Partial assignment of the calcium-bound calretinin I-II domain (residues 1-100):
1H, 13C and 15N backbone assignments (and partial sidechain assignment) for
structured elements

D29-E40 and D76-E87 constitute two 12-residue calcium-binding loops.
Both loops of calretinin I-II bind calcium.

Calcium-binding protein of the EF-hand family

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at http://wwpdb-remediation.rutgers.edu/downloads.html
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Structural and biochemical characterization of neuronal calretinin domain I-II (residues 1-100). Comparison to homologous calbindin D28k domain I-II (residues 1-93).

Calretinin (CR) is a calcium-binding, neuronal protein of undefined function.
Related proteins either buffer intracellular calcium concentrations or are
involved in calcium-signaling pathways. We transformed three CR gene fragment
sequences, corresponding to its three complementary domains (I-II, III-IV, and
V-VI), into Pichia pastoris. High yields of extracellular expression, of more
than 200 mg/liter, were achieved. Simple purification protocols provide high
yields of homogenous proteins: dialysis and DEAE-cellulose chromatography for
domains I-II and III-IV or ammonium sulfate precipitation and octyl-Sepharose
chromatography for domain V-VI. To our knowledge, this is the first report of
the expression of an EF-hand protein using P. pastoris. Direct comparison of
the purified yields of domain I-II indicates a approximately 20-fold
improvement over Escherichia coli. N-terminal amino acid sequencing confirmed
our gene products and two anti-calretinin antibodies recognized the appropriate
domains. All three CR domains bind (45)Ca and the domain containing EF-hands V
and VI seems to have a lower calcium capacity than the other domains. Circular
dichroism indicates a high helix content for each of the domains.
Calcium-induced structural changes in the first two domains, followed by
tryptophan fluorescence, correspond with previous studies, while tyrosine
emission fluorescence indicates calcium-induced structural changes also occur
in domain V-VI. The methods and expression levels achieved are suitable for
future NMR labeling of the proteins, with (15)N and (13)C, and
structure-function studies that will help to further understand CR function.

Palczewska M, Groves P, Kuznicki J.
Use of Pichia pastoris for the expression, purification, and characterization
of rat calretinin "EF-hand" domains.
Protein Expr Purif. 1999 Dec;17(3):465-76.

Use of Pichia pastoris for the expression, purification, and characterization of rat calretinin "EF-hand" domains.

Kay, L. E., Keifer, P. & Saarinen, T. (1992)
Pure Absorption Gradient Enhanced Heteronuclear Single Quantum Correlation
Spectroscopy with Improved Sensitivity,
J. Am. Chem. Soc. 114, 10663-10665.

Palmer, A. G., Cavanagh, J., Wright, P. E. & Rance, M. (1991)
Sensitivity Improvement in Proton-Detected 2-Dimensional Heteronuclear
Correlation Nmr-Spectroscopy,
J. Magn. Reson. 93, 151-170.

General pulse sequence elements that achieve sensitivity-enhanced coherence
transfer from a heteronucleus to protons of arbitrary multiplicity are
introduced. The building blocks are derived from the sensitivity-enhancement
scheme introduced by Cavanagh et al. ((1991) J. Magn. Reson., 91, 429-436),
which was used in conjunction with gradient coherence selection by Kay et al.
((1992) J. Am. Chem. Soc., 114, 10663-10665), as well as from a multiple-pulse
sequence effecting a heteronuclear planar coupling Hamiltonian. The building
blocks are incorporated into heteronuclear correlation experiments, in
conjunction with coherence selection by the formation of a heteronuclear
gradient echo. This allows for efficient water suppression without the need for
water presaturation. The methods are demonstrated in HSQC-type experiments on a
sample of a decapeptide in H2O. The novel pulse sequence elements can be
incorporated into multidimensional experiments.

Schleucher J, Schwendinger M, Sattler M, Schmidt P, Schedletzky O, Glaser SJ, Sorensen OW,
Griesinger C.
A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field
gradients.
J Biomol NMR. 1994 Mar;4(2):301-6.

A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients.

Grzesiek, S. & Bax, A. (1992)
Improved 3D Triple-Resonance NMR Techniques Applied to a 31 kDa Protein,
J. Magn. Reson. 96, 432-440.

Kay, L. E., Ikura, M., Tschudin, R. & Bax, A. (1990)
3-Dimensional Triple-Resonance NMR-Spectroscopy of Isotopically Enriched Proteins,
J. Magn. Reson. 89, 496-514.

Clubb, R. T., Thanabal, V. & Wagner, G. (1992)
A Constant-Time Three-Dimensional Triple-Resonance Pulse Scheme to Correlate Intraresidue
{+1}H{+N},{+15}N,and {+13}C' Chemical Shifts in {+15}N-{+13}C-Labeled Proteins,
J. Magn. Reson. 97, 213-217.

Kay, L. E., Xu, G. Y. & Yamazaki, T. (1994)
Enhanced-Sensitivity Triple-Resonance Spectroscopy with Minimal H2O Saturation,
J. Magn. Reson. Ser.A 109, 129-133.

Sattler, M., Schleucher, J. & Griesinger, C. (1999)
Heteronuclear multidimensional NMR experiments for the structure determination of
proteins in solution employing pulsed field gradients,
Prog. Nucl. Magn. Reson. Spect. 34, 93-158.

Muhandiram, D. R. & Kay, L. E. (1994)
Gradient-Enhanced Triple-Resonance 3-Dimensional Nmr Experiments With Improved Sensitivity,
J. Magn. Reson. Ser.B 103, 203-216.

Wittekind, M. & Mueller, L. (1993)
HNCACB, a High-Sensitivity 3D NMR Experiment to Correlate Amide-Proton and Nitrogen
Resonances with the Alpha-Carbon and Beta-Carbon Resonances in Proteins,
J. Magn. Reson. Ser.B 101, 201-205.

Grzesiek, S. & Bax, A. (1992)
An Efficient Experiment For Sequential Backbone Assignment of
Medium-Sized Isotopically Enriched Proteins,
J. Magn. Reson. 99, 201-207.

Measurement of one-bond 15N-13C' dipolar couplings in medium sized proteins.

Experiments and procedures are described that greatly alleviate the sequential
assignment process of uniformly 13C/15N-enriched proteins by determining the
type of amino acid from experiments that correlate side chain with backbone
amide resonances. A recently proposed 3D NMR experiment, CBCA(CO)NH, correlates
C alpha and C beta resonances to the backbone amide 1H and 15N resonances of
the next residue (Grzesiek, S. and Bax, A. (1992) J. Am. Chem. Soc., 114,
6291-6293). An extension of this experiment is described which correlates the
proton H beta and H alpha resonances to the amide 1H and 15N resonances of the
next amino acid, and a detailed product operator description is given. A simple
2D-edited constant-time HSQC experiment is described which rapidly identifies H
beta and C beta resonances of aromatic or Asn/Asp residues. The extent to which
combined knowledge of the C alpha and C beta chemical shift values determines
the amino acid type is investigated, and it is demonstrated that the combined C
alpha and C beta chemical shifts of three or four adjacent residues usually are
sufficient for defining a unique position in the protein sequence.

Grzesiek S, Bax A.
Amino acid type determination in the sequential assignment procedure of uniformly
13C/15N-enriched proteins.
J Biomol NMR. 1993 Mar;3(2):185-204.

Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins.

Two new methods are described for the measurement of three-bond JHNH alpha
couplings in proteins isotopically enriched with 15N. Both methods leave the
water magnetization in an unsaturated state, parallel to the z-axis, and
therefore offer significant enhancements in sensitivity for rapidly exchanging
backbone amide protons. The J couplings can be measured either from a set of
constant-time 2D 1H-15N HMQC spectra, which are modulated in intensity by JHNH
alpha, or from a water-flip-back version of the 3D HNHA experiment. The method
is demonstrated for a sample of calcium-free calmodulin. Residues Lys75-Asp80
have JHNH alpha values in the 6-7 Hz range, suggesting that a break in the
'central helix' occurs at the same position as previously observed in solution
NMR studies of Ca(2+)-ligated calmodulin.

Kuboniwa H, Grzesiek S, Delaglio F, Bax A.
Measurement of HN-H alpha J couplings in calcium-free calmodulin using new 2D and 3D
water-flip-back methods.
J Biomol NMR. 1994 Nov;4(6):871-8.

Measurement of HN-H alpha J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods.

Sattler, M., Maurer, M., Schleucher, J. & Griesinger, C. (1995)
A Simultaneous N-15,H-1-HSQC and C-13,H-1-HSQC With Sensitivity Enhancement
and a Heteronuclear Gradient-Echo,
J. Biomol. NMR 5, 97-102.

Time-sharing evolution and sensitivity enhancements in 2D HSQC-TOCSY and HSQMBC experiments.

YVEF N-terminal tag is the reuslt of extracellular expression from Pichia
pastoris, see ref 1.

calbindin 2, isoform CRA_c [Rattus norvegicus]

PREDICTED: calretinin-like [Macaca mulatta]

PREDICTED: calretinin [Mustela putorius furo]

PREDICTED: calretinin-like [Leptonychotes weddellii]

PREDICTED: calretinin isoform X2 [Equus przewalskii]

(Acq. points / acq. time nucleus Size of processed data (LP/ linear prediction))

experiment Dimension 1 Dimension 2 Dimension 3 Bruker pulse program

(512 / 102 ms 1H 1024) noesiietf3gsrdf[ref-5].

The pH was adjusted to pH 6.7 and a limited dataset was collected.