Structure and folding of glucagon-like peptide-1-(7

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2001; 39: 477–483
Structure and folding of glucagon-like
peptide-1-(7–36)-amide in aqueous trifluoroethanol
studied by NMR spectroscopy
Xiaoqing Chang,1† Danielle Keller,1 Søren Bjørn2 and Jens J. Led1∗
1
2
Department of Chemistry, University of Copenhagen, The H. C. Ørsted Institute, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark
Health Care Discovery, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsværd, Denmark
Received 21 February 2001; Revised 12 April 2001; Accepted 20 April 2001
The conformational changes of free, monomeric glucagon-like peptide-1-(7–36)-amide (GLP-1) in
aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR
spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in
water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes
were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from
292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained
energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around
G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil
(in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that
is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The
exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more
stable than the N-terminal part. Copyright  2001 John Wiley & Sons, Ltd.
KEYWORDS: NMR; 1 H NMR; 13 C NMR; GLP-1; solution structure; folding; trifluoroethanol
INTRODUCTION
Glucagon-like peptide-1-(7–36)-amide (GLP-1) is a 30 amino
acid peptide with the sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 . It is an incretin hormone that
can potentiate glucose-induced insulin secretion, stimulate insulin biosynthesis and inhibit glucagon secretion.1
Accordingly, it is a potential drug for the treatment of
non-insulin-dependent diabetes mellitus (NIDDM or type II
diabetes2 ). However, GLP-1 suffers from physical instability.
Thus it has been found that the conformation, aggregation
and solubility of GLP-1 depend on the purification procedure
and the in-process storage and handling.3,4 Efforts have been
made to characterize the structural properties of GLP-1 in
Ł Correspondence to: J. J. Led, Department of Chemistry, University
of Copenhagen, The H. C. Ørsted Institute, Universitetsparken 5,
DK-2100 Copenhagen Ø, Denmark. E-mail: [email protected]
†Present address: Ontario Cancer Institute, University of Toronto,
610 University Avenue, Toronto, Ontario, Canada M5G 2M9.
Contract/grant sponsor: Danish Technical Research Council;
Contract/grant number: 16-5028-1; 9601137.
Contract/grant sponsor: Danish Natural Science Research Council;
Contract/grant number: 9400351; 9502759; 9601648; 9801801.
Contract/grant sponsor: Ministry of Industry; Contract/grant
number: 85886.
Contract/grant sponsor: Julie Damm’s Studiefond.
Contract/grant sponsor: Direktør Ib Henriksens Fond.
Contract/grant sponsor: Carlsbergfondet.
Contract/grant sponsor: Novo Nordisk Fonden.
DOI: 10.1002/mrc.880
greater detail,4 including its aggregation behaviour in solution, but so far only limited structural information about
GLP-1 has been reported. However, the ˛-helix seems to be
an important structural motif of GLP-1 in solution. Thus it
was found that GLP-1 can form oligomers with a high helical
content,3 – 5 and that it is mainly helical in membrane-like
environments (dodecylphosphocholine micelle6 ). However,
ˇ-sheet formation also seems significant and can lead to less
soluble GLP-1 aggregates3 .
In order to obtain a more detailed insight into the
structure and folding propensity of GLP-1 in solution, we
studied the conformation of GLP-1 in mixtures of water
and 2,2,2-trifluoroethanol (TFE) using NMR spectroscopy.
Organic solvents such as TFE are known to promote helical
conformations in proteins and peptides while impeding
the specific tertiary interactions of native proteins. This
holds especially at high concentrations of TFE, possibly
because of its ability to enhance internal hydrogen bonding
in polypeptides and to lessen hydrophobic interactions
between residues distant in the amino acid sequence.7
Sonnichsen
et al.8 noted that, in general, TFE stabilizes
¨
helices in regions that have a high propensity for helix
formation. Furthermore, Hamada et al.9 suggested that the
intrinsic helical propensity of a peptide fragment elicited
by the addition of TFE is not necessarily related to the
secondary structure in the native state. It has also been
reported that TFE can stabilize the folded state,10 possibly by
Copyright  2001 John Wiley & Sons, Ltd.
478
X. Chang et al.
explicitly strengthening intramolecular hydrogen bonding.11
Alternatively, Kentsis and Sosnick12 proposed that TFE exerts
its helix-stabilizing effects by a mechanism that promotes
desolvation of the polypeptide backbone, rather than by
strengthening the hydrogen bonds.
On the basis of these helix-promoting and -stabilizing
effects of TFE, and the observed ˛-helix propensity of GLP-1,
a study of the structure and folding of GLP-1 in water–TFE
mixtures is interesting in order to obtain a more detailed
insight into the structure and folding of GLP-1 in solution.
EXPERIMENTAL
Sample preparation
Lyophilized, recombinant GLP-1 peptide was dissolved in
H2 O (with 10% D2 O) or in 99.96% D2 O. The concentration
of GLP-1 in the NMR samples was 1.4 mM. The pH was 2.5
(meter reading) in order to minimize the exchange rates of
the amide protons of the backbone.13 The TFE concentrations
varied from 0 to 50%. Perdeuterated TFE (TFE-d3 ) was used
in all cases.
NMR spectroscopy
The NMR spectra were recorded on Varian Unity Inova
500, 750 and 800 MHz spectrometers and on a Bruker
AM500 spectrometer. The three Varian spectrometers were
equipped with gradient probes. Standard NMR methods
based on 1 H DQF-COSY,14 TOCSY,15 NOESY,16 and 13 C
HSQC17 spectra were used for the sequential assignments
of the proton resonances of GLP-1 at different TFE concentrations and the assignment of the 13 C resonances at
35% TFE. All spectra were recorded at 300 K. Mixing times
of 30, 60 and 90 ms were used in the TOCSY experiments while 50, 100, 150 and 250 ms were used in the
NOESY experiments. The 1 H– 1 H correlated spectra were
recorded with a sweep width of 8000–10 000 Hz in both
dimensions. In the HSQC spectra the sweep width of the
carbon dimension was 30 000 Hz (at 800 MHz). All spectra
were recorded with 4096 data points in the t2 dimension
and the number of increments in the t1 dimension was
between 450 and 512. Qualitative discrimination between
slow- and fast-exchanging amide protons was achieved by
recording 11 h NOESY spectra immediately after dissolving
the sample in 99.96% D2 O with the desired TFE concentration, and defining the amide protons still giving rise to
cross peaks as slowly exchanging. Quantitative estimates
of the amide proton exchange rates were obtained from
the linewidth of the signals in the above NOESY spectrum, using a linear prediction-based method described
previously.18
Structure calculation of GLP-1
Interproton distance restraints were obtained from the
NOESY spectra with the mixing time m D 250 ms. The small
size of the GLP-1 peptide and the lower viscosity of the mixed
H2 O–TFE solvent as compared with pure water ensured that
spin diffusion plays only a minor role at this mixing time and
applied temperature (300 K). This conclusion is supported
by the observation that the cross peak intensities vary
Copyright  2001 John Wiley & Sons, Ltd.
approximately linearly in the applied range of mixing times
(50–250 ms), and extrapolate to zero at m D 0. The intensities
of the NOE cross peaks were determined quantitatively by a
combination of linear prediction analysis and least-squares
estimation19,20 and classified as strong, medium and weak by
comparison with the intensity of the well-resolved 1 Hυ – 1 Hε
˚ The corresponding
cross peak of Tyr13 (rHυ Hε D 2.49 A).
˚
upper bound distance restraints were 2.85, 3.56 and 5.00 A,
respectively. Torsion angle restraints were obtained from
secondary chemical shifts of the 1 H˛ , 13 C˛ , and 13 Cˇ nuclei
with the program TALOS.21 The average values of the 10 best
database matches were used as dihedral angle restraints and
the corresponding standard deviations were used as upper
and lower bounds.
The structures were calculated using X-PLOR 3.851.22
Initially a series of substructures were generated using the
DG-SUB-EMBED protocol. Hydrogen bonds in the helical
region were included only in this calculation. Subsequently
distance geometry simulated annealing calculations and
refinement by restrained energy minimization were carried
out as described previously.23 The force constants of the
˚ 2 and
NOE and dihedral angle terms were 50 kcal mol1 A
1
2
200 kcal mol rad , respectively.
RESULTS AND DISCUSSION
Initial folding of GLP-1 in TFE
Essential complete sequential assignments were accomplished for the proton resonances of GLP-1 at different TFE
concentrations. Figure 1(a), (b) and (c) show the amide proton regions of the NOESY spectra of GLP-1 in pure water
and in water with 20 and 35% TFE, respectively. As shown
in Fig. 1, there is a clear correlation between the NOE pattern
and the TFE concentration. In pure water solution only a
small number of NOEs are observed, indicating a random
coil conformation, whereas a large number of new NOEs
appear as the amount of TFE is increased, indicating the
formation of an increasing amount of secondary structure.
No further changes were observed in the spectra at TFE
concentrations higher than 35%. The 13 C˛ and 13 Cˇ nuclei
were assigned for GLP-1 in 35% TFE, using a 1 H– 13 C HSQC
spectrum obtained with 13 C in natural abundance.
The sequential and medium-range NOEs observed for
the GLP-1 in pure water and in the presence of 20 and
35% TFE are summarized in Fig. 2(a)–(c), together with the
1 ˛
H and 13 C˛ chemical shift indices24 (CSIs), and the slowly
exchanging amide protons. Primarily, the medium-range
NOEs in Fig. 2(c) indicate that a helical region ranging from
T7 to K28 is formed in 35% TFE, although the number of
NOEs connecting G16 to the rest of the helix is relatively
small. Second, the medium-range NOEs observed in 20%
TFE [Fig. 2(b)] show that the TFE-induced helix formation
starts at the C-terminal end of the peptide. Both of these
observations are supported by the 1 H˛ CSIs in Fig. 2(a)–(c).
It should be noted that, in general, the change of the
chemical shifts of the CH protons upon addition of TFE
is negligible,25 so that the CSIs observed here in the presence
of TFE reflect the formation of secondary structures in the
same way as in pure water. The helix formation is further
Magn. Reson. Chem. 2001; 39: 477–483
Structure and folding of GLP-1 in TFE
Figure 1. Amide proton region of the 1 H NOESY spectrum of GLP-1 in aqueous TFE (1.4 mM, pH 2.5, 300 K) with different H2 O/TFE
ratios (v/v), showing the correlation between the amide and aliphatic protons. (a) Pure water; (b) 20% TFE; (c) 35% TFE.
supported by the NH exchange rates. Thus, in pure water
all the amide protons are fast exchanging, in agreement
with the disordered structure of GLP-1 indicated by the
NOEs and CSIs. In contrast, 12 slowly exchanging amide
protons were observed in 35% TFE, all of which are located
within the helix and, predominantly, in the C-terminal
part.
A quantitative determination of the amide proton
exchange rates, kobs , of nine of the 12 slowly exchanging
amide protons (Table 1) indicates a relatively loose helix.
The rates were determined from the linewidths of the NH
correlated signals in a 2D NOESY spectrum in D2 O.18 For
S11, S12 and Y13 a reliable, quantitative determination of
the NH exchange rates was not possible because of severe
signal overlap. A comparison of the experimental and the
intrinsic exchange rates, kint , determined by the side-chains
Copyright  2001 John Wiley & Sons, Ltd.
of the adjacent amino acids,26 shows that the reduction of the
experimental exchange rates reflects a genuine protection
associated with ˛-helix formation. However, the size of
the calculated protection factors (Table 1) indicates that
the hydrogen bonding is relatively weak. Accordingly the
exchange rates are larger than 0.35 h1 (Table 1), resulting
in a complete exchange of all the NH protons within 12 h.
This was confirmed by the absence of all NH correlations in
a NOESY spectrum recorded 12 h after the dissolution of the
sample in D2 O.
More importantly, the NH exchange rates (Table 1) show
that the C-terminal end of the helical region is more stable
than the N-terminal end of the region. Thus seven of
the 12 residues of the C-terminal helical region are slow
exchanging whereas only five of the 11 residues in the
N-terminal helical region are slow exchanging [Fig. 2(c)].
Magn. Reson. Chem. 2001; 39: 477–483
479
480
X. Chang et al.
(a)
5
GLP-1:
dα N:
dNN:
dβ N:
dα N(i, i + 2):
dα N(i, i + 3):
dα N(i, i + 4):
dαβ (i, i + 3):
10
15
20
25
30
H A E G T F T S D V S S Y L E G Q A A K E F I A W L V K G R
CSI 1H:
(b)
GLP-1:
dα N:
dNN:
dβ N:
dα N(i, i + 2):
dα N(i, i + 3):
dα N(i, i + 4):
dαβ (i, i + 3):
H A E G T F T S D V S S Y L E G Q A A K E F I A W L V K G R
CSI 1H:
(c)
GLP-1:
Exchange:
dα N:
dNN:
dβN:
dα N(i, i + 2):
dα N(i, i + 3):
dα N(i, i + 4):
d αβ (i, i + 3):
H A E G T F T S D V S S Y L E G Q A A K E F I A W L V K G R
CSI 1H:
CSI 13C:
Figure 2. Summary of sequential and medium-range NOE connectivities, and schematic representation of the 1 H˛ and 13 C˛ CSIs of
GLP-1 in H2 O (1.4 mM, pH 2.5, 300 K) with different H2 O/TFE ratios (v/v). (a) Pure water; (b) 20% TFE; (c) 35% TFE. The observed
NOEs are indicated by bars connecting the two involved residues, and the intensities of the NOEs are indicated by the thickness of
the bars. The CSIs were calculated according to Wishart et al.24 Slowly exchanging backbone amide protons are indicated in (c) by
filled circles.
This difference in stability is further supported by the
observed protection factors (Table 1). A similar observation
of a more stable C-terminal region was made for GLP-16 and
for glucagon27 in the presence of a dodecylphosphocholine
micelle. In both cases the higher stability of the C-terminal
region was attributed to the interaction with the micelle.
However, the observation made here in the absence of a
micelle suggests that other factors must contribute to the
higher stability of the C-terminal helical region. One of
these factors could be the larger number of hydrophobic
Copyright  2001 John Wiley & Sons, Ltd.
residues in the C-terminal region, which increases the ˛-helix
propensity of the region. It should also be noted that the
correlation between the higher stability of the C-terminal
segment indicated by the NH exchange rates and the order
of appearance of the NOEs and CSIs with increasing TFE
concentration support the general view28,29 that the slowexchange core is the folding core, that is, the segment
of a protein that is the most rigid in the folded state is
also the segment that collapses early during the folding
process.
Magn. Reson. Chem. 2001; 39: 477–483
Structure and folding of GLP-1 in TFE
Table 1. Exchange rates for slowly exchanging amide protons in the GLP-1 monomer in D2 O with 35%
TFE
NH
Correlated to
 1
2
Leu L14
ˇ
,exch
(Hz)
 1 (Hz)
kobs (h1 )a
kint (h1 )b
Pc
2
CH2 (L14)
CH(L14)
121.0 š 12.0
110.0 š 10.0
29.0 š 1.8
16.0 š 1.7
0.62 š 0.08
0.63 š 0.07
2.92
2.92
4.6
4.6
Glu E15
˛
CH(E15)
115.0 š 16.0
19.7 š 1.1
0.64 š 0.11
7.40
11.6
Phe F22
ˇ
CH2 (F22)
108.0 š 10.0
16.2 š 1.6
0.62 š 0.07
8.62
13.9
Ile I23
˛
CH(I23)
CH(I23)
70.0 š 6.0
70.0 š 8.0
15.9 š 1.5
15.0 š 0.8
0.36 š 0.04
0.37 š 0.05
1.60
1.60
4.5
4.5
ˇ
Ala A24
˛
CH(A24)
100.0 š 8.0
32.0 š 2.0
0.46 š 0.06
6.20
13.5
Trp W25
˛
CH(W25)
68.5 š 5.0
16.3 š 1.1
0.35 š 0.03
7.23
20.7
Leu L26
˛
CH(L26)
68.0 š 9.0
10.7 š 1.7
0.39 š 0.06
2.27
5.8
Val V27
˛
CH(V27)
97.0 š 11.0
23.0 š 1.3
0.50 š 0.08
2.41
4.8
ˇ
CH(L26)
94.0 š 9.0
21.6 š 1.0
0.49 š 0.06
2.41
4.9
˛
CH(K28)
97.0 š 11.0
16.1 š 1.3
0.55 š 0.08
4.88
8.9
Lys K28
a
Experimental exchange rates at 300 K and pH 2.5 (meter reading) including 1 standard deviations. The
experimental rates were obtained from the linewidths of the NH cross peaks in a single NOESY spectrum
recorded immediately after dissolution of the protonated GLP-1 in D2 O.18 The individual amide protons and
is the linewidth in the F1 dimension of
their correlation partners are listed in the first two columns.  1
2
,exch
the cross peak below the diagonal and  1 is the linewidth in the F2 dimension of the symmetric cross peak
2
above the diagonal.
b
Intrinsic exchange rates in the random coil peptide calculated according to Bai et al.26
c
Protection factor P D kint /kobs .
Structure of GLP-1 in 35% TFE
Sixty structures were calculated from 280 NOEs, including
144 intra-residue, 84 sequential and 52 inter-residue sidechain NOEs, and 22 and 22 angle constraints, derived
from the secondary 1 H˛ , 13 C˛ and 13 Cˇ chemical shifts
using the program TALOS. Also 12 ˛-helix hydrogen bonds,
assigned on the basis of the slowly exchanging amide protons
and the NOE patterns observed for the GLP-1 monomer in
35% TFE, were included in the initial structure calculation
as described above. The 20 best structures with the lowest
total energies were chosen for further analysis. The best-fit
superposition of the backbone of the 20 structures is shown
in Fig. 3(a) and the structural statistics are given in Table 2.
˚ and no dihedral angle
No NOE violations larger than 0.2 A
violations larger than 5° were observed in any of the 20
structures. Table 2 indicates that the inclusion of the TALOS
derived dihedral angles in the structure calculation improves
the quality of the structure significantly. The correlation
between the and angles of the calculated structures and
the corresponding angles estimated with TALOS is shown
in Fig. 4.
The structures obtained show that GLP-1 in water with
35% TFE is predominantly helical, with a regular ˛-helix in
most of the region from T7 to K28. Only the first six and the
last two residues are disordered. However, the helix is less
well-defined in a region around G16. This is supported by
the chemical shift of the G16 1 H˛ that remains unchanged at
the random coil value at all TFE concentrations. In contrast,
the 13 C˛ resonance of G16 is shifted downfield, in agreement
with a helix conformation. A similar less well-defined region
Copyright  2001 John Wiley & Sons, Ltd.
(a)
N
C
(b)
N
C
(c)
N
C
Figure 3. Best-fit superposition of the 20 selected solution
structures of the GLP-1 monomer; only the C0 , C˛ , and N
backbone atoms are shown. (a) Residues 7–28 (the entire
helical segment); (b) residues 7–15 (N-terminal helical region);
(c) residues 18–28 (C-terminal helical segment).
Magn. Reson. Chem. 2001; 39: 477–483
481
482
X. Chang et al.
Table 2. Structural statistics for the GLP-1 monomer in H2 O
with 35% TFE
−45
−50
Number of constraints:
Total number of NOEs
Intra-residue NOEs
Sequential NOEs
Inter-residue NOEs
Dihedral angles
Hydrogen bonds
280
144
84
52
44
12
−55
−60
φ −65
−70
R.m.s.d. from NOE restraints and from the
idealized geometry used within X-PLOR:
˚
NOE (A)
Dihedral angles (° )
˚
Bond lengths (A)
°
Bond angles ( )
Improper dihedral angles (° )
0.012 š 0.003
1.06 š 0.38
0.0054 š 0.0002
0.44 š 0.04
0.43 š 0.04
−80
PROCHECK36 Ramachandran analysis:
Most favoured regions (%)
Additional allowed regions (%)
Generously allowed regions (%)
Disallowed regions (%)
89.0 (58)a
6.6 (33)a
2.6 (5)a
1.8 (4)a
−20
Average r.m.s.d. of the final ensemble:
˚
Backbone atoms (7–15) (A)
˚
Backbone atoms (18–28) (A)
˚
All backbone atoms (A)
˚
All heavy atoms (A)
0.49 (0.79)a
0.44 (0.99)a
2.55 (2.92)a
3.25 (3.94)a
−75
a
Numbers in parentheses correspond to structures calculated
without the TALOS-derived dihedral angle restraints.
around G16 was also suggested in the structure of the micellebound GLP-1.6 The two regular helical segments from T7 to
E15 [Fig. 3(b)] and from A18 to K28 [Fig. 3(c)] are both well
˚ for the backbone,
defined with r.m.s.d. of 0.49 and 0.44 A
respectively. Since the number of NOE constraints that
are observable for the side-chain atoms is relatively small,
as normally found for small single-stranded peptides, the
r.m.s.d. for the entire peptide is relatively high.
Overall, the GLP-1 structure obtained is similar to that
˚ resolution crystal
of micelle-bound GLP-16 and the 3 A
30
structure of glucagon (glucagon has a 50% sequence
homology with GLP-1), although the low resolution of the
last two structures prevents a detailed comparison. Two
features of the structure obtained here are worth noting.
First, the less well-defined structure around G16 indicates
an increased flexibility around this position. A similar flexible
region has been observed in other amphipathic helices31 – 33
and is presumably important for the function of the peptides.
In the case of the free GLP-1 in solution, the flexibility around
G16 undoubtedly contributes to the reported propensity of
the peptide to form aggregates with different ˛-helix and
ˇ-sheet conformations.3,4 In the case of the micelle-bound
GLP-1 it was suggested6 that the flexible region allows
the helix to assume a phase shift whereby the hydrophilic
residues will be located on one side of the strand and the
hydrophobic residues on the other side. This allows the helix
to bind to the membrane with a larger hydrophobic face. The
detailed structure including the flexible region around G16,
Copyright  2001 John Wiley & Sons, Ltd.
−85
10
15
20
25
Residue number
−15
−25
−30
ψ
−35
−40
−45
−50
−55
−60
10
15
20
25
Residue number
Figure 4. Comparison of the and angles estimated with
TALOS21 ž and the corresponding angles of the calculated
structures . The error bars correspond to the uncertainties of
the TALOS estimates and the variation of the calculated
structures, respectively. Incorporation of TALOS angles as
constraints in the structure calculations reduces the NOE
violations and improves the quality of the structure significantly
(see Table 2).
that is found here for the free monomeric GLP-1, support
this model for the binding of GLP-1 to its receptor.
Second, the homology of GLP-1 and glucagon, and
the fact that glucagon can replace receptor-bound GLP-1,34
prompt a comparison of the structures of the two peptides.
A high flexibility of the N-terminal region (residues 1–4), as
observed for GLP-1, was also found in the crystal structure
of glucagon30 and may be relevant for the function of
the two peptides. In both peptides this flexibility is most
likely due to the presence of a glycine in position 4. The
C-terminal fragment F22–V23–Q24–W25–L26 of glucagon,
that was suggested to be essential for the binding of the
hormone to its receptor,35 is partly preserved in GLP-1 as
F22–I23–A24–W25–L26. Indeed, this fragment is part of the
helix that binds to the membrane-like micelles.6 Therefore,
the C-terminal helix of GLP-1 is not only the most stable
part of the helix and the one that initiates the formation of
Magn. Reson. Chem. 2001; 39: 477–483
Structure and folding of GLP-1 in TFE
the helix in the folding pathway of the peptide chain (see
above), but may also play an important role in the receptor
recognition.
CONCLUSION
The results here show that the conformation of free
monomeric GLP-1 in water–TFE mixtures at pH 2.5 depends
strongly on the content of TFE. In pure water GLP-1 adopts
predominantly a flexible random coil, whereas in water with
35% TFE it forms a single-strand ˛-helix. The helix extends
from T7 to K28 with a less well-defined helical region around
G16 that increases the flexibility of the helix. The C-terminal
segment of the helix is formed first during the folding and
is considerably more stable than the N-terminal segment.
The propensity of free monomeric GLP-1 to form a helical
structure and the presence of the flexible region around G16
are both in agreement with the structure suggested for the
micelle-bound GLP-1, and support the model proposed for
the receptor binding of GLP-1.6
Acknowledgements
The 750 and 800 MHz NOESY and HSQC spectra were obtained at
the Danish Instrument Centre for NMR Spectroscopy of Biological
Macromolecules. We are grateful to Dr Jens Duus and Ms Else
Philipp for technical assistance. The coordinates for the GLP-1
monomer structures at 35% TFE and the NMR-derived restraints
have been deposited in the Protein Data Bank at RCSB (code:
1D0R). The 1 H chemical shifts of GLP-1 in aqueous TFE at nine
different TFE concentrations in the range from 0 to 50% TFE and
the 13 C chemical shifts of GLP-1 in 35% TFE have been deposited
in the BioMagResBank (http://www.bmrb.wisc.edu) under BMRB
accession number 4741.
This work was financially supported by the Danish Technical
Research Council (J. Nos 16-5028-1 and 9601137), the Danish Natural
Science Research Council (J. Nos 9400351, 9502759, 9601648 and
9801801), the Ministry of Industry (J. No. 85886), Julie Damm’s
Studiefond, Direktør Ib Henriksens Fond, Carlsbergfondet and Novo
Nordisk Fonden.
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