Methyl Side-Chain Dynamics in Proteins Using Selective Enrichment with a Single Isotopomer

Article abstract of DOI:10.1021/ja0368608

¹³C relaxation studies on side-chain methyl groups in proteins typically involve measurements on ¹³CHD₂ isotopomers, where the ¹³C relaxation mechanism is particularly straightforward in the presence of a single proton. While such isotopomers can be obtained in proteins overexpressed in bacteria by use of ¹³C enriched and fractionally deuterated media, invariably all possible ²H isotopomers are obtained. This results in a loss of both resolution and sensitivity, which becomes particularly severe for larger proteins. We describe an approach that overcomes this problem by chemical synthesis of amino acids containing a pure ¹³CHD₂ isotopomer. We illustrate the benefits of this approach in ¹³C side-chain relaxation measurements on the mouse major urinary protein selectively enriched with [γ ¹,γ²-¹³C₂,α,β ,γ¹,γ¹,γ²,γ ²-²H₆] valine. Relaxation measurements in the absence and presence of pyrazine-derived ligands suggest that valine side-chain dynamics do not contribute significantly to binding entropy.

Full text of DOI:10.1021/ja0368608

Published on Web 11/26/2003
Methyl Side-Chain Dynamics in Proteins Using Selective
Enrichment with a Single Isotopomer
†
†
†
†
Michael M. Chaykovski, Lynnette C. Bae, Minn-Chang Cheng, Jenny H. Murray,
†
†
‡
Kenneth E. Tortolani, Rui Zhang, Kothandaraman Seshadri,
John H. B. C. Findlay, Shih-Yang Hsieh, Arnout P. Kalverda,
‡
‡
‡
,
‡
†
Steve W. Homans,* and Jonathan Miles Brown
Contribution from the Astbury Centre for Structural Molecular Biology, School of Biochemistry
and Molecular Biology, UniVersity of Leeds, LS2 9JT U.K., and ProSpect Pharma Inc.,
6
480D Dobbin Road, Columbia, Maryland 21045
Received June 24, 2003; E-mail: s.w.homans@leeds.ac.uk
Abstract: 3C relaxation studies on side-chain methyl groups in proteins typically involve measurements
1
1
3
isotopomers, where the 13C relaxation mechanism is particularly straightforward in the presence
on CHD
of a single proton. While such isotopomers can be obtained in proteins overexpressed in bacteria by use
2
13
2
of C enriched and fractionally deuterated media, invariably all possible H isotopomers are obtained.
This results in a loss of both resolution and sensitivity, which becomes particularly severe for larger proteins.
We describe an approach that overcomes this problem by chemical synthesis of amino acids containing a
13
13
pure CHD
2
isotopomer. We illustrate the benefits of this approach in C side-chain relaxation
1
2
13
1
1
2
2 2
measurements on the mouse major urinary protein selectively enriched with [γ ,γ - C
2 6
,R,â,γ ,γ ,γ ,γ - H ]
valine. Relaxation measurements in the absence and presence of pyrazine-derived ligands suggest that
valine side-chain dynamics do not contribute significantly to binding entropy.
Introduction
While the entropic component to binding is multifaceted,
involving degrees of freedom of both the ligand, protein, and
solvent, NMR relaxation measurements can in principle provide
quantitative per residue information on the entropic contribution
Molecular recognition is fundamental to all biological pro-
cesses. However, despite enormous advances in structural
biology over recent decades, our understanding of the principles
that govern specificity and affinity in biomolecular interactions
is very limited. Simple thermodynamic arguments show that
the free energy of binding of two molecules is governed not
only by the enthalpy (loosely, structure) of the interacting
species, but also by the entropy of the system, which is in turn
related to dynamics. Consequently, there is currently much
interest in obtaining a measure of the elusive entropic component
to binding free energy using a variety of biophysical methods,
11-20
from each of these.
on the entropic contribution to binding of side-chain methyl
In particular, recent studies have focused
1
8,21-27
13
groups in proteins.
These studies have utilized either C
2
13
13
or H relaxation measurements in CHD2 or CH2D isoto-
(
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(
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(
(
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16) Bracken, C.; Carr, P. A.; Cavanagh, J.; Palmer, A. G. J. Mol. Biol. 1999,
†
ProSpect Pharma, Inc.
University of Leeds.
285, 2133-2146.
‡
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1
0539.
(
(
(
(
(
(
(
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3
2817.
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Soc. 1995, 117, 11536-11544.
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4
3-59.
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7
(24) Lee, A. L.; Flynn, P. F.; Wand, A. J. J. Am. Chem. Soc. 1999, 121, 2891-
6
2902.
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10) Schafer, H.; Daura, X.; Mark, A. E.; van Gunsteren, W. F. Proteins: Struct.,
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10.1021/ja0368608 CCC: $25.00 © 2003 American Chemical Society
J. AM. CHEM. SOC. 2003, 125, 15767-15771
9
15767

A R T I C L E S
Chaykovski et al.
Scheme 1. Synthesis of Isotopically Enriched Valine (6)
pomers, respectively. As discussed in detail by Muhandiram et
21
2
al., there are compelling reasons to choose H relaxation
13
measurements where possible. However, C relaxation mea-
surements are considerably more sensitive24 and may represent
the only viable approach in larger proteins.26 Indeed, during
studies on the mouse major urinary protein (MUP) as a model
system for studying the thermodynamics of ligand-protein
interactions, we have experienced considerable difficulty in the
2
measurement of accurate H relaxation rates for valine methyl
1
3
2
groups in methyl C, 50% H enriched protein due to the
combined effects of resonance overlap and relatively poor
sensitivity, despite the modest size of this protein (∼19 kDa).
The low sensitivity derives in part from the fact that only the
13
2
CH2D is detected in H relaxation measurements, whereas the
2
sample contains all possible H isotopomers, even with opti-
mized labeling schemes.28 Consequently, the effective protein
concentration is reduced considerably. Unfortunately, while
13
C
relaxation measurements on this system offered much higher
sensitivity, resonance overlap was very severe due in part to
the presence of contaminating resonances from 13CH3 isoto-
pomers in refocused INEPT experiments designed to selectively
detect resonances from 13CHD2 isotopomers. The former,
which resonate at a different chemical shift from the latter due
to the deuterium isotope effect, are incompletely suppressed in
refocused INEPT experiments as a consequence of the different
relaxation rates in proteins of the 3/2 and 1/2 spin manifolds of
the 13C spin in CH3 groups.
29
a
t
Reagents: (a) Mg/DCO2Me/Et2O. (b) HI/H2O. (c) (i) NaO Bu/CH3CN;
ii) Na/PhMe/MeOD. (d) 2 M HCl/MeOH.
(
This enantiomeric excess is adequate for biosynthetic purposes
since bacteria will only metabolize the “correct” enantiomer.
Full synthetic details can be found in the Supporting Informa-
tion.
Expression and Purification of [γ ,γ - C2,r,â,γ ,γ ,γ ,γ -
H6] Valine Enriched Mouse Major Urinary Protein. Full
details of the expression and purification of MUP can be found
in the Supporting Information. Briefly, M15 cells transformed
with the vector PQE30 MUP were grown in 1 liter of medium
containing initially unlabeled amino acids and nutrients. When
cell density had reached an OD of 1.2, the cells were transferred
To overcome these resonance overlap difficulties, we describe
a chemical synthetic approach for the preparation of valine
1
2 13
1
1
2
2
1
3
1
2 13
containing a pure CHD2 isotopomer, i.e., [γ ,γ - C2,R,â,-
2
1
1
2
2 2
γ ,γ ,γ ,γ - H6] valine. By inclusion of this material in an
appropriate bacterial growth medium,30 protein can be prepared
which contains isotopically enriched methyl groups only from
valine residues. In the case of MUP, all 12 valine methyl groups
are resolved, enabling highly accurate 13C relaxation times to
be measured. A comparison with relaxation data obtained in
the presence of the ligands 2-methoxy-3-isopropylpyrazine and
1
2 13
1
1
2
2
to an equivalent medium but in which [γ ,γ - C2,R,â,γ ,γ ,γ ,γ -
H6] valine was substituted for the unlabeled material. The
2
2
-methoxy-3-isobutylpyrazine suggests that valine side chains
concentrations of the relevant amino acids in this medium were
chosen to minimize isotopic scrambling which otherwise occurs
if glucose is used as the supplementary carbon source (unpub-
lished observations). After 30 min, protein expression was
induced by addition of IPTG to a final concentration of 0.1
mmol. After 6 h, the cells the culture was centrifuged at 4000
rpm for 20 min in a Sorvall RC-3B centrifuge. The cells were
broken by passing them through a French press, and the broken
cells were spun at 15000g for 20 min in Oakridge tubes. MUP
was purified from the supernatant by use of Ni-NTA im-
mobilized metal affinity column chromatography and further
purified by Sephadex G-25 size-exclusion chromatography and
Resource Q anion exchange chromatography. The protein was
dialyzed exhaustively at 4 °C versus 50 mM sodium phosphate
pH ) 7.0. The total yield was 90 mg. Throughout this work
do not contribute significantly to the entropy of binding.
Synthesis of [γ ,γ - C2,r,â,γ ,γ ,γ ,γ - H6] Valine (6). The
valine isotopomer (6, Scheme 1) was synthesized using a (S)-
1
2 13
1
1
2
2 2
2
-[N-(N′-benzylprolyl)amino]benzophenone (BPB) nickel(II)
31,32
complex as a chiral auxiliary.
The isotopically enriched
isopropyl side chain was installed using the appropriate second-
2
13
ary iodide (3), which was made from [ H2, C] methyl iodide
2
(
1) and methyl [ H] formate via a Grignard reaction, thus giving
secondary alcohol 2 which was subsequently iodinated with
hydroiodic acid. The valine R-hydrogen atom was exchanged
for deuterium under basic conditions prior to acidic cleavage
from the auxiliary which could be recovered in high yield.
Valine (6) was produced in an overall yield of 6% and 85% ee.
(
(
(
28) Ishima, R.; Louis, J. M.; Torchia, D. A. J. Biomol. NMR 2001, 21, 167-
71.
29) Ishima, R.; Louis, J. M.; Torchia, D. A. J. Am. Chem. Soc. 2000, 122,
33
the residue numbering of Abbate et al. was used.
1
13
2
Expression and Purification of Methyl- C, H Enriched
5
48-548.
13
2
30) Giesen, A. W.; Bae, L. C.; Barrett, C. L.; Chyba, J. A.; Chaykovsky, M.
M.; Cheng, M. C.; Murray, J. H.; Oliver, E. J.; Sullivan, S. M.; Brown, J.
M.; Dahlquist, F. W.; Homans, S. W. J. Biomol. NMR 2001, 19, 255-
Mouse Major Urinary Protein. Methyl- C, 50% H enriched
mouse major urinary protein was prepared as described in the
previous section, substituting 99.9% D2O medium containing
2
60.
(31) Belokon, Y. N.; Tararov, V. I.; Maleev, V. I.; Savel’eva, T. F.; Ryzhov,
M. G. Tetrahedron: Asymmetry 1998, 9, 4249-4252.
13
34
protonated 3- C-pyruvate, as described.
(
32) Belokon, Y. N.; Bakhmutov, V. I.; Chernoglazova, N. I.; Kochetkov, K.
A.; Vitt, S. V.; Garbalinskaya, N. S.; Belikov, V. M. J. Chem. Soc., Perkin
Trans. 1 1988, 2, 305-312.
(33) Abbate, F.; Franzoni, L.; Lohr, F.; Lucke, C.; Ferrari, E.; Sorbi, R. T.;
Ruterjans, H.; Spinsi, A. J. Biomol. NMR 1999, 15, 187-188.
15768 J. AM. CHEM. SOC.
9
VOL. 125, NO. 51, 2003

Side-Chain Dynamics in MUP
A R T I C L E S
1
3
1
2
1
2
C NMR Relaxation Measurements. Samples of [γ ,γ -
In contrast, an equivalent spectrum recorded on [γ ,γ -
1
3
1
1
2
2 2
13
1
1
2
2 2
C2,R,â,γ ,γ ,γ ,γ - H6] valine enriched MUP both alone and
C2,R,â,γ ,γ ,γ ,γ - H6] valine enriched MUP (Figure 1b) is
complexed with 2-methoxy-3-isopropylpyrazine and 2-methoxy-
essentially free from resonance overlap, and all 12 valine methyl
3
7
-isobutylpyrazine were prepared from a single sample at pH
.0 and a protein concentration of 1 mM. A further sample of
groups can be observed and assigned from the work of Abbate
3
3
et al. MUP exhibits significant promiscuity in binding small
hydrophobic ligands, and we have chosen to investigate the
thermodynamics of binding of 2-methoxy-3-isopropylpyrazine
and 2-methoxy-3-isobutylpyrazine on the basis of their global
thermodynamics of binding from isothermal titration calorimetry
experiments (data not shown). These ligands bind to MUP with
Kd’s of ∼1800 nM and ∼300 nM, respectively, at 308 K. Figure
1
3
2
methyl- C, H enriched MUP was prepared at pH 7.0 and a
protein concentration of 0.5 mM. Longitudinal (R1) and
transverse (R2) 13C relaxation rates were determined essentially
as described by Ishima et al.25 All spectra were recorded at a
proton frequency of 600 MHz and a probe temperature of 35
°
C. R1 rates were determined with relaxation delay times of
6, 64, 128, 240, 400, 720, 1120, 1600, 2240, and 2880 ms. R2
rates were determined with relaxation delay times of 16, 32,
8, 64, 96, 128, 160, 192, 240, and 288 ms and an effective
1
3
1
1
1c shows C, H HSQC spectra of complexes of these ligands
1
2 13
1
1
2
2 2
with [γ ,γ - C2,R,â,γ ,γ ,γ ,γ - H6] valine enriched MUP.
4
Significant shift perturbations are observed only for the cor-
γ1
γ2
field strength of 2 kHz. Relaxation data were fit to a single
exponential decay function I ) Io exp(-tR), where Io is the
initial resonance intensity, t is the relaxation delay time, and R
is the relaxation rate (R1 or R2). Isotropic rotational tumbling
was assumed on the basis of previous backbone N relaxation
measurements for this protein.17 Thus, relaxation data were fitted
to the Lipari-Szabo model-free spectral density3 of the form:
relations from Val 82 C and C , as anticipated since Val 82
3
7
is located within the binding pocket of MUP. Stereospecific
γ1
γ2
assignments of Val 82 C and C in these complexes were
15
confirmed by three-dimensional NOE measurements using N,
C enriched MUP and the relevant unlabeled ligands (data not
shown). In both complexes, all correlations are well-resolved,
in contrast to complexes with methyl- C, H enriched MUP
data not shown). Under these circumstances, measurement of
15
13
38
5
13
2
(
2
2
2
m
2
2 2
i
J(ω) ) S τ /(1 + ω τ ) + (1 - S )τ /(1 + ω τ ) (1)
m
i
13
C longitudinal and transverse relaxation rates (R1 and R2) can
where τi-1 ) τM-1 + τe-1, τM is the overall molecular tumbling
be undertaken in each sample at high sensitivity and with
minimal interference due to resonance overlap. Typical relax-
ation curves are shown in Figure 2. Values of Saxis were
correlation time, and τe is the effective internal correlation time.
This equation is valid for fast internal motions, τe , τM, under
which conditions the order parameter for methyl CH dipolar
2
derived from R1 and R2 data for each methyl group using the
2
axis
2
2
axis
3
5
2
model-free spectral density function given in the General
Methods section in the Supporting Information and are listed
in Table 1. From these data, it is immediately apparent that
relaxation is given by S ) S [P2(cos θH)] . S is the order
parameter of the methyl rotation axis and θH is the angle made
by this axis and the CH bond vector. The MODELFREE
program was used for all these calculations,36 kindly supplied
by Prof. Art Palmer (Columbia University) and modified in-
2
there are only minor changes in Saxis for any of the six valine
residues in MUP upon binding of either 2-methoxy-3-isopro-
pylpyrazine or 2-methoxy-3-isobutylpyrazine, most of which
13
house to include relaxation of C by the attached deuterons in
2
13
CHD2 isotopomers. A global rotational correlation time of 8.57
are within experimental error. An exception is Saxis for Val-70
γ
ns was used for these calculations, according to the previously
reported value.17 Errors in S values were estimated by Monte
Carlo simulations as implemented in the MODELFREE pack-
age. In all cases, good fits were obtained to the above spectral
density function without a contribution from exchange-broaden-
ing Rex. Although valine residues are perdeuterated at nonmethyl
positions, the protein is otherwise protonated, and dipolar
relaxation due to these “external” protons is not negligible for
C 2, which appears to increase dramatically on binding 2-meth-
2
oxy-3-isobutylpyrazine. However, this is an anomalous result:
γ
γ
Val-70 C 1 and C 2 possess similar shifts in both complexes
and differ by ∼3 Hz in the 2-methoxy-3-isobutylpyrazine
γ
γ
complex, under which conditions C 1 and C 2 will be strongly
coupled via the two bond homonuclear scalar coupling, which
is of similar magnitude. This results in a poor fit of R2 relaxation
γ
2
data for Val-70 C 2 (ø ) 49.7).
1
3
2
C relaxation. Thus, by analogy with the work of Ishima et
It is notable that measured S values for the two methyl
axis
al.,26 the contribution of these external protons to C R2 values
was estimated at 25% from the X-ray coordinates of MUP (R.
Bingham and S. E. V. Phillips, unpublished data). Consequently,
13
groups of certain valine residues are not identical within
experimental error. At first sight, this is inconsistent with the
requirement for their mobilities to be essentially the same, since
1
3
measured C R2 rates were multiplied by 0.75 to account for
they form part of the same isopropyl group. However, this is
2
axis
these external dipolar relaxation processes.
not necessarily reflected in equivalent S
previously,
values: as noted
2
2,39,40
order parameters of methyl groups from the
Results and Discussion
same isopropyl group may differ if the effective averaging axis
for this group makes different angles with the methyl 3-fold
13
1
Figure 1a shows a region from the C, H HSQC spectrum
of methyl 13C, 2H enriched mouse major urinary protein,
axes.
γ1
γ1
γ2
γ2
2
axis
containing valine C -H and C -H correlations. Significant
overlap is present in the spectrum, arising from the combined
effects of interference from resonances from 13CH3 isotopomers,
together with methyl resonances derived from residues other
than valine.
The constant values of S on complex formation for the
methyl groups of Val-82 is unexpected, since this residue is
(37) Timm, D. E.; Baker, L. J.; Mueller, H.; Zidek, L.; Novotny, M. V. Protein
Sci. 2001, 10, 997-1004.
(
38) Zwahlen, C.; Legault, P.; Vincent, S. J. F.; Greenblatt, J.; Konrat, R.; Kay,
L. E. J. Am. Chem. Soc. 1997, 119, 6711-6721.
(
34) Lee, A. L.; Urbauer, J. L.; Wand, A. J. J. Biomol. NMR 1997, 9, 437-
(39) Lemaster, D. M.; Kushlan, D. M. J. Am. Chem. Soc. 1996, 118, 9255-
4
40.
9264.
(
35) Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546-4559.
(40) Mittermaier, A.; Kay, L. E.; FormanKay, J. D. J. Biomol. NMR 1999, 13,
181-185.
(
36) Mandel, A. M.; Akke, M.; Palmer, A. G. Mol. Biol. 1995, 246, 144-163.
J. AM. CHEM. SOC.
9
VOL. 125, NO. 51, 2003 15769

A R T I C L E S
Chaykovski et al.
Figure 1. (a) Region of the 13C, H HSQC spectrum of methyl- C, 50% H enriched MUP showing valine methyl correlations. (b) Equivalent spectrum
1
13
2
1
2 13
1
1
2
2 2
13
1
1
2 13
1
1
2
2
of MUP selectively enriched with [γ ,γ - C2,R,â,γ ,γ ,γ ,γ - H6] valine. (c) Overlay of regions of the C, H HSQC spectra of [γ ,γ - C2,R,â,γ ,γ ,γ ,γ -
H6] valine enriched MUP in complex with 2-methoxy-3-isopropylpyrazine (black correlations) and 2-methoxy-3-isobutylpyrazine (gray correlations).
2
2
Table 1. S Values for Valine Methyl Groups Derived from 600
MHz 13C T1 and T2 Measurements on the Major Urinary Protein
1
2 13
1
1
2
2 2
Selectively Enriched with [γ ,γ - C2,R,â,γ ,γ ,γ ,γ - H6] Valine
ligand
2-methoxy-3-
2-methoxy-3-
none
isopropyl-pyrazine
isobutyl-pyrazine
2
2
2
residue
S
a
S
S
axis
axis
axis
γ1
γ2
γ1
γ2
γ1
γ2
γ1
γ2
γ1
γ2
γ1
γ2
Val-12 C
Val-12 C
Val-47 C
Val-47 C
Val-53 C
Val-53 C
Val-59 C
Val-59 C
Val-70 C
Val-70 C
Val-82 C
Val-82 C
0.88
0.77
0.91
0.86
0.74
0.90
0.73
0.98
0.78
0.77
0.53
0.46
0.90
0.69
0.84
0.90
0.80
0.85
0.68
0.86
0.85
0.77
0.50
0.48
0.86
0.66
0.86
0.88
0.78
0.86
0.63
0.90
0.81
(1.03)
0.53
0.53
γ1
1
2
Figure 2. Typical R2 relaxation curves for Val-12 C obtained from [γ ,γ -
13
1
1
2
2 2
C2,R,â,γ ,γ ,γ ,γ - H6] valine enriched MUP. Solid line: free protein;
dashed line: 2-methoxy-3-isopropylpyrazine complex; dash-dotted line:
-methoxy-3-isobutylpyrazine complex. For clarity, only the data points
corresponding to the free protein are shown. Error bars are smaller than
the symbols used for these data points.
a
2
axis
The average error in the reported S values is 0.045 ( 0.009. The
bracketed value is anomalous due to strong coupling between Val-70 C
γ2
2
γ1
and C .
ation pathways offered by ligand protons are offset by an equal
and opposite effective contribution due to increased mobility
of Val-82 side-chain atoms. In contrast, remaining valine methyl
groups are distal to the binding pocket, and ligand protons will
make a negligible contribution to relaxation in these cases, since
no significant structural changes in MUP are observed in the
crystal structures of these complexes (R. Bingham and S. E. V.
located within the binding pocket and is proximal to the bound
ligands (R. Bingham and S. E. V. Phillips, unpublished data).
Intuitively, the relevant Saxis values might be expected to
increase due to decreased mobility within the binding pocket
in the complexes. Since each ligand is protonated, there exists
the formal counterintuitive interpretation that additional relax-
2
15770 J. AM. CHEM. SOC.
9
VOL. 125, NO. 51, 2003

Side-Chain Dynamics in MUP
A R T I C L E S
Phillips, unpublished data). In the general case where significant
structural changes are observed, clearly it would be appropriate
demonstrated that incorporation into overexpressed MUP en-
1
3
13
ables measurement of C R1 and R2 relaxation times for
-
1
2 13
1
1
2
2 2
to incorporate [γ ,γ - C2,R,â,γ ,γ ,γ ,γ - H6] valine into a
perdeuterated protein, either by use of suitable auxotrophic
strains or by overexpression in media containing the relevant
CHD isotopomers that proved impossible in uniformly enriched
2
2
axis
material. Within experimental error, no changes in S can be
detected for each of the valine methyl groups both in the free
protein and in complexes with 2-methoxy-3-isopropylpyrazine
and 2-methoxy-3-isobutylpyrazine. This suggests that valine side
chains do not contribute significantly to the entropy of binding
2
H amino acids.
A number of researchers have developed approaches for the
derivation of thermodynamic parameters from the NMR-derived
11-13,15
generalized order parameter.
While all such approaches
13
on time scales detectable by C relaxation measurements. In
involve various assumptions such as motional independence of
bond vectors and are inherently limited in the motional time
scales that can be detected, they are proving to be very valuable
as a probe of binding thermodynamics. For example, studies
γ
the case of Val-70 C 2, R2 measurement was hampered in the
complex with 2-methoxy-3-isobutylpyrazine by strong coupling
γ
γ
between C 1 and C 2 due to near equivalence of chemical shifts.
Such complications can be overcome in a modified synthetic
strategy that involves selective enrichment of a single methyl
group. Alternatively, since the method is highly sensitive, lower
2
using H relaxation methods on calcium-saturated calmodulin
in complex with a peptide model of the calmodulin-binding
2
axis
domain have highlighted significant changes in methyl S
13
(10-20%) levels of C enrichment could be utilized. Develop-
values that cannot be predicted in a rational manner: changes
ment of related strategies for the synthesis of the analogous
methyl isotopomers of other methyl-containing amino acids is
in progress.
2
to S values were found at locations both proximal and distal
axis
2
+
18
from the Ca binding site. In contrast, in the present study
we found no changes in valine methyl Saxis values on ligand
2
binding within experimental error, suggesting that in the present
case the entropic contribution to binding is small. Clearly, it is
not possible to exclude entropic contributions derived from
motions on time scales outside those detectable by 13C relaxation
measurements. However, the dominant entropic contribution to
binding from MUP dynamics may derive from the backbone,
as has been suggested for the complex between MUP and a
small-molecule ligand unrelated to the pyrazine derivatives
Acknowledgment. Financial support is acknowledged from
the BBSRC, Grant No. 24/B12992 and the Wellcome Trust,
Grant No. 062164. We thank Dr. Richard Bingham and Prof.
S. E. V. Phillips (University of Leeds) for providing crystal
structure coordinates of MUP complexes prior to publication.
Supporting Information Available: Details of the synthesis
1
2 13
1
1
2
2 2
of [γ ,γ - C2,R,â,γ ,γ ,γ ,γ - H6] valine and the expression and
1
7
1
2 13
1
1
2
2 2
described here.
purification of [γ ,γ - C2,R,â,γ ,γ ,γ ,γ - H6] valine enriched
MUP (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
Conclusions
We have presented a chemical synthetic approach for the
1
2 13
1
1
2
2 2
preparation of [γ ,γ - C2,R,â,γ ,γ ,γ ,γ - H6] valine and have
JA0368608
J. AM. CHEM. SOC.
9
VOL. 125, NO. 51, 2003 15771