NMR mapping of the recombinant mouse major urinary protein I
binding site occupied by the pheromone
2-sec-butyl-4,5-dihydrothiazole

J 1999

NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole

ŽÍDEK, Lukáš, Martin J STONE, Susan M LATO, Mark D PAGEL, Zhongshan MIAO et. al.

Basic information

Original name

NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole

Authors

ŽÍDEK, Lukáš, Martin J STONE, Susan M LATO, Mark D PAGEL, Zhongshan MIAO, Andrew D ELLINGTON and Milos V NOVOTNY

Edition

Biochemistry, Washington, Amer. Chemical Soc. 1999, 0006-2960

Other information

Type of outcome

Článek v odborném periodiku

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 4.493

Organization unit

Faculty of Science

Keywords in English

CHEMICAL-SHIFT INDEX; GENE FAMILY; MUS-MUSCULUS; MOLECULAR HETEROGENEITY;IMPROVED SENSITIVITY; BACKBONE ASSIGNMENT; SECONDARY STRUCTURE; FEMALE MICE;SPECTROSCOPY; LIVER

Abstract

V originále

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. N-15-labeled and N-15,C-13-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4,5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. H-1, N-15, and C-13 chemical shifts of MUP-I were assigned using triple resonance and N-15-correlated 3D NMR experiments. Changes in protein H-1(N) and N-15(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping Ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.

Citovat

ŽÍDEK, Lukáš, Martin J STONE, Susan M LATO, Mark D PAGEL, Zhongshan MIAO, Andrew D ELLINGTON and Milos V NOVOTNY. NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole. Biochemistry. Washington: Amer. Chemical Soc., 1999, vol. 38, No 31, p. 9850-9861. ISSN 0006-2960.

@article{362176,
   author = {Žídek, Lukáš and Stone, Martin J and Lato, Susan M and Pagel, Mark D and Miao, Zhongshan and Ellington, Andrew D and Novotny, Milos V},
   article_location = {Washington},
   article_number = {31},
   keywords = {CHEMICAL-SHIFT INDEX; GENE FAMILY; MUS-MUSCULUS; MOLECULAR HETEROGENEITY;IMPROVED SENSITIVITY; BACKBONE ASSIGNMENT; SECONDARY STRUCTURE; FEMALE MICE;SPECTROSCOPY; LIVER},
   issn = {0006-2960},
   journal = {Biochemistry},
   title = {NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole},
   volume = {38},
   year = {1999}
}

TY  - JOUR
ID  - 362176
AU  - Žídek, Lukáš - Stone, Martin J - Lato, Susan M - Pagel, Mark D - Miao, Zhongshan - Ellington, Andrew D - Novotny, Milos V
PY  - 1999
TI  - NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole
JF  - Biochemistry
VL  - 38
IS  - 31
SP  - 9850
EP  - 9850
PB  - Amer. Chemical Soc.
SN  - 00062960
KW  - CHEMICAL-SHIFT INDEX
KW  - GENE FAMILY
KW  - MUS-MUSCULUS
KW  - MOLECULAR HETEROGENEITY;IMPROVED SENSITIVITY
KW  - BACKBONE ASSIGNMENT
KW  - SECONDARY STRUCTURE
KW  - FEMALE MICE;SPECTROSCOPY
KW  - LIVER
N2  - The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. N-15-labeled and N-15,C-13-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4,5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. H-1, N-15, and C-13 chemical shifts of MUP-I were assigned using triple resonance and N-15-correlated 3D NMR experiments. Changes in protein H-1(N) and N-15(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping Ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.
ER  -

ŽÍDEK, Lukáš, Martin J STONE, Susan M LATO, Mark D PAGEL, Zhongshan MIAO, Andrew D ELLINGTON and Milos V NOVOTNY. NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole. \textit{Biochemistry}. Washington: Amer. Chemical Soc., 1999, vol.~38, No~31, p.~9850-9861. ISSN~0006-2960.

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