NMR paper Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N

		BioNMR > Educational resources > Journal club
[NMR paper] Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N

Webservers

NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

Preditor

TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

Quality Control Check

NMR spectrum prediction:

FANDAS

MestReS

V-NMR

Flexibility from structure:

Backbone S2

Methyl S2

B-factor

Molecular dynamics:

Gromacs

Amber

Antechamber

Chemical shifts prediction:

From structure:

Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

From NMR-STAR 3.1

Validate NMR-STAR 3.1

NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

Solid-state NMR:

View First Unread

Thread Tools

Search this Thread

Rate Thread

Display Modes

08-21-2010, 11:12 PM

nmrlearner

Senior Member

Join Date: Jan 2005

Posts: 23,174

Points: 193,617, Level: 100

Level up: 0%, 0 Points needed

Activity: 50.7%

Last Achievements

Award-Showcase

NMR Credits: 0

NMR Points: 193,617

Downloads: 0

Uploads: 0

Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N

Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme.

Related Articles Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme.

J Biochem. 1991 Aug;110(2):295-300

Authors: Nakazawa T, Sakiyama F

The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [delta 1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GlcNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C]AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H-[2-13C]indol-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differently from Trp 62 on pH-titration of the 13C-label. These results suggest that a conformational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.

PMID: 1761525 [PubMed - indexed for MEDLINE]

Source: PubMed

Did you find this post helpful?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy Abstract Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of 13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and...	nmrlearner	Journal club	0	11-19-2011 07:51 AM
Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy Abstract Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of 13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on...	nmrlearner	Journal club	0	11-14-2011 08:45 AM
Site-specific labeling of proteins with NMR-active unnatural amino acids Site-specific labeling of proteins with NMR-active unnatural amino acids Abstract A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling...	nmrlearner	Journal club	0	01-09-2011 12:46 PM
[NMR paper] Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins. Related Articles Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins. J Biomol NMR. 2004 Nov;30(3):267-74 Authors: van Gammeren AJ, Hulsbergen FB, Hollander JG, de Groot HJ Partly biosynthetic site-directed isotopically (13)C enriched photosynthetic light-harvesting...	nmrlearner	Journal club	0	11-24-2010 10:03 PM
[NMR paper] Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and ir Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli. Related Articles Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli. J Biomol NMR. 2000 Aug;17(4):311-22 Authors: Sorkin DL, Miller AF We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide...	nmrlearner	Journal club	0	11-19-2010 08:29 PM
[NMR paper] 1H-NMR analysis of turkey egg-white lysozyme and comparison with hen egg-white lysozy 1H-NMR analysis of turkey egg-white lysozyme and comparison with hen egg-white lysozyme. http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif Related Articles 1H-NMR analysis of turkey egg-white lysozyme and comparison with hen egg-white lysozyme. Eur J Biochem. 1993 Jul 15;215(2):255-66 Authors: Bartik K, Dobson CM, Redfield C The complete main chain and approximately 75% of the side chain 1H-NMR assignments of the 129-residue protein, turkey...	nmrlearner	Journal club	0	08-22-2010 03:01 AM
[NMR paper] Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme. Related Articles Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme. J Biochem. 1991 Aug;110(2):295-300 Authors: Nakazawa T, Sakiyama F The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed...	nmrlearner	Journal club	0	08-21-2010 11:12 PM
Site-specific labeling of nucleotides for making RNA for high resolution NMR studies Abstract Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and...	nmrlearner	Journal club	0	08-14-2010 04:19 AM

« Previous Thread | Next Thread »

Posting Rules
You may not post new threads You may not post replies You may not post attachments You may not edit your posts BB code is On Smilies are On [IMG] code is On HTML code is On Trackbacks are Off Pingbacks are Off Refbacks are Off