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D M LeMaster

Publications and source records attributed to D M LeMaster.

27 records · Page 2Linked to original sources
PubMed1988

Protein NMR resonance assignment by isotropic mixing experiments on random fractionally deuterated samples.

The 108-residue protein E. coli thioredoxin has been uniformly enriched to 50% with deuterium at all carbon-bound hydrogen positions. Isotropic mixing (i.e. TOCSY) experiments have been conducted for both the deuterated and natural-abundance samples. Using a 54 ms mixing time correlation peaks can be seen for all four protons on the benzenoid ring of tryptophan in both samples. The deuteration results in an average decrease in cross-sectional area of a factor of 2-3 for the TOCSY cross-peaks. The cross-peak intensities for the deuterated sample systematically decrease as a function of the number of protons involved in the transfer process thus overcoming a common ambiguity in the TOCSY experiment.

Bacterial Proteins↗
PubMed1988

NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation.

All non-proline residues except for the N-terminal dipeptide have been assigned in the 108-residue protein Escherichia coli thioredoxin. Central to these experiments has been the use of protein samples in which all carbon-bound hydrogen positions are substituted to 75% with deuterium by bacterial growth on partially deuteriated carbon sources and media. The dilution of the local proton density gives rise to narrower line widths with little loss in sensitivity. In addition, passive or secondary coupling to protons not directly involved in the coherence transfer process of correlation experiments is largely suppressed, thus significantly improving the resolution for side-chain couplings. Simultaneous multiresidue-type assignments have been obtained by incorporation of several amino acids with differing selective alpha- and/or beta-deuteriation into a fractionally deuteriated background. Combined with several single residue type labeling experiments, these selective labelings have yielded direct residue type assignments for two-thirds of the protein. In addition to improved resolution, the amide to carbon-bound proton NOESY spectra offered equivalent sensitivity while the amide to amide NOESY spectra offered superior sensitivity to that observed for natural abundance samples. The resultant sequential assignment has an average number of nearest-neighbor NOE connectivities of 2.35 out of the possible 3 alpha-amide, beta-amide, and amide-amide connectivities.

Bacterial Proteins↗
PubMed1987

Chiral beta and random fractional deuteration for the determination of protein sidechain conformation by NMR.

Stereospecific assignments of the aspartic acid and asparagine beta-protons of the 108 residue protein E. coli thioredoxin have been obtained by the use of chiral deuteration. In addition protein samples have been prepared in which all carbon bound hydrogen positions are substituted to an extent of 75% with deuterium. These random fractionally deuterated samples significantly facilitate the measurement of coupling constants and intraresidue NOE intensities which combined with the stereospecific assignments have provided determination of the first sidechain dihedral angle chi 1 for all four asparagine residues and eight of the ten assigned aspartic acid residues.

Asparagine↗
PubMed1986

Nucleotide sequence and protein overproduction of bacteriophage T4 thioredoxin.

The bacteriophage T4 thioredoxin gene was cloned and physically mapped to 47.6 kilobases from the reference BamHI site. The DNA sequence is consistent with that reported from earlier protein sequence studies. The gene was subcloned into a lambda pL overexpression vector which allowed for the isolation of approximately 5 mg/liter.

Bacterial Proteins↗
PubMed1985

The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein.

A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene. Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP). Its interaction with the 3' splice site occurs rapidly even at 0 degrees C, does not require adenosine triphosphate, and is altered by certain mutations in the 3' splice site region. Binding is surprisingly insensitive to treatment of the extract with micrococcal nuclease. The U5 particle is the only abundant Sm snRNP with a capped 5' end that is equally resistant to micrococcal nuclease. This suggests that, in addition to the U1 and U2 snRNP's, U5 snRNP's participate in pre-mRNA splicing.

Base Sequence↗
PubMed1985

1H-15N heteronuclear NMR studies of Escherichia coli thioredoxin in samples isotopically labeled by residue type.

Ten samples of Escherichia coli thioredoxin were individually isotopically enriched by residue type via growth of an appropriate auxotrophic strain on media supplemented with one 2H, 15N-enriched amino acid. 1H observe-heteronuclear decoupling experiments were conducted on these samples making use of the 95-Hz 1H-15N amide J1 coupling. Subtraction of near-resonance from off-resonance 15N decoupled spectra generated difference patterns corresponding only to protons directly bonded to 15N nuclei. For the ten different enriched residue types observed to date, every labeled position (60) has been observed as a resolved resonance. The spectral dispersion in both the 1H and the 15N dimensions was roughly 1500 Hz (at 500-MHz field strength) with rather little apparent dependence on residue type. With the exception of the glycine-enriched sample, the range of the J1 coupling constants was not much greater than the precision of the measurements (1.5-2.0 Hz). However, for the glycine residues the J1 amide coupling values varied over a range of 10 Hz.

Bacterial Proteins↗
PubMed1982

Biosynthetic production of 13C-labeled amino acids with site-specific enrichment.

We have developed two Escherichia coli strains for the production of specifically labeled amino acids suitable for high resolution nuclear magnetic resonance experiments. The 13C atoms from the enriched carbon sources, [1-13C]lactate, [1,4-13C2]succinate, and [1-13C]-acetate, are incorporated into the amino acids producing multilabeled molecules with relatively few instances of adjacent enriched carbons. This greatly simplifies the resultant spectra as compared to the extensively spin-coupled spectra of uniformly enriched samples. No isotopic enrichment was found at carbon positions expected to be unenriched by consideration of the major biosynthetic pathways. Utilizing 90% enriched precursors, most positions were enriched to approximately 85% except for those positions derived from acetate and those affected by the carbon interchange of the pentose phosphate shunt. In both of these cases, the enrichment level fell to 70%. The only result enrichment at the delta-methyl carbon of isoleucine. For the bacterial strain in question, the main pathway of isoleucine biosynthesis appears not to be from threonine but from an alternate precursor, possibly glutamate.

Amino Acids↗