Biomedical subjects

M D Carr

Publications and source records attributed to M D Carr.

23 records · Page 2Linked to original sources

PubMed1991

The transcriptional control proteins c-Myb and v-Myb contain a basic region DNA binding motif.

A class of transcriptional control proteins, typified by c-Myb, are characterised by a highly conserved N-terminal DNA binding domain, which is composed of either 2 or 3 imperfect repeats of about 52 amino acids. A sequence homology search of the SWISSPROT protein sequence data bank with this region from mouse c-Myb has allowed us to identify an area near the C-terminus of repeat 2 that contains a short sequence motif, known as the basic region, which forms the DNA binding site in both leucine zipper and helix-loop-helix transcription factors. We therefore propose that this region of repeat 2 and the homologous part of repeat 3 will form the Myb DNA binding site.

Amino Acid Sequence↗

PubMed1990

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine.

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1990

A novel method of preparing totally alpha-deuterated amino acids for selective incorporation into proteins. Application to assignment of 1H resonances of valine residues in dihydrofolate reductase.

The pyridoxal/2H2O exchange reaction of the alpha-CH of amino acids is known to be accompanied by racemisation: Thus by using a D-amino acid as the starting material any L-amino acid formed in the reaction will be essentially fully deuterated at its alpha-position. We have used this method to prepare alpha-deuterated L-valine and incorporated this biosynthetically into L. casei dihydrofolate reductase. A comparison of the alpha CH-NH fingerprint regions of COSY spectra of deuterated and normal DHFR complexes allows one to identify cross-peaks from 15 of the 16 valine residues.

Amino Acids↗

PubMed1990

The 1H-NMR assignments of the aromatic resonances in complexes of Lactobacillus casei dihydrofolate reductase and the origins of their chemical shifts.

All the aromatic proton resonances in the 500-MHz NMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase. The conformation-dependent chemical shifts observed for many of the resonances could be explained qualitatively, but not quantitatively, in terms of ring-current shifts. The discrepancies between calculated ring-current shifts and the observed conformation-dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl-group anisotropic shielding contributions calculated using presently available models. In the case of the H delta 1, delta 2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.

Chemical Phenomena↗

PubMed1990

NMR and molecular dynamics studies of the mKr2 'zinc finger'.

We determined the structure of a 30-amino-acid 'zinc finger' motif from the mouse Kruppel-like gene mKr2 in solution in the absence of metal ions by two-dimensional NMR, distance geometry and restrained molecular dynamics methods. The most prominent secondary structural feature of the peptide is a helix extending from Ser14 to Ile20. The zinc-containing structure of the peptide was simulated by molecular dynamics calculations with restraints derived from the known geometry of the zinc ion and the ligating amino acid residues Cys6, Cys9, His22, His26. The latter structure does not deviate markedly from the set of structures determined for the zinc-free peptide, i.e. this peptide can accommodate a Zn2+ ion without major structural rearrangements. Thus, we propose that the metal ion stabilizes the existing three-dimensional peptide structural motif rather than introducing novel structural features.

Amino Acids↗