NMR Solution Structure of Domain 1 of Human Annexin I Shows
an Autonomous Folding Unit*
Jinhai
Gao,
Yue
Li, and
Honggao
Yan
From the Department of Biochemistry, Michigan State University,
East Lansing, Michigan 48824
 |
ABSTRACT |
Annexins are excellent models for studying the
folding mechanisms of multidomain proteins because they have
four-eight homologous helical domains with low identity in sequence
but high similarity in folding. The structure of an isolated domain 1 of human annexin I has been determined by NMR spectroscopy. The
sequential assignments of the 1H, 13C,
and 15N resonances of the isolated domain 1 were
established by multinuclear, multidimensional NMR spectroscopy. The
solution structure of the isolated domain 1 was derived from 1,099 experimental NMR restraints using a hybrid distance geometry-simulated
annealing protocol. The root mean square deviation of the ensemble of
20 refined conformers that represent the structure from the mean
coordinate set derived from them was 0.57 ± 0.14 Å and 1.11 ± 0.19 Å for the backbone atoms and all heavy atoms, respectively.
The NMR structure of the isolated domain 1 could be superimposed with a
root mean square deviation of 1.36 Å for all backbone atoms with the
corresponding part of the crystal structure of a truncated human
annexin I containing all four domains, indicating that the structure of
the isolated domain 1 is highly similar to that when it folded together
with the other three domains. The result suggests that in contrast to
isolated domain 2, which is largely unfolded in solution, isolated domain 1 constitutes an autonomous folding unit and interdomain interactions may play critical roles in the folding of annexin I.
 |
INTRODUCTION |
Most proteins in nature are large multidomain proteins (1). While
a great deal of knowledge on the folding properties of small
single-domain proteins has been acquired (2), our understanding of the
folding of multidomain proteins is still poor. It has been suggested
that the domains of large proteins fold independently and subsequently
assemble to form the native structures (3-5).
Annexins are a large family of ubiquitous proteins that bind to
phospholipids in the presence of calcium ions (6, 7). Although their
physiological functions are not clear, these proteins are implicated in
many important cellular processes (8) such as exocytosis (9, 10) and
ion channeling (11). All annexins contain four homologous repeats of
~70 residues (see Fig. 1, a and c) and a
variable N terminus, with the exception of annexin VI, which has four
additional repeats. The crystal structures of annexins I, II, III, IV,
V, VI, VII, and XII have been determined (12). As revealed by x-ray
crystallography, each repeat forms a compact domain consisting of five
helices. All the domains are highly similar in structure, as
illustrated in Fig. 1b with the four domains of annexin I. The four domains of each annexin are arranged in a planar-cyclic manner
with domain 4 in contact with domain 1, as depicted in Fig.
1a. Domains 1 and 4 as well as domains 2 and 3 have many
tight hydrophobic contacts, mainly involving helices B and E,
constituting two two-domain modules. The interactions between the two
modules are mostly hydrophilic via helices A and B of domains 2 and 4, forming a central hydrophilic channel.
With the well defined domains and the simple and elegant structure,
annexins are excellent models for studying the folding mechanisms of
multidomain proteins. Using synthetic peptides and more recently
recombinant peptides, Sanson and co-workers (13-16) have been
systematically studying the folding properties of domain 2 of human
annexin I. They have clearly shown, with CD and NMR, that isolated
domain 2 of annexin I is largely unfolded in aqueous solution (15). A
preliminary study on the folding properties of domain 1 has also been
reported (17).
Our approach to dissect the folding mechanism of annexin I is to
compare the folding properties of the intact protein and the four
isolated domains. We have expressed the entire annexin I and the four
individual domains in Escherichia coli. Using
multidimensional NMR techniques, we have determined the solution
structure of domain 1 (residues 14-86, according to the numbering of
the crystal structure of an N-terminal truncated human annexin I (18)).
The NMR structure of the isolated domain 1 is highly similar to the
corresponding part of the crystal structure of a truncated human
annexin I containing all four domains (18). The result shows that in
contrast to isolated domain 2, which is largely unfolded in solution,
isolated domain 1 constitutes an autonomous folding unit. Comparative
structural analysis suggests that interdomain interactions may play
critical roles in the folding of annexin I.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The E. coli clone containing the
cDNA encoding human annexin I was purchased from ATCC (ATCC number
65114, deposited by Joel Ernst). The expression vector pET-17b was
purchased from Novagen. DNA sequencing kit was obtained from United
States Biochemical. Enzymes for recombinant DNA experiments were
purchased from Life Technologies, Inc. or New England Biolabs.
15NH4Cl and
[13C6]-D-glucose were purchased
from ISOTEC. Other chemicals were analytical or reagent grade from
commercial sources.
Cloning--
The amino acid sequence of domain 1 of human
annexin I is shown in Fig. 1c.
The portion of human annexin I cDNA that encodes domain 1 was
cloned into the expression vector pET-17b by polymerase chain reaction
and other standard recombinant DNA techniques. The primers used for the
polymerase chain reaction cloning were 5'-GGAATTCCATATGACCTTCAATCCATCCTCG-3' (forward) and
5'-CCGGATCCTTATTTTAGCAGAGCTAAAACAAC-3' (reverse). The correct amino
acid sequence was verified by double-stranded DNA sequencing of the DNA
insert in the expression construct pET-17b-ANX1D1.

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Fig. 1.
a, ribbon diagram of the x-ray structure
of a truncated human annexin I that lacks N-terminal 31 residues (18).
The four homologous domains are indicated in different colors: domain
1, green; domain 2, yellow; domain 3, cyan; and domain 4, magenta. Except domain 1, only the helices involved in the interdomain interactions are labeled.
b, superposition of the four domains of annexin I: domain 1 (17-86), domain 2 (87-158), domain 3 (169-246), and domain 4 (247-319). Domains 1 to 4 are colored as in panel a. Only
the helices of each domain were used for the structural alignment.
c, sequence alignment of the four domains. The numbering is
according to the crystal structure of the truncated annexin I (18). The
hydrophobic core residues are shown in yellow and other
conserved residues are in blue. Panels
a and b were generated using the program MOLMOL
(43).
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Expression and Purification--
Unlabeled protein was produced
by growing the E. coli strain BL21(DE3) containing the
expression construct pET-17b-ANX1D1 in LB media in the presence of 100 µg/ml ampicillin at 37 °C without IPTG1 induction. Uniformly
15N-labeled protein was produced by growing the same
expression strain in M9 media with 15NH4Cl as
the sole nitrogen source, and uniformly
15N/13C-labeled protein was produced in M9
media with 15NH4Cl and
[13C6] D-glucose as the sole
nitrogen and carbon sources. Protein production in the M9 media was
induced by addition of IPTG to a final concentration of 0.4 mM when the cultures reached an A600 of ~1.0. The culture was incubated for 4 more h after addition of
IPTG. The bacterial cells were harvested by centrifugation and
suspended in buffer A (40 mM acetate, pH 5.3). The
bacterial suspension was sonicated on ice and centrifuged (27,000 × g) at 4 °C for 30 min. The supernatant was applied to
a CM-cellulose column equilibrated with buffer A. The column was washed
with buffer A until A280 of the eluent was less
than 0.05. Elution of the column was achieved by a linear NaCl gradient
(0-500 mM in buffer A) and monitored by
A280 and 15% SDS-PAGE. The fractions containing
domain 1 of annexin I were pooled and concentrated by an Amicon
ultrafiltration cell using a YM3 membrane. The protein preparations
were >95% pure as judged by SDS-PAGE. Isotopically labeled proteins
were further purified by a Sephadex G-50 column. The protein solutions
were dialyzed against double distilled water, lyophilized and stored at
80 °C.
NMR Spectroscopy--
NMR samples were prepared by dissolving
the lyophilized protein in 20 mM
[2-2H]acetate, pH 5.2 (pH meter reading without
correction for isotope effects), in
H2O/2H2O(9/1) or
2H2O. The protein concentrations of the NMR
samples were 2-5 mM. NMR spectra were acquired at 25 °C
on a Bruker DMX 600 spectrometer at The Ohio State University, a Bruker
DRX 600 spectrometer at Bruker, Billerica, MA, or a Varian INOVA 600 spectrometer at Varian Application Laboratories. Homonuclear
two-dimensional spectra recorded were DQF-COSY (D2O) (19,
20), TOCSY (D2O) (21-23), and NOESY (H2O) (24,
25). The heteronuclear double and triple resonance spectra acquired
included two-dimensional 1H-15N HSQC (26, 27),
three-dimensional 1H-15N TOCSY-HSQC (28),
three-dimensional 1H-15N NOESY-HSQC (28, 29),
HNCACB (30, 31), CBCA(CO)NH (31, 32), and HCCH-TOCSY (33, 34). The
spectra were processed with the program NMRPipe (35) and analyzed with
the program PIPP (36). Briefly, solvent suppression was improved by
convolution of time domain data (37). The data size in each indirectly
detected dimension of the three-dimensional data was extended by
backward-forward linear prediction (38). A 45°-shifted sine bell and
single zero-filling were generally applied before Fourier
transformation in each dimension.
Derivation of Structural Restraints--
Approximate interproton
distance restraints were derived from sequentially assigned NOEs. NOE
cross-peaks between aliphatic protons were picked from the homonuclear
two-dimensional NOESY spectrum, and those involving amide protons were
from the three-dimensional 1H-15N NOESY-HSQC
spectrum. The NOE intensities obtained by the program PIPP were
converted into approximate interproton distances by normalizing them
against the calibrated intensities of NOE peaks between backbone amide
protons (dNN) within the identified
-helices. The upper limits of the interproton distances were calibrated according
to the equation Va = Vb(rb/ra)6,
where Va and Vb were the
NOE intensities and ra and
rb were the distances. The distance bounds were
then set to 1.8-2.7 Å (1.8-2.9Å for NOE cross-peaks involving amide
protons), 1.8-3.3 Å (1.8-3.5 Å for NOE cross-peaks involving amide
protons), and 1.8-5.0 Å corresponding to strong, medium, and weak
NOEs, respectively. Pseudoatom corrections were made for
nonstereospecifically assigned methylene and methyl resonances (39). An
additional 0.5 Å was added to the upper bounds for methyl protons.
Structure Calculation--
NMR structures were calculated with a
hybrid distance geometry-simulated annealing protocol (40) using the
program X-PLOR (Version 3.1) (41) on an SGI Indigo II workstation. A
square-well potential function with a force constant of 50 kcal
mol
1 Å
2 was applied for the distance
restraints. The X-PLOR frepel function was used to simulate
van der Waals interactions, with atomic radii set to 0.80 times their
CHARMM values (42) and a force constant of 4.0 kcal
mol
1Å
4. A total of fifty structures were
generated using this protocol. The structures were inspected by the
programs MOMOL (43) and QUANTA96 (Molecular Simulations) and analyzed
by PROCHECK-NMR (Version 3.4.4) (44, 45). An iterative strategy was
used for the structure refinement. In each round of structure
refinement, newly computed NMR structures were employed to assign more
NOE restraints, to correct wrong assignments, and to loosen the NOE distance bounds if spectral overlapping was deduced. Then another round
of structure refinement was carried out with the modified NMR
restraints. All structures were converged after several rounds of such
refinement. An ensemble of 20 structures was selected according to
their best fit to the experimental NMR restraints and the low values of
their total energies.
 |
RESULTS |
Total sequential resonance assignments of the isolated domain 1 were achieved by the combined analysis of two-dimensional and
three-dimensional NMR data, including three-dimensional HNCACB, CBCA(CO)NH, and HCCH-TOCSY. The sequential assignments of the backbone
and side-chain amide resonances are shown in
Fig. 2. Stereospecific assignments were
made for about 70% of
-methylene protons and the methyl groups of
valine and leucine residues based on qualitative estimations of
3J
constants from the DQF-COSY spectrum
in conjunction with the NOE data (46).

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Fig. 2.
15N-1H HSQC
spectrum of domain 1 of human annexin I. Sequential assignments
are indicated with one-letter amino acid codes and residue numbers.
Pairs of cross-peaks resulting from the side chain NH2
groups of asparagine and glutamine residues are connected by
horizontal lines. The amino acid numbering is according to
the isolated domain 1 with residue 1 corresponding to residue 14 in the
crystal structure numbering.
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A total of 1099 structurally useful distance restraints were obtained
from the analyses of the homonuclear two-dimensional NOESY
(D2O) and three-dimensional 1H-15N
NOESY-HSQC spectra (Table I), 707 of
which were medium and long range NOEs. In average, each residue had
~15 NOE restraints. A superposition of 20 calculated structures with
no NOE restraint violations above 0.5 Å is shown in
Fig. 3a. The statistics of the
structures are summarized in Table I. The precision of the structures
(RMSD of the ensemble of the 20 NMR structures from its mean
coordinate) was 0.57 Å for the backbone (N, C
, C', O)
and 1.11 Å for all heavy atoms. The distribution of the average
backbone RMSDs is shown in Fig.
4a. The structure of domain 1 consists of five
-helices:
helix A, residues 5-15; helix B, residues 22-30; helix C, residues
34-47; helix D, residues 52-58; and helix E, residues 63-70
(numbering according to the isolated domain 1). Helices A, B, D, and E
are assembled in a bundle with two nearly parallel helix-loop-helix
motifs. Helix C lies approximately perpendicular to the helical bundle
with one end close to the N terminus and the other to the C terminus of
domain 1.

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Fig. 3.
a, superposition of the final 20 calculated NMR structures of domain 1 of annexin I. Only the backbone
atoms (N, C , and C') are superimposed and colored
according to the secondary structure: helices A (5-15) in
red, B (22-30) in green, C (34-47) in
cyan, D (52-58) in magenta, and E (63-70) in
yellow and the loops in gray. The amino acid
numbering is according to the isolated domain 1 with residue 1 corresponding to residue 14 in the crystal structure numbering.
b, superposition of the minimized average NMR structure
(red) and the x-ray structure (cyan) of domain
1.
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Fig. 4.
Distributions of the average backbone RMSDs
of the ensemble of the NMR structures from its mean coordinate
(a, top) and from the x-ray crystal
structure (b, bottom). The amino
acid numbering is according to the isolated domain 1 with residue 1 corresponding to residue 14 in the crystal structure numbering.
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 |
DISCUSSION |
Comparison with the Crystal Structure of Human Annexin I--
The
structure of a truncated human annexin I has been determined by x-ray
crystallography in the presence of 10 mM CaCl2
(18). The truncated annexin I lacks the N-terminal 32 residues but has four domains all intact (Fig. 1a). Six calcium ions are
found to bind to the truncated annexin I, two each in domains 1 and 4 and one each in domains 2 and 3. The solution structure of the isolated
domain 1 is highly similar to the corresponding part of the crystal
structure of the truncated annexin I containing all four domains. Thus,
the minimized average NMR structure of the isolated domain 1 can be
superimposed very well with the corresponding x-ray structure as shown
in Fig. 3b. There are 1-2 residue differences in the
lengths of some helices but the five helices are assembled in the same
way. The distribution of the average backbone RMSDs of the ensemble of
the 20 NMR structures from the corresponding x-ray structure is shown
in Fig. 4b. The largest differences are found at the N
terminus and in the AB loop. It should be noted that the NMR structure
of the isolated domain 1 was determined in the absence of
Ca2+. The difference in the conformations of the AB loop
could be because of binding of Ca2+ because the carbonyls
of Gly-32 and Val-33 in the AB loop along with the carboxylate of
Glu-35 at the N terminus of helix B form a calcium-binding site.
However, binding of Ca2+ to the second calcium-binding site
apparently does not cause any significant conformational change because
the conformation of the DE loop that constitutes the second site is
essentially the same as that found in the crystal structure, probably
because the second site has lower affinity for Ca2+ than
the first site.
Implications for Protein Folding--
As described earlier, the
four domains of annexin I are highly homologous in structure when
folded together (Fig. 1, a and b). The
hydrophobic cores are highly conserved among all annexin domains.
Surprisingly, isolated domain 2 is largely unfolded in aqueous solution
and thus is not an independent folding unit (15). Its helical content
is less than 25% compared with ~80% when the domain is folded
together with the rest of the protein. In contrast to domain 2, our
work presented here clearly demonstrates that the isolated domain 1 is
fully folded in solution with little change in structure from that in
the native state, and thus constitutes an autonomous folding unit. The
results present an interesting question of why the domains with high
sequential and structural homologies exhibit totally different folding behaviors.
The failure of the isolated domain 2 to form its native structure is
likely because of the removal of the interdomain interactions that
exist in the whole protein. As mentioned earlier, according to the
crystal structure of annexin I (18), domains 2 and 3 form a modular
structure with many hydrophobic interactions, and so do domains 1 and
4. Thus, it is unlikely that the removal of the hydrophobic contacts
with domain 3 is the cause for the folding failure of the isolated
domain 2. By default then, the removal of the interactions with domain
4 may be the cause for the failure of the isolated domain 2 to fold to
its native structure. Indeed, there are many interactions between
domain 2 and domain 4 as shown in Fig. 5.
This explanation is supported by the NMR studies of the isolated domain
2 and its components helices A and B (14, 15).

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Fig. 5.
The hydrophobic core structure of domain 2 and the interface between domains 2 and 4. The drawing is based on
the x-ray structure of the truncated human annexin I containing four
domains (18). The main-chains of domain 2 and domain 4 (partial) are
represented by blue and cyan ribbons,
respectively. The residues involved in the nonnative cap and the
cluster of acidic residues as well as Arg-117 in domain 2 are shown in
magenta. The residues within 5 Å distance of Leu-96 are
shown in yellow, and other core residues are in
gray. The residues of domain 4 are in green.
Hydrogen bonds are indicated by dotted lines. The amino acid
numbering is according to the crystal structure of the truncated
annexin I.
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It has been shown by NMR that a stable nonnative N-terminal cap, with
the sequence
F91D92A93D94E95L96
(numbering according to the crystal structure of the truncated annexin
I), is formed in helix A in a peptide fragment containing helices A and
B of domain 2 (14). With the carboxyl groups of Asp-92 and Glu-95 hydrogen-bonded to their reciprocal backbone amides and many
hydrophobic contacts between Phe-91 and Leu-96, it is a canonical
N-terminal cap (47, 48). Furthermore, the nonnative cap persists in
isolated domain 2 (15, 16). It has been suggested that the nonnative N-terminal cap serve as a very potent initiation site for folding (14).
However, it may be more likely that the formation of the nonnative
N-terminal cap prevents the isolated domain 2 from reaching the native
state for two reasons although its role in the folding of entire
annexin I is not known. First, it disrupts a pair of hydrogen bonds
between the carboxyl group of Asp-92 and the guanidinium group of
Arg-117 that helps to lock helices A and B in place (18) (Fig. 5). The
breakage of the hydrogen bond also makes it possible for Arg-117 to
form nonnative salt bridges as found in the isolated domain 2 (16).
Second, as shown in Fig. 5, in the native structure, Leu-96 is roughly
at the center of the hydrophobic core. It is surrounded by as many as
seven core residues: Met-100 from helix A; Leu-110, Ile-113, and
Ile-114 from helix B; Ile-125 and Tyr-129 from helix C; and Leu-137
from helix D. On the other hand, the side-chains of Phe-91 and Leu-96
are >10 Å apart. Thus, the nonnative hydrophobic interactions between
Phe-91 and Leu-96 in the isolated domain may not only take out the
side-chain of Leu-96 from the hydrophobic core structure but also
disrupt the packing of the other hydrophobic core residues. The
nonnative conformation of the isolated domain 2, however, may not
necessarily have a lower energy than the native conformation. The
nonnative N-terminal cap may act as a kinetic trap that keeps the
isolated domain 2 from reaching the native structure.
Why does the nonnative N-terminal cap form in the isolated domain 2?
The separation of domain 2 from the rest of the protein has two
structural consequences that may bear on the formation of the nonnative
N-terminal cap as shown in Fig. 5. First, it breaks four hydrogen bonds
between domains 2 and 4, namely Glu-95/Lys-267, Asp-108/Lys-254, and
Glu-112/Arg-271 (two hydrogen bonds). The salt bridge between Glu-107
of domain 2 and Lys-235 of domain 3 is also broken. This leaves a
cluster of negatively charged residues without positively charged
partners, including Glu-95, Asp-106, Glu-107, Asp-108, and Glu-112. The
carboxyl group of Glu-95 is ~6.7 Å away from that of Asp-106 and
~7.1 Å away from that of Glu-112. It is likely that the negative
charge potential generated by the cluster of acidic residues may push
away the carboxyl group of Glu-95 so that it forms a hydrogen bond to
the backbone amide of Asp-92. Second, Phe-91 is almost completely buried in the whole protein but its side-chain becomes mostly exposed
to solvent in the isolated domain 2. Thus, Phe-91 in the isolated
domain 2 seeks hydrophobic partners, and it finds Leu-96. It is noted
that Phe-91 and Glu-95 are replaced by a serine and an alanine,
respectively, in domain 1 (Fig. 1c). Therefore, the nonnative N-terminal cap is unlikely to form in the folding process of
the isolated domain 1. The hypothesis may be tested by replacing Phe-91
and Glu-95 of domain 2 with the corresponding amino acids of domain 1 by site-directed mutagenesis. Refolding at a higher salt concentration
may also help the isolated domain 2 to reach the native conformation by
reducing the effects of the negative charges of the cluster of acidic
residues and strengthening the hydrophobic interactions to drive
formation of the hydrophobic core.
For multidomain proteins, the formation of a native structure requires
not only the correct folding of each domain but also the appropriate
assembly of the domains via interdomain interactions. However, little
is known about the roles of interdomain interactions during the folding
process. As discussed above, interdomain interactions may play a
critical role in the folding of domain 2 of annexin I. It is
interesting to note that among the four domains of annexin I, only
domain 1 is folded and soluble when expressed in E. coli. Domain 2 is soluble but largely unfolded. Expression of separated domains 3 and 4 in E. coli results in inclusion bodies (data
not shown). It has been reported that domain 3 is easily degraded, but
domain 4 forms inclusion bodies when expressed as fusion proteins of
glutathione transferase (17). It appears that only domain 1 is an
autonomous folding unit, although it is not known at present whether
domains 3 and 4 can be solubilized and refolded to their native
structures. As described earlier, annexin I is composed of two modules.
One module consists of domains 1 and 4, and the other domains 2 and 3. Each module has a hydrophobic interface between its constituents. The
two modules are assembled with mostly hydrophilic interactions between
domains 2 and 4. It is tempting to speculate that folding of annexin I
follows a sequential process with domain 1 as an autonomous initial
folding unit. The folded structure of domain 1 facilitates the folding
of domain 4 through the hydrophobic interface. Then, the hydrogen bonds
and hydrophobic interactions between domains 4 and 2 help domain 2 to
get rid of the nonnative cap and reach the native structure. Domain 2, in turn, assists the folding of domain 3 through many hydrophobic interdomain interactions. This proposal can be tested by systematic studies of the folding properties of the entire protein and separated domains of annexin I.
 |
ACKNOWLEDGEMENTS |
We are indebted to Drs. Charles Cottrell,
Clemens Anklin, and George Gray for assistance in acquiring the NMR
data. We thank Drs. Xiangwei Weng and Sung-Hou Kim for providing us the
unpublished coordinate of the refined crystal structure of annexin I at
1.8-Å resolution.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant GM51901. This study made use of a Bruker DMX-600 NMR spectrometer at The Ohio State University funded by National Institutes of Health Grant RR08299 and National Science Foundation Grant BIR-9221639.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1bo9) have
been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
To whom correspondence should be addressed. Tel.: 517-353--8786;
Fax: 517-353-9334; E-mail: yanh{at}pilot.msu.edu.
The abbreviations used are:
IPTG, isopropyl-1-thio-
-D-galactopyranoside; DQF-COSY, double
quantum filtered correlation spectroscopy; HSQC, heteronuclear single
quantum coherence; NOE, nuclear Overhauser effect; NOESY, NOE
spectroscopy; PAGE, polyacrylamide gel electrophoresis; RMSD, root mean
square deviation; TOCSY, total correlation spectroscopy; HCCH-TOCSY, proton-carbon-carbon-proton correlation using carbon TOCSY; HNCACB, amide proton to nitrogen to
/
carbon correlation; CBCA(CO)NH,
/
proton to
/
carbon (via carbonyl carbon) to nitrogen to
amide proton correlation.
 |
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