N-Ac-(3R) and (3S)-fluoroproline-OMe.

Full text of DOI:10.1039/b821952d

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3S-Fluoroproline as a probe to monitor proline isomerization during
19
protein folding by F-NMRw
Colin A. Thomas,ab Erach R. Talatya and James G. Bann*a
Received (in College Park, MD, USA) 8th December 2008, Accepted 29th April 2009
First published as an Advance Article on the web 19th May 2009
DOI: 10.1039/b821952d
Variable-temperature inversion transfer NMR is used to determine
the kinetic and thermodynamic parameters of cis–trans iso-
merization of N-Ac-(3R) and (3S)-fluoroproline-OMe.
model compounds will provide a basis for future studies in
which biosynthetic incorporation of 3S and 3R fluoro-
proline will be used for monitoring cis–trans isomerization of
proline during protein folding.
The use of 19F-NMR is unique for protein folding studies
since a 1-D 19F spectrum provides residue-specific information
on the folding of proteins, and the development of stopped-
flow NMR methods has allowed time-dependent folding
information to be obtained with dead times less than 2
seconds.8 The 19F signal to noise ratio is as good as hydrogen,
which is approximately 8 times as sensitive as carbon.9 Fluorine
is only slightly larger than hydrogen (0.15 A), and in most
cases is non-structurally perturbing.10 Finally, London and
co-workers have shown that the peptide [p-fluoro-Phe]bradykinin,
which contains a -Pro-p-FPhe- peptide bond, exhibited well
resolved cis and trans resonances that could be used to
monitor the catalysis of cis–trans isomerization by the enzyme
cyclophilin.11
Understanding how proteins change in structure from an
unfolded to a folded state is complicated, in many cases, by
the cis to trans isomerization of prolyl peptide bonds.1 While
dependent upon the residues surrounding a particular proline,2
it is assumed that in the unfolded state, proline will isomerize to
equilibrium values approximated by model peptides (typically
B80% trans, 20% cis).3 Although this assumption is valid for
some proteins, model peptides cannot a priori predict the
population of cis–trans isomers in the unfolded state. The
ability to directly measure this population in a protein would
have distinct advantages, in particular in monitoring how this
population changes during the folding process.4
The idea to monitor directly the population of proline
cis–trans isomers in the unfolded state is not new, and was
pioneered initially by Torchia and co-workers, through the
biosynthetic incorporation of [4-13C] proline into collagen,
and later applied to the study of staphylococcal nuclease.5
Although methods and instrumentation (cryogenic probes)
have been developed to improve sensitivity, carbon remains
one of the least sensitive nuclei for detection and thus has
limited its use for monitoring processes which occur in real time.
As an alternative to carbon detection of proline cis–trans
isomerization, we report here the kinetics and thermo-
dynamics of isomerization of the simple compounds Ac-(3R)
and (3S)-fluoroproline-OMe (compounds 1 and 2, respectively)6
in water–D2O solution using 19F-NMR.7 Studies on these
The Ac-(3S) and (3R)-fluoroproline-OMe analogs also
exhibit clearly resolvable cis (E) and trans (Z) resonances—for
2 the resonances are separated by 0.8 ppm, while for 1 the
resonances are separated by nearly 2 ppm. This large differ-
ence in chemical shift allowed us to monitor the kinetics of
cis–trans isomerization using inversion-transfer NMR.12
Eyring analysis of 1 and 2 is shown in Fig. 1, and the
activation parameters derived from this plot are summarized
in Table 1. The calculated equilibrium constant from the
kinetic data is consistent with the observed ratio of integrated
signal intensities of cis and trans isomers—at 37 1C for 1, KZ/E
is 8.2 ꢀ 0.2 (90% trans, 10% cis) and for 2 KZ/E is 4.12 ꢀ 0.04
(80% trans, 20% cis). In addition, the rate constants for 2 in
water are similar to that reported for Ac-proline-OMe (37 1C)
in sodium phosphate buffer, pH 7.4.13a
The data in Table 1 indicate that the transition state
enthalpic differences between compounds 1 and 2 are small,
suggesting that water-mediated hydrogen bonding to the
prolyl peptide bond has not been perturbed. However, the
entropic barrier of 1 is approximately twofold greater than 2,
with values that are negative, which is unusual for proline and
other amides,13 and for proteins in general.14 One of the
reviewers has aptly pointed out that a similar trend is also
observed when comparing the Ac-(4R) and (4S)-fluoroproline-
OMe derivatives,13a,15 suggesting that a fluorine in the syn
configuration of the pyrrolidine ring may sterically hinder the
barrier to rotation around the C–N bond.
a Department of Chemistry, Wichita State University, Wichita,
KS 67260-0051, USA. E-mail: jim.bann@wichita.edu;
Fax: +1 316-978-3431; Tel: +1 316-978-7373
b Department of Chemistry, Carroll College, 1601 N Benton Ave.,
Helena, MT 59625, USA
w Electronic supplementary information (ESI) available: 19F-NMR
spectra of 1 and 2 at 35 1C in 90% H2O/10% D2O, synthesis of
(3S)-fluoro-L-proline and N-acetyl-(3S)-3-fluoro-L-proline methyl
ester, and determination of the kinetic and thermodynamic
parameters. See DOI: 10.1039/b821952d
Raines and co-workers first showed that synthesis of
a
collagen-like peptide with the sequence H-(Pro-(4R)-
fluoroproline-Gly)10-OH resulted in a triple-helical collagen
with greatly enhanced thermal stability, which was attributed
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3366 | Chem. Commun., 2009, 3366–3368

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displays similar kinetic isomerization parameters as both
natural and 3-hydroxy-substituted prolines.17 The study
presented here indicates that biosynthetic incorporation of
(3S)-fluoroproline into proteins would have little impact on
the natural population of cis and trans isomers. Indeed, the
biosynthetic incorporation of (3S)-fluoroproline, which was
reported recently in an elegant study by Conticello and
co-workers,18 and more recently into the protein ribonuclease
T1 (Carl Frieden, personal communication), should display
similar kinetics of cis–trans isomerization as that of the
wild-type protein, and if there are effects on the structure,
activity or folding kinetics, it will be most likely due to other
mechanisms.10
Fig. 1 Eyring analysis of the temperature dependence of isomeriza-
tion for 1 (closed symbols) and 2 (open symbols). Data were analyzed
according to ref. 15. The symbols (K) (J) correspond to trans (Z) to
cis (E) isomerization, whereas the symbols, (’) (&) correspond to cis
to trans isomerization. Linear least squares fits of the data (-) are
shown.
We thank Dr Takashi Mimura and Dr Akio Ozaki
(Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan) for the
generous gift of cis-3-hydroxy-L-proline, which was used as a
precursor for the synthesis of (3S)-fluoroproline. We also
thank Dr Larry Brethorst (Washington University in
St. Louis) for help with Bayes Analysis. J.G.B was supported
by an ACS PRF grant (PRF348233-G4) and an NIH
COBRE-PSF award to the University of Kansas, and
C.A.T. was supported by an RSEC (Research Sites For
Educators) postdoctoral fellowship award.
Table 1 Eyring parameters for compounds 1 and 2
a
Ea
DHza
DSza
kb/sꢂ1
1
2
E to Z
Z to E
E to Z
Z to E
81.3 (2.1)c
73.5 (1.1)
85.9 (2.7)
80.5 (2.9)
78.8 (2.0)
70.9 (1.1)
83.4 (2.6)
77.9 (2.8)
ꢂ20.1 (0.7)
ꢂ28.7 (0.6)
ꢂ10.8 (0.5)
ꢂ16.5 (0.8)
0.028 (0.002)
0.229 (0.008)
0.016 (0.001)
0.065 (0.005)
Notes and references
a
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which is in J molꢂ1 Kꢂ1
parentheses.
.
Data recorded at 37 1C. Error is in
b
c
to a stereoelectronic inductive effect of the fluorine.16 The
inductive effect changed the kinetics and equilibrium values of
cis and trans isomers of Ac-Pro-OMe, from B80% trans, 20%
cis to 90% trans, 10% cis. Independently, Renner and
co-workers showed for the first time the ability to bio-
synthetically incorporate fluoroproline analogs into barstar
C40A/C82A/P27A, which has only one cis proline (Pro 48), in
Escherichia coli. Incorporation of (4R)-fluoroproline
decreased the stability of the protein, whereas incorporation
of (4S)-fluoroproline increased the thermal stability, since
(4S)-fluoroproline favors the cis isomer.15 Incorporation of
the difluoro analog (4,4-F2), which exhibits a cis–trans isomer
ratio similar to proline, leads to a protein in which the stability
was unchanged.
5 (a) S. K. Sarkar, P. E. Young, C. E. Sullivan and D. A. Torchia,
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6 Synthesis of (3S)-fluoroproline was carried out using as a precursor
cis-3-hydroxy-L-proline derived from bacteria: H. Mori,
T. Shibasaki, K. Yano and A. Ozaki, J. Bacteriol., 1997, 179,
5677. Conversion of either cis or trans-3-hydroxy-L-proline into
1 and 2 was done in a manner similar to ref. 16, with the
exception that the conversion of cis-3-fluoro-L-proline methyl
ester to the N-acetyl derivative was done by adding 1 molar
equivalent of triethylamine in chloroform to the fluoroproline,
followed by addition of excess acetic anhydride (see ESIw for
synthesis).
7 NMR experiments were carried out on a Varian INOVA400
spectrometer with a tunable inverse detection probe. Samples were
at a concentration of B0.2 mg mlꢂ1 in 90% H2O/10% D2O.
Temperature was corrected using the change in chemical shift of
methanol methyl and hydroxyl resonances. The data at 35 1C
(ESIw) were referenced to neat TFA which was present in a coaxial
insert in the NMR tube.
However, because of the overlap of the signals from the cis
and trans isomers for the 4-fluoroproline derivatives,
obtaining isomerization rates or equilibrium values from
simple 1-D 19F-NMR experiments could not be achieved,
and more complicated two-dimensional 19F-NMR methods
were required.15 Based on our study using the small model
compounds 1 and 2, the cis and trans isomers are easily
distinguishable, and may provide a useful alternative for
measuring the ratio of cis and trans isomers in the unfolded
state and real-time kinetics of cis–trans proline isomerization
by 19F-NMR.
Raines and co-workers have shown that hydroxylation at
the 3-position of Ac-(3S)-hydroxyproline-OMe has little
influence on the kinetics of cis–trans isomerization, and thus
it is perhaps not surprising that Ac-(3S)-fluoroproline-OMe
8 (a) S. D. Hoeltzli and C. Frieden, Proc. Natl. Acad. Sci. U. S. A.,
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a
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18 W. Kim, K. I. Hardcastle and V. P. Conticello, Angew. Chem., Int.
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12 Inversion transfer experiments were carried out essentially as
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ꢁc
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3368 | Chem. Commun., 2009, 3366–3368